KR100716444B1 - Neutrokine-alpha and neutrokine-alpha splice variant - Google Patents

Abstract

The present invention relates to a novel cytokine named Neutrokine-alpha. The present invention also provides nucleic acid molecules encoding Neutrokine-alphaSV polypeptides, which are apparent splicing variants of Neutrokine-alpha and Neutrokine-alpha, vectors for producing them, host cells, and recombinant methods.

Neutrokine-alpha, Neutrokine-alphaSV polypeptide.

Description

Neutrokine-alpha and neutrokine-alpha splice variants             

The present invention relates to a novel cytokine named Neutrokine-alpha ("Neutrokine-alpha"). In addition, an apparent splicing variant of Neutrokine-alpha was identified and named Neutrokine-alphaSV. In certain embodiments, the invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alphaSV polypeptides. In a further aspect, there is also provided Neutrokine-alpha and Neutrokine-alphaSV polypeptides and vectors, host cells, and recombinant methods for producing them.

Human tumor necrosis factor (TNF-alpha) and (TNF-beta or lymphotoxin) are related members of a broad class of polypeptide mediators including interferons, interleukins and growth factors collectively referred to as cytokines. Beutler, B and Cerami, A., Annu. Rev. Immunol. 7: 625-655 (1989). Sequence analysis of cytokine receptors revealed several subfamily membrane proteins: (1) Ig superfamily, (2) hematopoietin (cytokine receptor superfamily), and (3) tumor necrosis factor ( TNF) / nerve growth factor (NGF) receptor superfamily has been identified (Gruss and Dower, Blood 85 (12): 3378-3404 (1985) and Aggarwal and Natarajan, Eur. Cytokine Netw., 7 (for TNF superfamily). 2): 93-124 (1996)). The TNF / NGF receptor superfamily contains at least 10 different proteins (Gruss and Dower, supra). Ligands for these receptors have been identified and identified as belonging to two or more cytokine superfamily (Gruss and Dower, supra).

Tumor necrosis factor (a mixture of TNF-alpha and TNF-beta) was initially discovered as a result of its anti-tumor activity, but is now a number of biological agents, including mediating apoptosis, cell activation and proliferation in some transgenic cell lines. It is recognized as a pleiotropic cytokine that can exhibit activity and plays an important role in immune regulation and inflammation.

To date, known members of the TNF-ligand superfamily include TNF-alpha, TNF-beta (lymphotoxin-alpha), LT-beta, OX40L, Fas ligand, CD30L, CD27L. CD40L and 4-IBBL are included. Ligands of the TNF ligand superfamily are acidic TNF-like molecules with about 20% sequence homology (range: 12% to 36%) in the extracellular domain and are primarily in membrane-bound form, wherein the biologically active form is Trimer / multimer complex. So far, soluble forms of the TNF ligand superfamily have been identified only for TNF, LT-beta and Fas ligands (Gruss, H. and Dower, SK, Blood, 85 (12): 3378-3404 (1995) for general review. ), The entire contents of these documents are incorporated herein by reference). These proteins are involved in the regulation of cell proliferation, activation and differentiation, including the regulation of cell survival or death by apoptosis or cytotoxicity. See also Armitage, R. J., Curr. Opin. Immunol. 6: 407 (1994) and Smith, C. A., Cell 75: 959 (1994)].

Tumor necrosis factor-alpha (also referred to as TNF-alpha; carnitine, hereinafter referred to as "TNF") is a soluble allotrimer of the 17 kD protein subunit by monocytes and macrophages that respond to endotoxins or other stimuli Is secreted mainly as a sieve. Smith, RA et al., J. Biol. Chem. 262: 6951-6954 (1987). The membrane-bound 26 kD precursor form of TNF is also described (Kriegler, M. et al., Cell 53: 45-53 (1988)).

According to much evidence, TNF is a regulatory cytokine with pleiotropic biological activity. These activities include inhibition of lipoprotein lipase synthesis (“cachettin” 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)], inhibiting cell growth or stimulating cell growth [Vilcek, J. et al., J. Exp. Med. 163: 632 (1986); Sugarman, B. J. et al., Science 230: 943 (1985); Lachman, L.B. et al., J. Immunol. 138: 2913 (1987)], cytotoxic action on certain transgenic cell types [see, Lachman, L.B. et al .; Darzynkiewicz, Z. et al., Canc. Res. 44:83 (1984)], antiviral activity [Kohase, M. et al., Cell 45: 659 (1986); Wong, G.H.W. et al., Nature 323: 819 (1986)], stimulation of bone resorption [Bertolini, D.R. et al., Nature 319: 516 (1986); Saklatvala, J., Nature 322: 547 (1986)], stimulation of collagenase and prostaglandin E2 production [Dayer, J.M. et al., J. Exp. Med. 162: 2163 (1985), and T cells (Tokota, S. et al., J. Immunol. 140: 531 (1988)], B cells (Kehl, J.H. et al., J. Exp. Med. 166: 786 (1987)], monocytes (Philippines, R. et al., Nature 323: 86 (1986)), thymic cells [Ranges, G.E. et al., J. Exp. Med. 167: 1472 (1988)] and stimulation of cell-surface expression of major histocompatibility complex (MHC) class I and class 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 its inflammatory process leading to tissue injury, for example, induction of coagulation stimulatory activity against vascular endothelial cells [Pober, J.S., et al., J. Immunol. 136: 1680 (1986)], and increased adsorption of neutrophils and lymphocytes [Pober, J.S., et al., J. Immunol. 138: 3319 (1987)) and stimulation of platelet activator secretion from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166: 1390 (1987).

Recent evidence suggests the development of many infectious diseases [Caeami, A. et al., Immunol. Today 9:28 (1988)], autoimmune diseases, oncological pathologies, such as some malignancies involving cachexia (Oliff, A. et al., Cell 50: 555 (1987)) and autoimmunity Pathology and Graft-versus-Host Pathology [Piguet, PF, et al., J. Exp. Med. 166-1280 (1987), to TNF. The association of TNF with cancer and infectious pathology is mainly related to the catabolic state of the host. The main problem for cancer patients is weight loss, which is usually associated with loss of appetite. The enormous breakdown that results is known as "cachexia" [Kern, K. A. et al. J. Parent. Enter. Nutr. 12: 286-298 (1988). Cachexia includes gradual weight loss, loss of appetite and persistent decrease in body mass in response to malignant growth. Thus, cachexia is associated with effective morbidity and is responsible for the majority of cancer mortality. Many studies have suggested that TNF is a critical mediator of cachexia in cancer, pathologies of infection and other catabolic conditions.

TNF is considered to play a key role in the pathophysiological consequences of Gram-negative sepsis and toxic shock, including fever, malaise, loss of appetite and cachexia. Michie, H. R. et al., Br. J. Surg. 76: 670-671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p. 463-466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5: 27-47 (1989). Endotoxins are described in TNF [Kornbluth, S.K. et al., J. Immunol. 137: 2585-2591 (1986)] and other cytokines are potent monocyte / macrophage activators that stimulate the production and secretion. Since TNF can mimic many of the biological effects of endotoxins, it is inferred to be a key mediator of the clinical symptoms of endotoxin-associated diseases. TNF and another monocyte-induced cytokine mediate metabolic and neurohormonal responses to endotoxins. Michie, H.R. et al., N. Eng. J. Med. 318: 1481-1486 (1988). Administering endotoxins to human volunteers causes acute diseases of cold-like symptoms, including fever, tachycardia, increased metabolic rate and stress hormone release. Revhaug, A. et al., Arch. Surg. 123: 162-170 (1988). Elevated levels of circulating TNF have also been found in patients with Gram-negative sepsis (Waage, A. et al., Lancet 1: 355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17: 489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161: 982-987 (1990).

Passive immunotherapy focusing on neutralizing TNF has beneficial effects in these pathologies based on increased TNF production and elevated TNF levels in the pathologies of Gram-negative sepsis and endotoxin, as discussed above. Can be represented. See: Cerami et al; EPO Patent Publication No. 0,212,489, filed Mar. 4, 1987, describes antibodies against a "modulator" substance identified as capetine (later found to be the same as TNF). Such antibodies are known to be useful for diagnostic immunoassays and shock treatment in bacterial infections. See Rubin et al; EPO Patent Publication No. O, 218,868, filed April 22, 1987, discloses monoclonal antibodies against human TNF, hybridomas secreting such antibodies, methods of producing such antibodies, and the immunoassay of these antibodies in the immunoassay of TNF. Uses are described. See, eg, Yone et al .; EPO Patent Publication No. 0,288,088, filed October 26, 1988, describes anti-TNF antibodies and pathologies comprising mAbs, in particular the use of these antibodies in the diagnosis of Kawasaki's pathology and immunoassay of bacterial infections. . Kawasaki disease (infant acute febrile mucosal skin lymph node syndrome) [Kawasaki, T., Allergy 16: 178 (1967); Kawasaki, T., Shonica (Pediatrics) 26: 935 (1985)] is considered to contain the elevated TNF levels associated with the progression of the disease [Yone et al., Supra]. .

Other researchers have described mAbs specific for recombinant human TNF with neutralizing activity in vitro. Liang, C-M. et al. Biochem. Biophys. Res. Comm. 137: 847-854 (1986); Meager, A. et al., Hybridoma 6: 305-311 (1987); Fendly et al., Hybridoma 6: 359-369 (1987); Bringman, T S et al., Hybridoma 6: 489-507 (1987); Hirai, M. et al., J. Immunol. Meth. 96: 57-62 (1987); Moller, A et al., (Cytokine 2: 162-169 (1990)) Some of these mAbs are used to map epitopes of human TNF and to develop enzymatic immunoassays [Fendly et al., Supra; Hirai et al, supra; Moller et al., Supra], are used to aid in the purification of recombinant TNF (Bringman et al., Supra) .However, these studies lack immunogenicity, specificity and / or medicament Due to their chemical suitability, they do not provide a basis for generating TNF neutralizing antibodies that can be used for diagnostic or therapeutic purposes in vivo in humans.

Neutralizing antiserum or mAb against TNF has been shown to resolve harmful physiological changes and prevent death after experimentally endotoxin and bacteremia in non-human mammals. This effect has been demonstrated, for example, in rodent lethal assays and primate pathology model systems. Mathison, J.C. et al., J. Clin. Invest. 81: 1925-1937 (1988); Beutler, B. et al., Science 229: 869-871 (1985); Tracey, K. J. et al., Nature 330: 662-664 (1987); Shimamoto, Y. et al., Immunol. Lett. 17: 311-318 (1988); Silva, A. T. et al., J. Infect. Dis. 162: 421-427 (1990); Opal, S. M. et al., J. Infect. Dis. 161: 1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30: 279-292 (1990).

To date, experiments using anti-TNF mAb therapy in humans have been limited, but have shown beneficial therapeutic results, for example in arthritis and sepsis. Elliott, M. J. et al., Baillieres Clin. Rheumatol. 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., Crit. Care. Med. 21: S 436-40 (1993); Tracey K. J., et al., Crit. Care Med. 21: S415-22 (1993).

The development of mammals depends not only on the proliferation and differentiation of cells but also on programmed cell death that occurs through apoptosis (Walker, et al., Methods Achiev. Exp. Pathol. 13:18 (1988). Apoptosis plays a crucial role in the destruction of immune thymic cells that recognize autoantigens. Failure of this normal clearance process may play a role in autoimmune diseases (Gammon et al., Immunology Today 12: 193 (1991)).

Itoh et al., Cell 66: 233 (1991) describe Fas / CD95, a cell surface antigen that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in ovaries of neutrophils, thymus, liver, heart, lungs and mature mice, in addition to activated T-cells, B-cells. Watanabe-Fukunaga et al., J. Immunol. 148: 1274 (1992). In experiments in which monoclonal Abs are cross-linked to Fas, apoptosis is induced [Yonehara et al., J. Exp. Med. 169: 1747 (1989); Trauth et al., Science 245: 301 (1989). In addition, there is an example that the binding of monoclonal Abs to Fas is a stimulus for T-cells under certain conditions [Alderson et al., J. Exp. Med. 178: 2231 (1993).

Fas antigen is a cell surface protein with a relative MW of 45 Kd. Both human and mouse genes for Fas have been cloned by Watanabe-Fukunaga et al., J. Immunol. 148: 1274 (1992) and Itoh et al., Cell 66: 233 (1991). Proteins encoded by these genes are transmembrane structurally homologous to two TNF receptors, low affinity neuronal growth factor receptors and neuronal growth factor / tumor necrosis factor receptor superfamily including CD40, CD27, CD30 and OX40 It is both a transmembrane protein.

Recently, a Fas ligand has been described (Suda et al., Cell 75: 1169 (1993)). The amino acid sequence is a type II transmembrane protein whose Fas ligand belongs to the TNF family. Thus, the Fas ligand polypeptide comprises three major domains: the short intracellular domain at the amino terminus and the longer extracellular domain at the carboxy terminus, linked by hydrophobic transmembrane domains. Fas ligand is expressed in splenocytes and thymus cells that are closely related to T-cell mediated cytotoxicity. The MW of the purified Fas ligand is 40 kD.

Recently, it has been demonstrated that Fas / Fas ligand interactions are required for apoptosis after activation of T-cells. Ju et al., Nature 373: 444 (1995); Brunner et al., Nature 373: 441 (1995). Activation of T-cells induces both proteins on the cell surface. Subsequent ligand-receptor interactions lead to apoptosis of the cell. This supports a possible regulatory role for apoptosis induced by Fas / Fas ligand interactions during normal immune responses.

Thus, there is a need to provide cytokines similar to TNF that are associated with pathological symptoms. Such novel cytokines can be used to prepare novel antibodies or other antagonists that bind to the TNF-like cytokines for the diagnosis and treatment of TNF-like cytokine related diseases.

Summary of the Invention

According to one aspect of the invention, the novel extracellular domain of the Neutrokine-alpha polypeptide, the novel extracellular domain of the Neutrokine-alphaSV polypeptide, and their biologically active, diagnostic or therapeutically useful fragments , Analogs and derivatives are provided.

According to another aspect of the invention, human Neutrokine-alpha or Neutrokine-alpha, including mRNA, DNA, cDNA and genomic DNA, as well as analogues and biologically active and diagnostic or therapeutically useful fragments and derivatives thereof An isolated nucleic acid molecule is provided that encodes an SV.

The present invention includes isolated nucleic acids comprising or alternatively consisting of polynucleotides encoding cytokines and apparent splice variants thereof that are structurally similar to TNF and related cytokines and exhibit similar biological effects and activities. Provide a molecule. Said cytokine is named Neutrokine-alpha and the present invention is directed to the amino acid sequence of SEQ ID NOS: 1A and 1B (SEQ ID NO: 2) or cDNA clone (HNEDU15), deposited on October 22, 1996, assigned ATCC No. 97768. A Neutrokine-alpha polypeptide having at least a portion of an amino acid sequence that is encoded. The nucleotide sequence measured by sequencing the deposited Neutrokine-alpha clone, shown in FIGS. 1A and 1B (SEQ ID NO: 1), consists of an N-terminal methionine, an estimated intracellular domain consisting of about 46 amino acid residues, about 26 amino acids. It contains an open reading frame that encodes a complete polypeptide of 285 amino acid residues comprising an estimated transmembrane domain consisting of an estimated extracellular domain consisting of about 213 amino acids and an estimated extracellular domain consisting of about 213 amino acids. In the case of another type II transmembrane protein, the soluble form of Neutrokine-alpha is a complete Neutrokine-alpha polypeptide that lacks a transmembrane domain and all or a portion of the extracellular domain cleaved from the transmembrane domain, ie Polypeptides comprising extracellular domains linked to intracellular domains.

The apparent splice variant of Neutrokine-alpha is termed Neutrokine-alphaSV and the present invention has been assigned the amino acid sequence of SEQ ID NOS: 5A and 5B (SEQ ID NO: 19) or Dec. 10, 1998, assigned ATCC No. 203518. a Neutrokine-alphaSV polypeptide comprising at least a portion of an amino acid sequence encoded by a cDNA clone HDPMC52 or alternatively consisting of such sequence. The nucleotide sequence (SEQ ID NO: 18) measured by sequencing the deposited Neutrokine-alphaSV clone, shown in FIGS. 5A and 5B, is N-terminal methionine, putative intracellular domain consisting of about 46 amino acid residues, about 26 amino acids. An open reading frame encoding an intact transmembrane domain consisting of an estimated extracellular domain of about 194 amino acids and an intact polypeptide consisting of 266 amino acid residues having an estimated molecular weight of about 29 kDa. . In the case of another type II transmembrane protein, the soluble form of Neutrokine-alphaSV is a complete Neutrokine-alphaSV polypeptide that lacks all or a portion of the extracellular domain and the transmembrane domain cleaved from the transmembrane domain. Ie, polypeptides comprising extracellular domains linked to intracellular domains.

Thus, one aspect of the present invention is (a) full length Neutrokine- having the complete amino acid sequence of SEQ ID NOS: 1A and 1B (SEQ ID NO: 2) or encoded by a cDNA clone contained in a deposit of ATCC Accession No. 97768. Nucleotide sequence encoding an alpha polypeptide, (b) a neutron sequence having the amino acid sequence at positions 73-285 in FIGS. 1A and 1B (SEQ ID NO: 2) or encoded by a clone contained in a deposit of ATCC Accession No. 97768 Nucleotide sequence encoding a putative extracellular domain of a keen-alpha polypeptide, (c) a nucleotide sequence encoding a fragment of the polypeptide of (b) having a Neutrokine-alpha functional activity (eg, biological activity), (d ) Neutrokine-alpha intracellular domain (presumed to consist of amino acid residues from about 1 to about 46 of Figures 1A and 1B (SEQ ID NO: 2)) or ATCC Accession No. 97768 A nucleotide sequence encoding a polypeptide encoded by a clone contained in a deposit of (e) about 47 to about 72 amino acid residues of the Neutrokine-alpha transmembrane domain (FIGS. 1A and 1B (SEQ ID NO: 2)) Nucleotide sequence encoding a polypeptide encoded by a clone contained in the deposit of ATCC Accession No. 97768, (f) having extracellular and intracellular domains but lacking a transmembrane domain A nucleotide sequence encoding a soluble Neutrokine-alpha polypeptide, and (g) a nucleotide sequence complementary to any one of (a), (b), (c), (d), (e) or (f) above. Include, or alternatively include, polynucleotides having a nucleotide sequence selected from the group consisting of nucleotide sequences It provides an isolated nucleic acid molecule consisting of OTA Jethro.

A further aspect of the invention is a nucleotide sequence of any of (a), (b), (c), (d), (e), (f) or (g) with 80%, 85% or 90% Or more preferably, at least 95%, 96%, 97%, 98%, or 99% of the same nucleotide sequences having the same nucleotide sequence or stringent hybridization conditions under (a), (b), (c or polynucleotides that hybridize with the polynucleotides of (d), (e), (f) or (g), or alternatively include isolated nucleic acid molecules consisting of such polynucleotides. Polynucleotides that hybridize do not hybridize under strict hybridization conditions with polynucleotides consisting of only A residues or having nucleotide sequences consisting of only T residues.

Another aspect of the invention is (a) by a cDNA clone having the complete amino acid sequence of SEQ ID NOS: 5A and 5B (SEQ ID NO: 19) or contained in an ATCC deposit of ATCC Accession No. 203518 deposited on 10 December 1998. Nucleotide sequence encoding the full-length Neutrokine-alphaSV polypeptide to be encoded, (b) an ATCC agent having an amino acid sequence from positions 73 to 266 of FIGS. 1A and 1B (SEQ ID NO: 2) or deposited on 10 December 1998; Nucleotide sequence encoding the putative extracellular domain of the Neutrokine-alphaSV polypeptide encoded by the cDNA clone contained in 203518, (c) Neutrokine-alphaSV intracellular domain (Figures 5A and 5B (SEQ ID NO: 19)). Nucleotides encoding polypeptides encoded by the cDNA clones contained in ATCC 203518 deposited on Dec. 10, 1998, or deposited on Dec. 10, 1998). De sequence, (d) comprising the Neutrokine-alphaSV transmembrane domain (presumed to consist of about 47 to about 72 amino acid residues of FIGS. 5A and 5B (SEQ ID NO: 19)) or deposited on 10 December 1998. A nucleotide sequence encoding a polypeptide encoded by a cDNA clone contained in ATCC 203518, (e) encoding a soluble Neutrokine-alphaSV polypeptide having extracellular and intracellular domains but lacking a transmembrane domain A nucleotide sequence and (f) a polynucleotide having a nucleotide sequence selected from the group consisting of a nucleotide sequence complementary to any one of (a), (b), (c), (d) or (e) Alternatively, an isolated nucleic acid molecule consisting of such polynucleotides is provided.

Another aspect of the invention, at least 80%, 85% or 90%, more preferably any one of the nucleotide sequence of (a), (b), (c), (d), (e) or (f) Preferably a polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence or (a), (b), (c), (d), (e) or (f) Polynucleotides that hybridize under stringent hybridization conditions with polynucleotides of or alternatively include isolated nucleic acid molecules consisting of such polynucleotides. Polynucleotides that hybridize do not hybridize under stringent hybridization conditions with polynucleotides consisting of only A residues or having nucleotide sequences consisting of only T residues.

In one embodiment, the amino acid sequence of Gly-142 to Leu-266 as shown in FIGS. 5A and 5B (SEQ ID NO: 19) or by cDNA clone HDPMC52, deposited on December 10, 1998, assigned ATCC Accession No. 203518 Obvious splice variants of Neutrokine-alpha are provided which comprise or alternatively consist of at least a portion of the amino acid sequence to be encoded.

In a further embodiment, the nucleic acid molecule of the invention is Neutrokine-alpha or Neutrope having the amino acid sequence of (a), (b), (c), (d), (e), (f) or (g) above. It comprises, or alternatively consists of, a polynucleotide encoding the amino acid sequence of an epitope-bearing portion of a keen-alphaSV polypeptide. Further nucleic acid embodiments of the present invention comprise amino acid sequences containing at least one to 50, preferably 40, more preferably 30, even more preferably up to 20 amino acid additions, substitutions and / or deletions. A polynucleotide encoding the amino acid sequence of a Neutrokine-alpha or Neutrokine-alphaSV polypeptide having or alternatively relates to an isolated nucleic acid molecule consisting of such polynucleotides. Of course, as the order of increasing the preferred degree, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than 1 or 1 to 100, 1 to 50, 1 to 25, 1 to 20 Encoding the amino acid sequence of a Neutrokine-alpha or Neutrokine-alphaSV polypeptide having an amino acid sequence containing the addition, substitution and / or deletion of dogs, 1-15, 1-10 or 1-5 amino acids Polynucleotides are very preferred. Preservation substitutions are preferred.

The present invention also relates to recombinant vectors comprising the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, as well as methods of making such vectors and host cells and using them by means of recombinant techniques and Neutrokine-alpha polypeptides. It relates to a method of manufacturing.

In a further aspect of the invention, the prokaryotic and / or eukaryotic host cell containing the Neutrokine-alpha or Neutrokine-alphaSV nucleic acid sequence of the invention is cultured under conditions that promote expression of said polypeptide and then said poly Provided are methods for preparing the polypeptide by recombinant techniques, including recovering the peptide.

In addition, the present invention (a) is a full length Neutrokine- having the complete amino acid sequence shown in FIGS. 1A and 1B (positions 1 to 285 of SEQ ID NO: 2) or encoded by a cDNA plasmid contained in a deposit of ATCC Accession No. 97768 Amino acid sequence of an alpha polypeptide, (b) an amino acid sequence of a full length Neutrokine-alpha polypeptide having the complete amino acid sequence set forth in SEQ ID NO: 2, excluding N-terminal methionine (positions 2 to 285 of SEQ ID NO: 2), (c) A fragment of the polypeptide of (b) having Neutrokine-alpha functional activity (eg, biological activity), (d) having an amino acid sequence at positions 73 to 285 shown in FIGS. 1A and 1B (SEQ ID NO: 2) or having an ATCC accession number Amino acid sequence of the putative extracellular domain of the Neutrokine-alpha polypeptide encoded by the cDNA plasmid contained in the deposit of No. 97768, (e) the amino acid sequence of positions 134-285 shown in FIGS. 1A and 1B (SEQ ID NO: 2) Nucleotide sequence encoding a Neutrokine-alpha polypeptide having (f) a Neutrokine-alpha intracellular domain (presumed to contain about 1 to 46 amino acid residues shown in FIGS. 1A and 1B (SEQ ID NO: 2)) An amino acid sequence encoded by a cDNA plasmid containing or contained in a deposit of ATCC Accession No. 97768, (g) about 47 to about 72 described in Neutrokine-alpha transmembrane domain (FIGS. 1A and 1B (SEQ ID NO: 2)) Amino acid sequence encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768, or (h) extracellular and intracellular domains, but lacking a transmembrane domain Amino acid sequence of the soluble Neutrokine-alpha polypeptide, wherein each of the domains mentioned is as defined above and (i) the above (a), (b), (c), (d), (e) , (f), ( An isolated Neutrokine-alpha polypeptide comprising an amino acid sequence selected from the group consisting of fragments of the polypeptide of g) or (h), or alternatively consisting of such amino acid sequence. Polypeptides of the present invention may also be used in combination with the polypeptides described in (a), (b), (c), (d), (e), (f), (g), (h) or At least 80%, preferably at least 85% or 90%, more preferably at least 95% to 99% polypeptide having the same amino acid sequence, as well as at least 80%, 85% or 90% similarity to the above, more preferably Polypeptides having an amino acid sequence having at least 95% similarity. Further embodiments of the present invention comprise amino acid sequences described in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above. It relates to a polypeptide comprising or alternatively consisting of the amino acid sequence of the epitope-containing portion of a Neutrokine-alpha polypeptide having. Polypeptides having the amino acid sequence of the epitope-containing portion of the Neutrokine-alpha polypeptide according to the present invention may be 4-8 or more, preferably 9-15 or more, 20 or more, 25 or more, 30 or more, A portion of said polypeptide consisting of at least 40, at least 50, more preferably from about 30 to 50 amino acids, and comprises a whole amino acid sequence of the polypeptide of the invention Epitope-containing polypeptides are included in the present invention.

Very preferred embodiments of the present invention are polynucleotide sequences encoding Neutrokine-alpha polypeptides having amino acid sequences at positions 134 to 285 of Figures 1A and 1B (SEQ ID NO: 2) and 80%, 85%, 90% or more, more preferably Preferably it relates to a nucleic acid molecule comprising or alternatively consisting of polynucleotides having the same nucleotide sequence of 95%, 96%, 97%, 98%, 99% or 100%. Preferred embodiments of the invention comprise polynucleotides having a nucleotide sequence that is at least 90% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134 to 285 of Figures 1A and 1B (SEQ ID NO: 2); Alternatively, to a nucleic acid molecule consisting of such polynucleotides. More preferred embodiments of the invention include polynucleotides having a nucleotide sequence that is at least 95% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134 to 285 of FIGS. 1A and 1B (SEQ ID NO: 2). Or alternatively, to a nucleic acid molecule consisting of such polynucleotides. More preferred embodiments of the present invention include polynucleotides having a nucleotide sequence that is at least 96% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 of FIGS. 1A and 1B (SEQ ID NO: 2). Or alternatively, to a nucleic acid molecule consisting of such polynucleotides. In addition, a more preferred embodiment of the invention is a poly having a nucleotide sequence that is at least 97% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising or alternatively consisting of such polynucleotides. Additionally, more preferred embodiments of the present invention have a nucleotide sequence that is at least 98% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134 to 285 shown in Figures 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising, or alternatively consisting of, polynucleotides. Additionally, more preferred embodiments of the invention have a nucleotide sequence that is at least 99% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising, or alternatively consisting of, polynucleotides.

In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also included in the present invention.

The invention also relates to (a) cDNA clones contained in ATCC Accession No. 203518 having the complete amino acid sequence shown in FIGS. 5A and 5B (positions 1 to 266 of SEQ ID NO: 19) or deposited December 10, 1998. Full length Neutrokine-alphaSV poly having the amino acid sequence of full length Neutrokine-alphaSV polypeptide encoded by (b) the complete amino acid sequence shown in SEQ ID NO: 19, excluding N-terminal methionine (positions 2 to 266 of SEQ ID NO: 19) Amino acid sequence of the peptide, (c) by a cDNA clone having an amino acid sequence at positions 73 to 266 shown in FIGS. 5A and 5B (SEQ ID NO: 19) or contained in ATCC Accession No. 203518 deposited December 10, 1998 Amino acid sequence of the putative extracellular domain of the Neutrokine-alphaSV polypeptide encoded, (d) about 1 to 46 amino acid residues of the Neutrokine-alphaSV intracellular domain (FIGS. 5A and 5B (SEQ ID NO: 19)) Allegedly) Amino acid sequence encoded by a cDNA clone comprising or contained in ATCC Accession No. 203518, (e) amino acid residues from about 47 to about 72 of the Neutrokine-alphaSV transmembrane domain (FIGS. 5A and 5B (SEQ ID NO: 19)) Amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 203518 deposited on Dec. 10, 1998, or (f) an extracellular and intracellular domain, but including a membrane Amino acid sequence of the soluble Neutrokine-alphaSV polypeptide lacking a penetrating domain, wherein each domain mentioned is as defined above; and (g) (a), (b), (c), (d) An isolated Neutrokine-alphaSV polypeptide comprising an amino acid sequence selected from the group consisting of fragments of the polypeptides of (a), (e) or (f), or alternatively consisting of such amino acid sequences. Polypeptides of the invention can also be used in combination with the amino acid sequences described in (a), (b), (c), (d), (e), (f) or (g) above 80%, 85% or 90%. Or more preferably more than 95% to 99% of polypeptides having identical amino acid sequences, as well as polypeptides having an amino acid sequence having at least 80%, 85% or 90% similarity, more preferably at least 95% similarity . A further aspect of the invention is a Neutrokine-alphaSV polypeptide having the amino acid sequence described in (a), (b), (c), (d), (e), (f) or (g) above. A polypeptide comprising or alternatively comprising an amino acid sequence of an epitope-containing portion of a. Peptides or polypeptides having the amino acid sequence of the epitope-containing portion of the Neutrokine-alphaSV polypeptide according to the present invention are 4 to 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 Above, more preferably an epitope-containing poly that comprises a portion of said polypeptide consisting of about 30 to 50 amino acids, and which comprises the entire amino acid sequence of the polypeptide of the invention described above and is less than or equal to Peptides are included in the present invention.

Certain common embodiments of the present invention comprise the amino acid sequences described in A polypeptide having an amino acid sequence of an epitope-containing portion of a Neutrokine-alpha or Neutrokine-alphaSV polypeptide. In another embodiment, the present invention provides an amino acid sequence as described in (a), (b), (c), (d), (e), (f), (g), (h) Provided are isolated antibodies that bind specifically (ie, specifically) to Neutrokine-alpha or Neutrokine-alphaSV polypeptides having.

The present invention also provides a method of separating an antibody that specifically (ie, specifically) binds to a Neutrokine-alpha or Neutrokine-alphaSV polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.

The invention also relates to soluble Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides, in particular human Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or anti-Neutrokine-alpha antibodies and / or Anti-Neutrokine-alphaSV antibodies and include, for example, treating tumors and tumor metastases, infections of bacteria, viruses and other parasites, immunodeficiency, inflammatory diseases, lymphadenopathy, autoimmune diseases, graft-versus-host disease Used to prevent, prognose and / or diagnose, stimulate peripheral resistance, destroy some transgenic cell lines, mediate cell activation, survival and proliferation, mediate immune regulation and inflammatory responses, or enhance or suppress immune responses. It provides a pharmaceutical composition that can be.

In certain embodiments, the soluble Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or agonists thereof of the present invention are immunodeficiency (eg, severe combined immunodeficiency (SCID) -reflective, SCID-autosomal, adenosine desings). Aminase deficiency (ADA deficiency), reflex anzamagglobulinemia (XLA), Bruton's disease, congenital agammagglobulinemia, congenital infantile agammagglobulinemia, acquired agammagglobulinemia, adult-onset agammaglobulin Hyperglycemia, late-onset gamma globulinemia, dysgammaglobulinemia, reduced maglobulinemia, transient reduced maglobulinemia in infants, non-specific reduced maglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiscott-Aldrich syndrome (WAS), IgM hyperimmune immunodeficiency, IgM hyperimmune immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without accompanying IgA deficiency) ), Normal or elevated Ig bearing antibody deficiency, immunodeficiency with thymic cancer, Ig heavy chain deletion, kappa chain deficiency, B cell lymphocytic proliferative disease (BLPD), selective IgM immunodeficiency disease, recessive agammaglobulinemia (Swiss type) ), Delusional dysplasia, neonatal neutropenia, severe congenital leukopenia, thymic lymphocytic dysplasia with acute immunodeficiency- aplasia or dysplasia, ataxia-capillary dilatation, complex dwarfism, seminal lymphocytic syndrome (XLP), negelof Treating, preventing, prognostic and treating symptoms associated with syndrome-combinative Ig immunodeficiency, purine-based nucleoside phosphorylase deficiency (PNP), MHC type II deficiency (bare lymphocyte syndrome) and severe combined immunodeficiency syndrome) or immunodeficiency And / or to diagnose.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, prognose and / or diagnose a variety of conventional immunodeficiencies.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, prognostic and / or diagnose semi-agammaglobulinemia.

In another specific embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, prognostic and / or diagnose severe combined immunodeficiency syndrome (SCID). do.

In another particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof treat, prevent, prognose Wiskott-Aldrich syndrome. And / or to diagnose.

In another particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof treat, prevent, prognose and / or diagnose semi-Ig deficiency with excess IgM. Is administered.

In another embodiment, Neutrokine-alpha antagonists and / or Neutrokine-alphaSV antagonists (eg, anti-Neutrokine-alpha antibodies) are autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, idiopathic thrombocytopenia) Retinopathy, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune thrombocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy) , Multiple sclerosis, neuritis, uveitis keratoconjunctivitis, polyendocrine dysplasia, purpura (e.g., Henro-Skoenlein purpura), Later's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barr syndrome, insulin-dependent diabetes and autoimmunity Inflammatory eye, autoimmune thyroiditis, hypothyroidism (ie Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, receptor autoimmunity, e.g. Cotton, (a) Grave's Disease, (b) Myasthenia Gravis and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed Sexual connective tissue disease, polymyositis / dermatitis, pernicious anemia, idiopathic Edison disease, infertility, glomerulonephritis (eg primary glomerulonephritis and IgA nephropathy), ileal swelling, Sjogren's syndrome, diabetes and adrenergic drug resistance (asthma or cystic) Adrenergic drug resistance with fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine glands, vitiligo, vasculitis, post-MI, heart incision syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy and Other inflammatory, granulomatous, degenerative and atrophic diseases) or autoimmune diseases related symptoms, is administered to treat, prevent, prognostic and / or diagnose. In a direct embodiment, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention are used to treat, prevent, prognose and / or diagnose rheumatoid arthritis. In certain preferred embodiments, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention are used to treat, prevent, prognostic and / or diagnose systemic lupus erythematosus. In another preferred particular embodiment, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention are used to treat, prevent, prognostic and / or idiopathic thrombocytopenic purpura. Diagnose In another preferred particular embodiment, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention are used to treat, prevent, prognostic and / or diagnose IgA nephropathy. In another preferred specific embodiment, the autoimmune diseases and disorders and / or the diseases and disorders described above using the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention. Treating, preventing, prognosis and / or diagnosing symptoms associated with the disease.

The invention also provides for administration to in vitro cells, ex vivo cells, in vivo cells or multicellular organisms, with Neutrokine-alpha or Neutrokine-alphaSV polynucleotides, Neutrokine-alpha or Neutrokine-alphaSV polypeptides and And / or a composition comprising an anti-Neutrokine-alpha antibody or an anti-Neutrokine-alphaSV antibody. In a preferred embodiment, the compositions of the present invention comprise Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides for the expression of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides in a host organism for the treatment of a disease. Include. In a most preferred embodiment, the compositions of the present invention comprise Neutrokine-alpha for the expression of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides in a host organism for the treatment of immunodeficiency and / or immunodeficiency related conditions. Or Neutrokine-alphaSV polynucleotides. Particularly preferred in this respect is the expression in human patients (eg, by increasing B-cell numbers or prolonging B cell life) for the treatment of disorders associated with abnormal endogenous activity of Neutrokine-alpha or Neutrokine-alphaSV genes. Expression to enhance normal B-cell function).

The invention also provides a standard cellular response in which cells expressing Neutrokine-alpha and / or Neutrokine-alphaSV are contacted with a candidate compound, the cellular response is analyzed, and the cellular response is made in the absence of the candidate compound. A screening method is provided for identifying compounds capable of enhancing or inhibiting the cellular response induced by Neutrokine-alpha and / or Neutrokine-alphaSV, including comparing with Cellular response indicates that the compound is an agonist and reduced cellular response compared to standard cellular response indicates that the compound is an antagonist.

In another aspect, methods are provided for identifying Neutrokine-alpha and / or Neutrokine-alphaSV receptors, as well as assays for using such receptors to select agonists and antagonists. The assay includes measuring the effect of the candidate compound on Neutrokine-alpha and / or Neutrokine-alphaSV that the candidate compound binds to Neutrokine-alpha and / or Neutrokine-alphaSV receptors. In particular, the method comprises contacting the Neutrokine-alpha and / or Neutrokine-alphaSV receptors with the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and candidate compounds of the invention and neutralizing Neutrokine-alpha and / or Neutrokine. Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides that bind to the kin-alphaSV receptor include determining whether there is an increase or decrease due to the presence of the candidate compound. Antagonists are septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-versus-host rejection, bone resorption, rheumatoid arthritis, cachexia (weakness or malnutrition), immune system function, lymphoma, and autoimmune diseases (eg : Can be used to prevent rheumatoid arthritis and systemic lupus erythematosus.

We found that Neutrokine-alpha was found in cells of mononuclear lineage as well as in kidney, lung, peripheral leukocytes, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages and umbilical cord blood tissue. It was found that it is expressed. We also found that Neutrokine-alphaSV is highly expressed only in primary dendritic cells. Many diseases of these tissues and cells, such as tumors and tumor metastases, infections of bacteria, viruses and other parasites, immunodeficiency (e.g. chronic impaired immunodeficiency), septic shock, inflammation, cerebral malaria, activation of the HIV virus Patients suffering from such grafts, graft-versus-host rejection, bone resorption, rheumatoid arthritis, autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, and cachexia (weakness or malnutrition) In body fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid, or certain tissues (e.g. bone marrow)) obtained from Significantly higher or lower levels of Neutrokine-alpha and / or Neutrokine-al compared to Neutrokine-alpha and / or Neutrokine-alphaSV expression levels in body fluids or tissues from individuals who are not It is contemplated that the present invention may detect (a) Neutrokine-alpha and / or Neutrokine-alphaSV gene expression levels in cells or body fluids of individuals and (b) Neutro Diagnostic methods useful during the diagnosis of a disease, including comparing kin-alpha and / or Neutrokine-alphaSV gene expression levels to standard Neutrokine-alpha and / or Neutrokine-alphaSV gene expression levels, wherein An increase or decrease in Neutrokine-alpha and / or Neutrokine-alphaSV gene expression levels assayed compared to standard expression levels indicates the presence of a disease.

A further aspect of the present invention comprises administering to a subject a composition comprising a therapeutically effective amount of an isolated Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or an agonist thereof in an individual. A method of treating an individual in need of increased or constitutive levels of Neutrokine-alpha and / or Neutrokine-alphaSV activity.

A still further aspect of the invention comprises administering to the individual a composition comprising a therapeutically effective amount of an isolated Neutrokine-alpha and / or Neutrokine-alphaSV antagonist according to the invention to a Neutrokine-alpha in the body. And / or methods of treating an individual in need of reduced levels of Neutrokine-alphaSV activity. Preferred antagonists for use in the present invention are Neutrokine-alpha-specific and / or Neutrokine-alphaSV-specific antibodies.

The following drawings illustrate aspects of the invention and do not limit the scope of the invention described in the claims.

1A and 1B show the nucleotide (SEQ ID NO: 1) and putative amino acid (SEQ ID NO: 2) sequences of Neutrokine-alpha. Amino acids 1-46 represent putative intracellular domains, amino acids 47-72 represent putative transmembrane domains (doubled underlined sequences), and amino acids 73-285 represent putative extracellular domains (resting sequences). Potential asparagine-linked glycosylation sites are shown in FIGS. 1A and 1B and are bold symbols over the first nucleotide encoding the asparagine residue in the Neutrokine-alpha nucleotide sequence, as symbol N in bold in the Neutrokine-alpha amino acid sequence. It is indicated by the symbol (#). Potential N-linked glycosylation sequences are found at the following positions of the Neutrokine-alpha amino acid sequence: N-124 to Q-127 (N-124, S-125, S-126, Q-127) and N- 242 to C-245 (N-242, N-243, S-244, C-245).

The region of high identity between Neutrokine-alpha, Neutrokine-alphaSV, TNF-alpha, TNF-beta, LT-beta, and closely related Fas ligands (these sequences are shown in FIG. 2) is shown in FIGS. Underline 1B. These regions are not limited and conserved domain (CD) -I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD-VIII in FIGS. 1A and 1B. , CD-IX, CD-X and CD-XI.

2A and 2B show Neutrokine-alpha (SEQ ID NO: 2) and Neutrokine-alphaSV (SEQ ID NO: 19), TNF-alpha (FIG. 2A) measured by the "MegAlign" routine, which is part of a computer program named "DNA * STAR". And "TNFalpha" in 2B; Genbank No. Z15026; SEQ ID NO: 3), TNF-beta ("" TNFbeta "in Gen. 2A and 2B; Genbank No. Z15026; SEQ ID NO: 4), Lymphotoxin-beta (Fig. 2A). And region of identity between "LTbeta; GenBank L11016; SEQ ID NO: 5) and FAS ligand (" FASL "in FIGS. 2A and 2B; GenBank U11821; SEQ ID NO: 6). Residues that match the conserved region are shaded.

3 shows the analysis results of the Neutrokine-alpha amino acid sequence. Alpha, beta, turn and coil regions by estimating the amino acid sequence of SEQ ID NO: 2 using the default parameters of the computer program described above; Hydrophilic and hydrophobic; Amphoteric region; Variable region; Antigen index and surface probability. The "Antigenic Index-Jameson-Wolf" graph shows the position of the high antigenic region of Neutrokine-alpha, the region from which the epitope-containing peptide of the present invention can be obtained. The antigenic polypeptide may comprise amino acid residues of SEQ ID NO: 2 about Phe-115 to about Leu-147, about Ile-150 to about Tyr-163, about Ser-171 to about Phe-194, about Glu-223 to about Tyr-246 and From about Ser-271 to about Phe-278.

The data presented in FIG. 3 is also shown in Table 1. Columns are indicated by the header "Res", "position" and Roman numerals I to XIV. The column headings characterize the amino acid sequences described in Figure 3 and Table 1 as follows: "Res": amino acid residues of SEQ ID NO: 2 and Figures 1A and 1B; "Position": position of the corresponding residue in SEQ ID NO: 2 and FIGS. 1A and 1B; I: alpha, region-Garnier-Robson; II: beta, domain-Chou-Fasman; III: beta, region-Garnier-Robson; IV: beta, region-Chow-Pasman; V: turn, region-Garnier-Robson; VI: turn, area-Chow-Pasman; VII: coil, region—Garnier-Robson; VIII: hydrophilic float-Kyte Doolittle; IX: hydrophobicity float-Hop-Woods; X: alpha, amphoteric region-Eisenberg; XI: beta, amphipathic region-Eisenberg; XII: variable region-Karplus-Schlz; XIII: Antigen Index-James-Wolf; And XIV: Surface Probability Float-Emini.

4A, 4B and 4C show the Neutrokine-alpha nucleotide sequence measured from human cDNA deposited with ATCC No. 97768 and the relevant persons of the present invention designated HSOAD55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8), and HLTBM08 (SEQ ID NO: 9). Represents an array of cDNA clones.

5A and 5B show the nucleotide (SEQ ID NO: 18) and putative amino acid (SEQ ID NO: 19) sequences of Neutrokine-alphaSV proteins. Amino acids 1-46 represent putative intracellular domains, amino acids 47-72 represent putative transmembrane domains (doubled underlined sequences), and amino acids 73-266 represent putative extracellular domains (resting sequences). Potential asparagine-linked glycosylation sites are shown in FIGS. 5A and 5B and are asparagine bold symbols N in the Neutrokine-alphaSV amino acid sequence, bold over the first nucleotide encoding that asparagine residue in the Neutrokine-alphaSV nucleotide sequence. It is indicated by pound signs (#). Potential N-linked glycosylation sequences are found at the following positions of the Neutrokine-alphaSV amino acid sequence: N-124 to Q-127 (N-124, S-125, S-126, Q-127) and N -223 to C-226 (N-223, N-224, S-225, C-226). The antigenic polypeptide may comprise about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro-218 to about Tyr-227 of the amino acid sequence of SEQ ID NO: 19 , About Ala-232 to about Gln-241, about Ile-244 to about Ala-249 and about Ser-252 to about Val-257.

The regions of high identity between Neutrokine-alpha, Neutrokine-alphaSV, TNF-alpha, TNF-beta, LT-beta, and closely related Fas ligands (the arrangement of these sequences are shown in Figure 2) are shown in Figures 1A and 1B. Underline These conserved regions (of Neutrokine-alpha and Neutrokine-alphaSV) are conserved domains (CD) -I, CD-II, CD-III, CD-V, CD-VI, CD-VII, in FIGS. 5A and 5B. Labeled as CD-VIII, CD-IX, CD-X and CD-XI. Neutrokine-alphaSV does not contain the sequence of CD-IV described in the legend of FIGS. 1A and 1B.

Additional arrangements of APRIL, TNF alpha and LT alpha with Neutrokine-alpha polypeptide sequence (SEQ ID NO: 2) are shown in FIG. 7A. In FIG. 7A, the beta sheet region is indicated as described below in FIG. 7A legend.

6 shows analysis of Neutrokine-alphaSV amino acid sequence. Alpha, beta, turn, and coil regions by estimating the amino acid sequence of SEQ ID NO: 19 using default parameters of the computer program described above; Hydrophilic and hydrophobic; Amphoteric region; Variable region; Antigen index and surface probability. The "Antigen Index-James-Wolf" graph indicates the location of the high antigenic region of the Neutrokine-alpha protein (ie, the region from which the epitope-containing peptide of the present invention can be obtained). Antigenic polypeptides include, but are not limited to, amino acid residues of SEQ ID NO: 19 about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro-218 A polypeptide comprising from about Tyr-227, about Ser-252 to about Thr-258, about Ala-232 to about Gln-241, about Ile-244 to about Ala-249 and about Ser-252 to Val-257 Include.

The data shown in FIG. 6 can be easily displayed in a tabular form similar to the data shown in Table 1. The tabular form of the exact data described in FIG. 6 can be created using the MegAlign component of the DNA * STAR computer sequencing package set as default parameter. This is the same program used to prepare the FIGS. 3 and 6 herein.

Figure 7A. The amino acid sequence of Neutrokine-alpha and its estimated ligand-binding domains and APRIL, TNF-alpha and LT-alpha (specifically, human APRIL polypeptides (SEQ ID NO: 20; amino acid residues 115-250 of ATCC Accession No. AF046888) , Amino acid residues 88 to 233 of TNF alpha (SEQ ID NO: 3; Genbank Accession No. Z15026) and LT alpha (also named amino acid residues 62 to 205 of SEQ ID NO: 4; Genbank Accession No. Z15026) ) Array. The estimated membrane transit region of Neutrokine-alpha is indicated and the cleavage site of Neutrokine-alpha is shown by an arrow. The sequences represented by lines A through H represent the estimated beta-pleat sheet regions.

Figure 7B. Expression of Neutrokine-alpha mRNA. Northern hybridization is performed using Neutrokine-alpha orf as a probe for blots of poly (A) + RNA (clonetech) from human tissue type spectra and as a probe for the selection of cancer cell lines. Neutrokine-alpha mRNA of 2.6 kb is detected at high levels in the placenta, heart, lung, fetal liver, thymus and pancreas. In addition, 2.6 kb of Neutrokine-alpha mRNA was detected in HL-60 and K562 cell lines.

8A and 8B. Neutrokine-alpha expression increases after activation of human monocytes by IFN-gamma. 8A. Flow cytometry analysis of Neutrokine-alpha protein expression in monocytes cultured in vitro. Purified mononuclear cells are cultured for 3 days in the presence or absence of IFN-gamma (100 U / ml). Cells were then stained with Neutrokine-alpha-specific mAb (2E5) (solid line) or isotype-matched control (IgG1) (dotted line). Comparative results are obtained using mononuclear cells purified from three different donors in three independent experiments. 8B. Neutrokine-alpha-specific TaqMan primers are prepared and used to assess the relative Neutrokine-alpha mRNA expression levels in unstimulated monocytes and IFN-gamma (100 U / mL) treated monocytes. The nucleotide sequence of the TaqMan primer is as follows: (a) Probe: 5'-CCA CCA GCT CCA GGA GAA GGC AAC TC-3 '(SEQ ID NO: 24); (b) 5 'amplification primers: 5'-ACC GCG GGA CTG AAA ATC T-3' (SEQ ID NO: 25); And (c) 3 'amplification primers: 5'-CAC GCT TAT TTC TGC TGT TCT GA-3' (SEQ ID NO: 26).

9A and 9B. Neutrokine-alpha is an effective B lymphocyte stimulating factor. Figure 9A. The biological activity of Neutrokine-alpha is assessed in a standard B-lymphocyte co-stimulation assay using Staphylococcus aureus cowan 1 SAC (priming agent) as a priming agent. The SAC itself provides a base coefficient of 1427 +/- 316. Values are reported as mean +/- standard deviation of three wells. Similar results are obtained using recombinant Neutrokine-alpha purified from stable CHO transfectants and transiently transfected HEK 293T cells. Figure 9B. Proliferation of tonsil B cells by Neutrokine-alpha and co-stimulation with anti-IgM. Biological assays are performed as described for SAC, except that each well is precoated with goat anti-human IgM antibody (10 micrograms / mL) in PBS.

10A and 10B. Neutrokine-alpha receptor expression in peripheral blood mononuclear cells and tumor cell lines of normal persons. 10A. Human peripheral blood nucleated cells are obtained from normal volunteers and separated by density gradient centrifugation. Cells are stained with biotinylated Neutrokine-alpha and then stained with PE-conjugated streptavidin and FITC or PerPC bound CD3, CD20, CD14, CD56 and CD66b specific mAbs. Cells are analyzed on a Becton Dicknson FACScan using CellQuest software. The data represent one of four independent experiments. 10B. Neutrokine-alpha that binds to histiocyte cell line U-937 and melanoma line IM-9.

11A, 11B and 11C. In vivo effect of Neutrokine-alpha administration in BALB / cAnNCR mice. Figure 11A. Formalin-fixed spleens are impregnated in paraffin and 5 micron sections are stained with hematoxylin and eosin (top panel). The lower panel was obtained from the same animal stained with anti-CD45R (B220) mAb and with horseradish-peroxidase coupled rabbit anti-rat Ig (mouse adsorbed) and substrate diaminobenzidine tetrahydrochloride (DAB). It is an expanded intercept. Slides are contrast stained with Meyer Hematoxylin. CD45R (B220) expressing cells appear brown. Figure 11B. Flow cytometry analysis of normal (left panel) mice and Neutrokine-alpha treated (right panel) mice stained with PE-CD45R (B220) and FITC-ThB (Ly6D). Figure 11C. Serum IgM, IgG and IgA Levels in Normal and Neutrokine-alpha Treated Mice.

The present invention provides isolated nucleic acid molecules comprising polynucleotides determined by encoding Neutrokine-alpha polypeptides having the amino acid sequences shown in FIGS. 1A and 1B (SEQ ID NO: 2) and sequencing the cDNA clones. The nucleotide sequences shown in FIGS. 1A and 1B (SEQ ID NO: 1) were deposited on October 22, 1996 with an institution [American Type Culture Collection, 10801, Universuty Boulevard, Manassas, Virginia 20110-2201] to assign ATCC Accession No. 97768. Obtained by sequencing the received HNEDU15 clone. The deposited clones are contained in the pBluescript SK (-) plasmid (Stratagene, La Jolla, Calif.).

The present invention also provides an isolated nucleic acid molecule comprising a polynucleotide measured by encoding a Neutrokine-alphaSV polypeptide having the amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19) and sequencing the cDNA clone. The nucleotide sequences described in FIGS. 5A and 5B (SEQ ID NO: 18) are obtained by sequencing HDPMC52 clones deposited on December 10, 1998 in the American Type Culture Collection and assigned ATCC Accession No. 203518. The deposited clones are contained in the pBluescript SK (-) plasmid (Stratagene, La Jolla, Calif.).

Neutrokine-alpha and Neutrokine-alphaSV polypeptides of the invention share sequence homology with translation products of human mRNA for TNF-alpha, TNF-beta, LTbeta, Fas ligand, APRIL and LTalpha (see : 2A, 2B and 7A). As noted above, TNF-alpha plays a role in the regulation of cytotoxicity, necrosis, apoptosis, costimulation, proliferation, lymph node formation, immunoglobulin class conversion, differentiation, antiviral activity and adsorption molecules and other cytokines and growth factors. It is considered to be an important cytokine.

Nucleic acid molecule

Unless otherwise specified, all nucleotide sequences determined by sequencing DNA molecules are measured using automated DNA sequencing (e.g., Model 373, Applied Biosystems, Ind., Foster City, CA) and measured herein. All amino acid sequences of the polypeptides encoded by the DNA molecules were estimated by translation of the DNA sequences measured as above. Thus, as is known in the art for DNA sequences measured by such automated methods, all nucleotide sequences measured herein may contain some error. The nucleotide sequence measured by automation is typically at least 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be measured more accurately by other methods, including manual DNA sequencing, which are well known in the art. As is also known in the art, one insertion or deletion in the nucleotide sequence measured compared to the actual sequence causes a frame shift in the translation of the nucleotide sequence and thus the putative amino acid sequence encoded by the measured nucleotide sequence Will be completely different from the amino acid sequence actually encoded by the DNA molecule sequenced from the point of insertion or deletion.

A “nucleotide sequence” of a nucleic acid molecule or polynucleotide is a DNA molecule or polynucleotide (a sequence of deoxyribonucleotides), and each thymidine deoxyribonucleotide (T) of the deoxyribonucleotide sequence specified above is ribonucleotide uridine. An RNA molecule or polynucleotide corresponding to a ribonucleotide (A, G, C and U) sequence substituted with (U).

When using the information provided herein, eg, the nucleotide sequences of FIGS. 1A and 1B, Neutrokine- according to standard cloning and selection methods, eg, cloning cDNA using mRNA as starting material. Nucleic acid molecules of the invention encoding alpha polypeptides can be obtained. As an example of the invention, the nucleic acid molecules described in FIGS. 1A and 1B (SEQ ID NO: 1) are found in cDNA libraries derived from neutrophils. Expressed sequence tags corresponding to portions of Neutrokine-alpha cDNA are also found in kidney, lung, peripheral leukocytes, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages and spinal cord blood tissue do. In addition, the present invention encodes Neutrokine-alphaSV polypeptides using the nucleotide information provided in FIGS. 5A and 5B and according to standard cloning and screening methods, such as cloning cDNA using mRNA as starting material. Nucleic acid molecules can be obtained. As an example of the present invention, the nucleic acid molecules described in FIGS. 5A and 5B (SEQ ID NO: 18) are found in cDNA libraries derived from primary dendritic cells.

Neutrokine-alpha plasmid HNEDU 15, deposited as ATCC Accession No. 97768, is a protein of about 285 amino acid residues, about 1 to 46 of an estimated intracellular domain of about 46 amino acids (FIGS. 1A and 1B (SEQ ID NO: 2)). Amino acid residues), an estimated transmembrane domain of about 26 amino acids (about 47 to about 72 amino acid residues underlined in FIGS. 1A and 1B (SEQ ID NO: 2)) and an estimated extracellular domain of about 213 amino acids ( Amino acid residues from about 73 to about 285 of FIGS. 1A and 1B (SEQ ID NO: 2) and contain an open reading frame with an estimated molecular weight of about 31 kDa. The Neutrokine-alpha polypeptides shown in FIGS. 1A and 1B (SEQ ID NO: 2) are about 20% similar and about 10% identical to human TNF-alpha available as Gen. No. 339764 in GenBank.

Neutrokine-alphaSV plasmid HDPMC52 deposited as ATCC Accession No. 203518 is a protein of about 266 amino acid residues, about 1 to 46 in the estimated intracellular domain of about 46 amino acids (FIGS. 5A and 5B (SEQ ID NO: 19)). Amino acid residues), the estimated transmembrane domain of about 26 amino acids (about 47 to about 72 amino acid residues underlined in FIGS. 5A and 5B (SEQ ID NO: 19)) and the estimated extracellular domain of about 194 amino acids ( 5A and 5B (SEQ ID NO: 19 amino acid residues from about 73 to about 266) and contain an open reading frame with an estimated molecular weight of about 29 kDa. The Neutrokine-alphaSV polypeptides shown in FIGS. 5A and 5B (SEQ ID NO: 19) are about 33.9% similar and about 22.0% identical to human TNF-alpha, available under Genbank No. 339764.

As will be appreciated by those skilled in the art, due to the possibility of sequencing errors discussed above, in fact complete Neutrokine-alpha and / or Neutrokine encoded by deposited cDNAs comprising about 285 and 266 amino acids, respectively AlphaSV polypeptides may be slightly shorter. In particular, the measured Neutrokine-alpha and Neutrokine-alphaSV coding sequences are shown in the nucleotide positions 210 to 212 of FIGS. 1A and 1B (SEQ ID NO: 1) and the nucleotide positions 64 to 66 of FIGS. 5A and 5B (SEQ ID NO: 18). It contains a common second methionine codon which can serve as another starting codon for the decoding of an open reading frame. More typically, the actual open reading frame is ± 20 amino acids as estimated from the first or second methionine codons from the N-terminus shown in FIGS. 1A and 1B (SEQ ID NO: 1) and 5A and 5B (SEQ ID NO: 18). , With a higher probability, there may be any position in the range of ± 10 amino acids. Since the polypeptide domains described herein were estimated by computer analysis, the extracellular, intracellular and transmembrane of Neutrokine-alpha and Neutrokine-alphaSV polypeptides according to the analytical criteria used to identify several functional domains It will also be appreciated that the exact "address" of the domain may vary slightly. For example, the exact location of Neutrokine-alpha and Neutrokine-alphaSV extracellular domains in FIGS. 1A and 1B (SEQ ID NO: 2) and FIGS. 5A and 5B (SEQ ID NO: 19) is slightly dependent on the criteria used to define the domain. (Eg, the address may shift by about 1 to about 20 residues, with a higher probability about 1 to 5 residues). In this case, the end point of the transmembrane domain and the start point of the extracellular domain were estimated based on the identification of hydrophobic amino acid sequences at the positions described above, as shown in FIGS. 3 and 6 and Table 1. In any case, as discussed further below, the present invention also contemplates the N-terminus of the extracellular domain described herein, which constitutes a soluble form of the extracellular domains of the Neutrokine-alpha and Neutrokine-alphaSV polypeptides. Polypeptides having multiple residues deleted from the N-terminus and / or C-terminus of a complete polypeptide are provided, including polypeptides from which one or more amino acids are deleted from.

As noted, the nucleic acid molecules and polynucleotides of the present invention may be in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or generated synthetically, or in the form of RNA, such as mRNA. DNA may be double stranded or single stranded. Single-stranded DNA or RNA can be a coding chain, also known as a sense chain, or a non-coding chain, also known as an antisense chain.

By "isolated" nucleic acid molecule is meant a nucleic acid molecule (DNA or RNA) that has been removed from its natural environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Still other examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or DNA molecules purified (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules according to the invention. However, isolation from clones or cells or cell lysates that are members of the library (eg, genome or cDNA libraries) that are not isolated from other members of the library (eg, in the form of a homogeneous solution containing clones and another member of the library). Or nucleic acid contained in a removed chromosome (eg, a "dispersed chromosome" as in karyotype) is not isolated for the purposes of the present invention. As discussed further herein, isolated nucleic acid molecules according to the present invention can be prepared naturally, recombinantly or synthetically.

Isolated nucleic acid molecules of the invention comprise an open reading frame (ORF) with, or alternatively, an open reading frame with an initiation codon at positions 147-149 in the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1). A constructed DNA molecule. In addition, the isolated nucleic acid molecules of the present invention comprise DNA molecules which comprise sequences substantially different from those described above, or alternatively consist of such sequences, but which encode a Neutrokine-alpha protein due to the degeneracy of the genetic code. Include. Of course, genetic code is well known in the art. Thus, it is common for those skilled in the art to prepare the degenerate variants described above. In another embodiment, the present invention comprises or alternatively consists of a sequence encoding a Neutrokine-alpha polypeptide having an amino acid sequence encoded by a cDNA contained in a plasmid of ATCC Accession No. 97768. Provided are isolated nucleic acid molecules. Preferably, the nucleic acid molecule comprises or alternatively comprises a sequence encoding an extracellular domain or a mature or soluble polypeptide sequence of a polypeptide encoded by a cDNA contained in a plasmid of ATCC Accession No. 97768. Is made of.

Isolated nucleic acid molecules of the invention also include DNA molecules comprising an open reading frame (ORF) with an initiation codon at positions 1 to 3 of the nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18). In addition, the isolated nucleic acid molecules of the invention comprise sequences substantially different from the sequences described above, or alternatively consist of such sequences, but due to the degeneracy of the genetic code, the DNA encoding Neutrokine-alphaSV polypeptides. It includes molecules. Of course, genetic code is well known in the art. Thus, it is common for one skilled in the art to prepare the degenerate variants described above. In another embodiment, the present invention comprises or alternatively consists of a sequence encoding a Neutrokine-alpha SV polypeptide having an amino acid sequence encoded by a cDNA contained in a plasmid of ATCC Accession No. 203518. Provided are isolated nucleic acid molecules. Preferably, the nucleic acid molecule comprises or alternatively comprises a sequence encoding an extracellular domain or a mature or soluble polypeptide sequence of a polypeptide encoded by a cDNA contained in a plasmid of ATCC Accession No. 203518. Is made of.

The invention also relates to a nucleic acid molecule having the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) or the nucleotide sequence of Neutrokine-alpha cDNA contained in the plasmid of ATCC Accession No. 97768 or a sequence complementary to one of the above sequences. Or alternatively provide isolated nucleic acid molecules consisting of such molecules. The present invention also provides a nucleic acid having a nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18) or a nucleotide sequence of Neutrokine-alphaSV cDNA contained in the plasmid of ATCC Accession No. 203518 or a sequence complementary to one of these sequences. Isolated nucleic acid molecules are provided which comprise or alternatively consist of such molecules. Such isolated molecules, particularly DNA molecules, include, but are not limited to, probes for gene mapping by in situ hybridization with chromosomes and Neutrokine in human tissue, for example by Northern or Western blot analysis. It has use as a probe for detecting the expression of alpha and Neutrokine-alphaSV.

In one embodiment, the polynucleotides of the invention comprise or alternatively consist of the sequence set forth in SEQ ID NO: 22. The sequence provided as SEQ ID NO: 22 is constructed from several duplicate mouse EST sequences (AI182472, AA422749, AA254047 and AI122485) obtained from Genebank. The EST sequence is aligned to produce the Neutrokine-alpha-like polynucleotide sequence provided as SEQ ID NO: 22. The amino acid sequence obtained from the translation of SEQ ID NO: 22 is provided as SEQ ID NO: 23. Fragments, variants, and derivatives of the sequences provided as SEQ ID NO: 22 and SEQ ID NO: 23 are also included in the present invention.

In another embodiment, the polynucleotides of the invention comprise, or alternatively consist of, those polynucleotides comprising, or fragments, variants and derivatives thereof that encode the sequence shown in SEQ ID NO: 27 and / or the amino acid sequence shown in SEQ ID NO: 28 Nucleotides are also included in the present invention. For example, certain embodiments of the invention are polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: 28 It relates to a polynucleotide comprising or alternatively consisting of a sequence encoding a sequence. The amino acid sequence obtained from the translation of SEQ ID NO: 27 is provided as SEQ ID NO: 28. Polypeptides comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 28 and the sequences provided as SEQ ID NO: 28, are also included in the present invention. For example, certain embodiments of the invention are polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: 28 It relates to a polypeptide comprising or alternatively consisting of such sequence. A nucleic acid molecule having the sequence provided as SEQ ID NO: 27 is obtained using RT-PCR from cyanomologous monkey (ie, Macacayrus) PBMC using two degenerate primers. In summary, total RNA is prepared from cyanomoloccus monkey PBMC using Trizol (Life Technologies, Inc., Rockville, MD) according to the manufacturer's protocol. Subsequently, single-chain cDNA is synthesized from cyanohomologus monkey PBMC preparations according to standard methods using oligo-dT primers. Neutrokine-alpha-specific primers are designed based on the conserved region between mouse and human Neutrokine-alpha molecules (SEQ ID NOS: 22 and 1, respectively). The cyanohomococcus monkey Neutrokine-alpha nucleic acid molecule is then prepared by PCR using a cDNA template in combination with the following two degenerate oligonucleotide primers. 5 'primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3 'primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequences of degenerate primers (SEQ ID NOs: 35 and 36), "I" represents deoxyinosine or dideoxyinosine.

In another embodiment, a polynucleotide of the invention comprises or alternatively consists of a sequence encoding a sequence shown in SEQ ID NO: 29 and / or an amino acid sequence set forth in SEQ ID NO: 30, fragments, variants and derivatives thereof. . These polynucleotides are also included in the present invention. For example, certain embodiments of the invention are polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: 30. It relates to a polynucleotide comprising or alternatively consisting of a sequence encoding a sequence. The amino acid sequence obtained from the translation of SEQ ID NO: 29 is provided as SEQ ID NO: 30. Polypeptides comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 30 and the sequences provided as SEQ ID NO: 29 and SEQ ID NO: 30, alternatively consisting of these, are also included in the present invention. For example, certain embodiments of the invention are polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: 30. It relates to a polypeptide comprising or alternatively consisting of such sequence. Nucleic acid molecules having the sequence provided as SEQ ID NO: 29 are obtained using RT-PCR from rhesus monkey PBMC using two degenerate primers. In summary, total RNA is prepared from the rhesus monkey PBMC using Trizol (Life Technologies, Inc., Rockville, MD) according to the manufacturer's protocol. Subsequently, single-chain cDNA is synthesized from the rhesus monkey PBMC preparation according to standard methods using oligo-dT primers. Neutrokine-alpha-specific primers are designed based on the conserved region between mouse and human Neutrokine-alpha molecules (SEQ ID NOS: 22 and 1, respectively). A rhesus monkey Neutrokine-alpha nucleic acid molecule is then prepared by PCR using a cDNA template in combination with the following two degenerate oligonucleotide primers. 5 'primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3 'primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequences of degenerate primers (SEQ ID NOs: 35 and 36), "I" represents deoxyinosine or dideoxyinosine.

The present invention also provides nucleic acid molecules having nucleotide sequences associated with a broad portion of SEQ ID NO: 1 and SEQ ID NO: 18 determined from the following related cDNA clones: HSOAD55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8), and HLTBM08 (SEQ ID NO: 9). .

The invention also relates to nucleic acid molecules encoding portions of the nucleotide sequences described herein and fragments of the isolated nucleic acid molecules described herein. In one embodiment, the present invention provides a polynucleotide having a nucleotide sequence that represents a portion of SEQ ID NO: 1 consisting of nucleotides from positions 1 to 1001 in SEQ ID NO: 1. In another aspect, the present invention provides a polynucleotide having a nucleotide sequence that represents a portion of SEQ ID NO: 18 consisting of positions 1 through 798 of SEQ ID NO: 18.

The invention also relates to fragments of the nucleic acid molecules (ie polynucleotides) described herein. For example, the nucleotide sequence of the cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence encoding the polypeptide encoded by the cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: The nucleotide sequence encoding the polypeptide sequence, the nucleotide sequence of the cDNA contained in the plasmid of ATCC Accession No. 203518, the nucleotide sequence encoding the polypeptide encoded by the cDNA contained in the plasmid of ATCC Accession No. 203518, SEQ ID NO: 18 A fragment of a nucleic acid molecule having a nucleotide sequence, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 20, or a complementary chain thereof, is at least 15 nt in length, preferably at least 20 nt or at least 25 nt, more preferably at least 30 nt, even more Preferably 40, 50 and means 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 or at least 500 nt fragment. These fragments have numerous uses, including but not limited to diagnostic probes and primers as discussed herein. Of course, larger fragments, for example, fragments 501-1500 nt in length, may include the nucleotide sequence of cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence of SEQ ID NO: 1, the cDNA contained in the plasmid of ATCC Accession No. 203518. The nucleotide sequence of and the nucleotide sequence of SEQ ID NO: 18, in most cases but in most cases, are useful in accordance with the invention as well as the corresponding fragments. Preferred nucleic acid fragments of the invention are those of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides identified in FIGS. 1A and 1B (SEQ ID NO: 2) and FIGS. 5A and 5B (SEQ ID NO: 19), respectively, described in detail below. Nucleic acid molecules comprising an epitope-containing moiety, or alternatively encoding a polypeptide consisting of such moiety. Polypeptides encoded by these polynucleotide fragments are also included in the present invention.

Also, for example, the nucleotide sequence of SEQ ID NO: 21, the nucleotide sequence of SEQ ID NO: 22, the nucleotide sequence of SEQ ID NO: 27, the nucleotide sequence of SEQ ID NO: 29, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 23, the polypeptide sequence of SEQ ID NO: 28 A fragment of a nucleic acid molecule having a nucleotide sequence, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 30, or a complementary chain thereof, is at least 15 nt in length, preferably at least 20 or 25 nt, more preferably at least 30 nt, even more preferably 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 or 500nt or more fragments. These fragments have numerous uses, including but not limited to many uses, including diagnostic probes and primers as discussed herein. Of course, larger fragments, eg, 501-1500 nt in length, may also contain the nucleotide sequence of SEQ ID NO: 21, the nucleotide sequence of SEQ ID NO: 22, the nucleotide sequence of SEQ ID NO: 27, the nucleotide sequence of SEQ ID NO: 29, or the polypeptide sequence of SEQ ID NO: 23 Useful in accordance with the present invention, as in all but not all of the corresponding fragments, the nucleotide sequence encoding, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 28, the nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 30, or its complementary chain, in most cases Do. Polypeptides encoded by these polynucleotide fragments are also included in the present invention.

Representative examples of the Neutrokine-alpha polynucleotide fragments of the present invention include about 1 to 50, 51 to 100, 101 to 146, 147 to 200, 201 to 250, 251 of the nucleotide of SEQ ID NO: 1 # 300-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 600-650, 651- 700, 701 to 750, 751 to 800, 800 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050 and / or 1051 to Fragments comprising or alternatively consisting of the cDNA contained in sequence 1082, its complementary chain, or the plasmid of ATCC Accession No. 97768. In the context of the present invention, "about" includes the specifically mentioned ranges and includes ranges as large or small as several (5, 4, 3, 2, 1) nucleotides at either or both ends.

Representative examples of the Neutrokine-alphaSV polynucleotide fragment of the present invention include the nucleotides of SEQ ID NO: 18 about 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651 Or cDNA contained in the sequence of No. 700, No. 701 to 750, No. 751 to 800, No. 800 to 850 and / or No. 851 to 900, its complementary chain or the plasmid of ATCC Accession No. 203518, Alternatively, fragments consisting of such sequences are included. In the context of the present invention, "about" includes the specifically mentioned ranges and includes ranges as large or small as several (5, 4, 3, 2, 1) nucleotides at either or both ends. .

In certain preferred embodiments, the polynucleotides of the invention comprise nucleotide residues 571-627, 580-627, 590-627, 600-627, 610-627, 571-620 of the nucleotide residues of SEQ ID NO: 1 Times, 580 to 620, 590 to 620, 600 to 620, 571 to 610, 580 to 610, 590 to 610, 571 to 600, 580 to 600 and And / or 571 to 590, or alternatively consist of such moieties.

In certain other preferred embodiments, the polynucleotides of the invention may comprise a nucleotide residue of SEQ ID NO: 18.

Or alternatively consist of such moieties.

In certain further preferred embodiments, the polynucleotide of the present invention is a nucleotide residue of SEQ ID NO: 1

Or alternatively consist of such moieties.

In a further preferred embodiment, the polynucleotide of the invention is a nucleotide residue of SEQ ID NO: 1

Or alternatively consist of such moieties.

Preferably, the polynucleotide fragment of the present invention encodes a polypeptide demonstrating Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. A polypeptide demonstrating "functional activity" means a polypeptide that can exhibit one or more known functional activities associated with full length and / or secreted Neutrokine-alpha polypeptides and / or Neutrokine-alphaSV polypeptides. Such functional activities include, but are not limited to, biological activity (eg, the ability to stimulate B cell proliferation, survival, differentiation and / or activation), antigenicity [anti-Neutrokine-alpha and / or anti-Neutrokine- The ability to bind (or compete with Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides for binding) alphaSV antibodies; and immunogenicity (Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides) The ability to form antibodies to bind), the ability to form multimers with the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention and the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides The ability to bind to a receptor or ligand is included.

In a further particular embodiment, the polynucleotide fragment of the present invention comprises the putative intracellular domain of Neutrokine alpha (amino acids 1 to 46 of SEQ ID NO: 2), the putative transmembrane domain (amino acids 47 to 72 of SEQ ID NO: 2), the putative cell Encodes a polypeptide comprising an extra domain (amino acids 73 to 285 of SEQ ID NO: 2) or the putative TNF conserved domain (amino acids 191 to 284 of SEQ ID NO: 2), or alternatively consisting of such a domain. In a further embodiment, the polynucleotide fragments of the present invention comprise polypeptides comprising one, two, three or all four of the domains described above in combination, or alternatively consisting of such combinations. Polypeptides encoded by these polynucleotides are also included in the present invention.

In a further particular embodiment, the polynucleotide fragment of the present invention comprises the putative intracellular domain of Neutrokine alphaSV (amino acids 1 to 46 of SEQ ID NO: 19), the putative transmembrane domain (amino acids 47 to 72 of SEQ ID NO: 19), the putative The polypeptide comprises an extracellular domain (amino acids 73 to 266 of SEQ ID NO: 19) or an putative TNF conserved domain (amino acids 172 to 265 of SEQ ID NO: 19), or alternatively encodes a polypeptide consisting of such a domain. In a further embodiment, the polynucleotide fragments of the present invention comprise polypeptides comprising one, two, three or all four of the domains described above in combination, or alternatively consisting of such combinations. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises residues Met-1 to Lys-113, Leu-114 to Thr-141, Ile-142 to Lys-160, Gly-161 to Gln-198, Val- of SEQ ID NO: 2. Polynucleotides encoding an amino acid sequence selected from 199 to Ala-248 and Gly-250 to Leu-285, or alternatively consist of such polynucleotides. In addition, polynucleotides encoding the combination of two, three, four, five or more of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragments of the invention comprise residues Met-1 to Lys-113, Leu-114 to Thr-141, Gly-142 to Gln-179, Val-180 to Ala-229 and Gly- of SEQ ID NO: 19. Polynucleotides encoding an amino acid sequence selected from 230 to Leu-266, or alternatively consist of such polynucleotides. In addition, polynucleotides encoding the combination of two, three, four, five or more of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragments of the invention comprise residues Met-1 to Lys-106, Leu-107 to Thr-134, Glu-135 to Asn-165, Ile-167 to Lys-184, Gly- of SEQ ID NO: 23. Polynucleotides encoding an amino acid sequence selected from 185 to Gln-224, Val-225 to Ala-272 and Gly-273 to Leu-309, or alternatively consist of such polynucleotides. In addition, polynucleotides encoding the combination of two, three, four, five or more of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragments of the invention comprise residues Tyr-1 to Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln-132, Val- of SEQ ID NO: 28. Polynucleotides encoding an amino acid sequence selected from 133 to Ala-182 and Gly-183 to Ala-219, or alternatively consist of such polynucleotides. In addition, polynucleotides encoding the combination of two, three, four, five or more of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragments of the invention comprise residues Tyr-1 to Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln-132, Val- of SEQ ID NO: 30. Polynucleotides encoding an amino acid sequence selected from 133 to Ala-182 and Gly-183 to Ala-219, or alternatively consist of such polynucleotides. In addition, polynucleotides encoding the combination of two, three, four, five or more of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotides of the present invention comprise or alternatively consist of the sequences set forth in SEQ ID NO: 21. The sequence described as SEQ ID NO: 21 encodes a polypeptide consisting of starting methionine residues linked to residues Ala-134 to Leu-285 of the Neutrokine-alpha polypeptide sequence described as SEQ ID NO: 2. Polypeptides encoded by these polynucleotides are also included in the present invention.

In a further particular preferred embodiment, the polynucleotide of the invention is a nucleotide residue of SEQ ID NO: 21.

Or alternatively consist of such moieties. Polypeptides encoded by these polynucleotides are also included in the present invention.

Thus, certain embodiments of the invention comprise, or alternatively comprise, the amino acid sequences of the beta pleat sheet regions A, A ', B, B', C, D, E, F, G, or H described in FIGS. 7A and 6 The present invention relates to polynucleotides encoding polypeptides consisting of such sequences. Additional aspects of the invention include a combination of one, two, three, four, five, six, seven, eight, nine, or ten of the beta pleat sheet regions AH described in FIGS. 7A and Example 6, or alternatively The present invention relates to polynucleotides encoding Neutrokine-alpha polypeptides consisting of these combinations. A further preferred embodiment of the invention comprises the Neutrokine-alpha amino acid sequence of the beta pleat sheet regions A, A ', B, B', C, D, E, F, G or H described in Figure 7A and Example 6. Or alternatively relates to a polypeptide consisting of such sequences. Additional aspects of the invention include a combination of one, two, three, four, five, six, seven, eight, nine or ten of the beta pleat sheet regions A to H described in FIGS. 7A and Example 6, or Alternatively, to a Neutrokine-alpha polypeptide consisting of such a combination.

In certain other preferred embodiments, the polynucleotides of the invention comprise nucleotide residues 34-57, 118-123, 133-141, 151-159, 175-216, 232-255, 280-315, 328-357, 370 of SEQ ID NO: 21. -393 and / or 430-456 or alternatively consist of such moieties. Polypeptides encoded by these polynucleotides are also included in the present invention. These polynucleotides and polypeptide fragments correspond to the putative beta-pleat sheet regions described in Figure 7A. In certain embodiments, the polynucleotides of the present invention comprise 90% of the polynucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the beta-fleet sheet regions described above, It comprises or consists of at least 95%, 96%, 97%, 98% or 99% identical polynucleotide sequences. The invention also encompasses such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotide sequences are also included in the present invention. In another embodiment, the invention includes polynucleotides that hybridize under stringent hybridization conditions to one, two, three, four, five, six, seven, eight, nine, or ten of the beta-pleat sheet regions described above. To provide an isolated nucleic acid molecule. As used herein, the meaning of the term "strict conditions" is described below.

In a further preferred embodiment, the polynucleotides of the invention comprise nucleotide residues 576-599, 660-665, 675-683, 693-701, 717-758, 774-803, 822-857, 870-899, 912 of SEQ ID NO: 1 -935 and / or 972-998, or alternatively consist of such moieties. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotides and polypeptide fragments correspond to the putative beta-pleat sheet regions described in Figure 7A.

In another further preferred aspect, the polynucleotides of the invention comprise nucleotide residues 457-462, 472-480, 490-498, 514-555, 571-600, 619-654, 667-696, 699-732 and And / or 769-795 or alternatively consist of such residues. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotides and polypeptide fragments correspond to the putative beta-pleat sheet regions described in Figure 7A.

In yet another preferred embodiment, the polynucleotides of the invention comprise nucleotide residues 124-129, 139-147, 157-165, 181-222, 238-267, 286-321, 334-363, 376-399 And / or 436-462, or alternatively consists of such moieties. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotides and polypeptide fragments correspond to the putative beta-pleat sheet regions described in Figure 7A. Polypeptides comprising amino acid sequences combined into one, two, three, four, five, six, seven, eight, nine or ten of these regions, or alternatively consisting of such sequences, are included in the present invention.

Relative positions of several intron / exon boundaries relative to mouse Neutrokine-alpha (SEQ ID NOs: 22 and 23) were determined based on sequencing of mouse genomic DNA. The apparent second exon (temporarily named "exon 2") from the 5 'end of the mouse Neutrokine-alpha genomic clone consists of Tyr-187 to Gln-222 of the sequence described in SEQ ID NO: 23. An apparent third exon (temporarily named "exon 3") from the 5 'end of the mouse Neutrokine-alpha genomic clone comprises Val-223 to Gly-273 of the sequence set forth in SEQ ID NO: 23.

Thus, in one embodiment, the present invention provides a polynucleotide comprising the amino acid sequence of residues Tyr-187 to Gln-222 in SEQ ID NO: 23, or alternatively encoding a polypeptide consisting of such sequence. The present invention also provides a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mouse Neutrokine-alpha polypeptide described above. Or alternatively relates to a nucleic acid molecule consisting of such sequences. The invention also encompasses such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention.

In another embodiment, the present invention provides a polynucleotide comprising the amino acid sequence of residues Val-223 to Gly-273 in SEQ ID NO: 23, or alternatively encoding a polypeptide consisting of such sequence. The present invention also provides a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the mouse Neutrokine-alpha polypeptide described above. Or alternatively relates to a nucleic acid molecule consisting of such sequences. The invention also encompasses such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention.

In addition, relative positions of the corresponding intron / exon boundaries relative to human Neutrokine-alpha (SEQ ID NO: 1 and SEQ ID NO: 2) were determined based on the arrangement of the mouse and human Neutrokine-alpha polypeptide sequences. The apparent second exon (temporarily named "exon 2") from the 5 'end of human Neutrokine-alpha consists of Tyr-163 to Gln-198 of the sequence set forth in SEQ ID NO: 2. The apparent third exon (temporarily named "exon 3") from the 5 'end of human Neutrokine-alpha consists of Val-199 to Gly-249 of the sequence described in SEQ ID NO: 2.

Thus, in one embodiment, the present invention provides a polynucleotide comprising the amino acid sequence of residues Tyr-163 to Gln-198 in SEQ ID NO: 2 or alternatively encoding a polypeptide consisting of such sequence. The present invention also provides a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide described above. Or alternatively relates to nucleic acid molecules consisting of such sequences. The invention also encompasses such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention.

In another aspect, the present invention provides a polynucleotide comprising the amino acid sequence of residues Val-199 to Gly-249 in SEQ ID NO: 2 or alternatively encoding a polypeptide consisting of such sequence. The present invention also provides a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide described above. Or alternatively relates to nucleic acid molecules consisting of such sequences. The invention also encompasses such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention.

The functional activity of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and fragments, variant derivatives and analogs thereof can be assayed by several methods described herein and well known in the art.

For example, binding with anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies or with Neutrokine-alpha receptor (s) and / or Neutrokine-alphaSV receptor (s) on B cells. In one embodiment that assays for the ability to bind or compete with full-length Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides for binding, radioimmunoassay, ELISA (enzyme linked immunosorbent assay) ), "Sandwich" immunoassay, immunoradiation assay, gel diffusion sedimentation reaction, immunodiffusion assay, in situ immunoassay (using gelatinous gold, enzyme or radioisotope labeling), western blot, precipitation reaction, aggregation reaction (E.g., gel aggregation assays, hemagglutination assays), competitive and non-competitive assay systems using techniques such as complement fixation assays, immunofluorescence assays, Protein A assays and immunoelectrophoresis assays There can be a number of immunoassay known. In one embodiment, antibody binding is detected by detecting a label in the first antibody. In another embodiment, the first antibody is detected by detecting binding of the second antibody or reagent to the first antibody. In further embodiments, the second antibody is labeled. Many means for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In another embodiment, reducing and non-reducing gel chromatography, protein, when identifying Neutrokine-alpha and / or Neutrokine-alphaSV ligands and assessing the ability of the polypeptide fragments, variants or derivatives of the invention to multimerize Binding can be assayed by means known in the art, such as affinity chromatography and affinity blotting (General Reference: Phizicky, E. et al., 1995, Microbiol. Rev. 59: 94-123). In another embodiment, the physiological correlations of binding (signal transduction) of Neutrokine-alpha and / or Neutrokine-alphaSV to its substrate can be assayed.

In addition, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and fragments, variants, derivatives thereof, and the like described herein (see Examples 6 and 7) are commonly applied in the art. The analog stimulates or otherwise stimulates B cell proliferation, differentiation and / or activation in the case of Neutrokine-alpha and / or Neutrokine-alphaSV associated biological activities (eg, Neutrokine-alpha and / or Neutrokine-alphaSV antagonists). Ability to inhibit and / or prolong B cell survival in vitro or in vivo).

Other methods are well known to those skilled in the art and these methods are within the scope of the present invention.

In a further embodiment, the polynucleotides of the invention encode polypeptides that comprise or alternatively comprise the functional attributes of Neutrokine-alpha and Neutrokine-alphaSV. In this respect, preferred embodiments of the present invention are the alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions (") of Neutrokine-alpha and Neutrokine-alphaSV polypeptides. Beta-region "), turn and turn-forming regions (" turn-regions "), coils and coil-forming regions (" coil-regions "), hydrophilic regions, hydrophobic regions, alpha amphoteric regions, beta amphoteric regions, Or a fragment comprising, or alternatively consisting of, a flexible region, a surface-forming region, and a high antigenic region.

At least one of the beta pleat sheet regions of Neutrokine-alpha described in FIG. 7 is believed to be important for dimerization and interaction between Neutrokine-alpha and its ligands.

Specific preferred areas in this regard are described in FIG. 3 (Table 1). The data described in FIG. 3 and Table 1 merely provide that the format differs from that obtained when analyzing the amino acid sequence of SEQ ID NO: 2 using the default parameters of the DNA * STAR computer algorithm and the results are identical.

The preferred regions described above in FIG. 3 and Table 1 include but are not limited to those regions of this type identified by analysis of the amino acid sequences described in FIGS. 1A and 1B. Such preferred regions, as described in FIG. 3 and Table 1, include Garnier-Robson alpha-regions, beta-regions, turn-regions and coil-regions, Chow-Passman alpha-regions, beta-regions and coil-regions, Kite-dulit hydrophilic and hydrophobic regions, Eisenberg alpha- and beta-bilateral regions, Carplus-Shultz flexible regions, Emini surface-forming regions and high antigenic Jameson-wolf regions. Highly preferred polynucleotides in this regard are regions of Neutrokine-alpha and / or Neutrokine-alphaSV that combine several structural features such as several of the features described above (eg, 1, 2, 3 or 4). Or alternatively polynucleotides encoding polypeptides consisting of such regions. Polypeptides encoded by these polynucleotides are also included in the present invention.

In addition, the data described in columns VIII, IX, XIII and XIV in Table 1 can be used routinely to determine regions of Neutrokine-alpha that exhibit a high degree of potential for antigenicity (column VIII in Table 1 Hydrophilicity according to kite-dulit; column IX in Table 1 shows hydrophobicity according to Hof-woods; column XIII in Table 1 shows the antigenic index of James-Wolf; column XIV in Table 1 shows the surface according to Emini Probability). Highly antigenic regions are assigned to columns VIII, IX, XIII and / or IV by selecting values representing regions of the polypeptide that are likely to be exposed to the surface of the polypeptide in an environment where antigen recognition may occur during the initiation of an immune response. Measure from the data provided. The data provided in FIG. 6 may also be commonly provided in a similar tabular format by simply examining the amino acid sequences described in FIG. 6 (SEQ ID NO: 19) using modules and algorithms of DNA * STAR immobilized with default parameters. As above, the amino acid sequences provided in FIG. 6 can also be used to measure regions of Neutrokine-alpha that exhibit a high degree of potential for antigenicity provided as a figure (as in FIG. 6) or as a table (as in Table 1). Can be.

Further preferred nucleic acid fragments according to the invention comprise a nucleic acid molecule comprising or alternatively comprising a sequence encoding at least one epitope-containing portion of Neutrokine-alpha. In particular, such nucleic acid fragments of the invention have an amino acid sequence from SEQ ID NO: 2 about Phe-115 to about Leu-147, about Ile-150 to about Tyr-163, about Ser-171 to about Phe-194, about Glu-223 to about A nucleic acid molecule comprising a sequence encoding a polypeptide selected from Tyr-246 and about Ser-271 to about Phe-278, or alternatively consisting of such sequence. Here, "about" means a range that is specifically cited and ranges as large or small as several, ie 5, 4, 3, 2 or 1, on either or both of the amino and carboxy termini. Polypeptides encoded by these nucleic acid molecules are also included in the present invention. Polypeptide fragments containing antigenic epitopes of Neutrokine-alpha can be readily determined by those skilled in the art using the assay described above of the Jameson-Wolf antigen index as described in FIG. 3. Methods of measuring such other epitope-containing portions of Neutrokine-alpha are described in detail below.

Further preferred nucleic acid fragments according to the invention comprise a nucleic acid molecule comprising or alternatively comprising a sequence encoding one or more epitope-containing portions of Neutrokine-alphaSV. In particular, the nucleic acid fragments of the present invention comprise about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro-218 to about Encoding a polypeptide selected from Tyr-227, about Ser-252 to about Thr-258, about Ala-232 to about Gln-241, about Ile-244 to about Ala-249, and about Ser-252 to about Vla-257 Nucleic acid molecules comprising or alternatively consisting of such sequences. Here, "about" means a range that is specifically cited and ranges as large or small as several, ie 5, 4, 3, 2 or 1, on either or both of the amino and carboxy termini. Polypeptides encoded by these nucleic acid molecules are also included in the present invention. Polypeptide fragments containing antigenic epitopes of Neutrokine-alphaSV can be readily determined by one skilled in the art using the assay described above of the Jameson-Wolf antigen index as described in FIG. 3. Methods of measuring such other epitope-containing portions of Neutrokine-alphaSV are described in detail below.

In certain embodiments, the polynucleotides of the present invention have a length of 100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb. , 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb or less than 5 kb.

In a further embodiment, the polynucleotides of the present invention comprise at least 15, at least 30, at least 50, at least 100, at least 250, at least 500 or at least 1000 consecutive nucleotides of the Neutrokine-alpha coding sequence, 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 of genomic DNA flanking the 5 'or 3' coding nucleotides described in FIGS. 1A and 1B (SEQ ID NO: 1) or 5A and 5B (SEQ ID NO: 18). kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb or less than 5 kb or the same. In a further embodiment, the polynucleotides of the present invention comprise at least 15, at least 30, at least 50, at least 100, at least 250, at least 500 or at least 1000 consecutive nucleotides of the Neutrokine-alpha coding sequence, Does not contain all or part of Neutrokine-alpha introns. In another embodiment, the nucleic acid comprising the Neutrokine-alpha coding sequence does not contain the sequence of the genomic flanking gene (ie, 5 'or 3' of the Neutrokine-alpha gene in the genome). In another embodiment, the polynucleotides of the invention do not contain the coding sequence of 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2 or more than one genomic flanking genes. Do not.

In another embodiment, the invention deposits a nucleic acid molecule of the invention described above, eg, a sequence complementary to the coding and / or noncoding sequences described in FIGS. 1A and 1B (SEQ ID NO: 1), ATCC Accession No. 97768. The sequence of the cDNA clone contained in water, the sequence complementary to the coding and / or non-coding sequence deposited in FIGS. 5A and 5B (SEQ ID NO: 18), the sequence, sequence of the cDNA clone contained in the deposit of ATCC Accession No. 203518 The sequence complementary to the coding sequence and / or non-coding sequence described in (21) (ie, transcribed, non-translated), the coding sequence described in SEQ ID NO: 22 and / or the sequence complementary to the non-coding sequence, A sequence complementary to a coding sequence and / or a non-coding sequence, a sequence complementary to a coding sequence and / or a non-coding sequence shown in SEQ ID NO: 29, or a fragment of those sequences as described herein (eg, an open reading frame or fragment thereof) Of polynucleotides It provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions minutes. "Strict hybridization conditions" include 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate and 20 micrograms Incubated overnight at 42 ° C. in a solution containing / ml denatured and truncated salmon sperm DNA, then washing the filter at about 65 ° C. in 0.1 × SSC.

The polynucleotides that hybridize with the “part” of the polynucleotides are at least about 15 nucleotides (nt), preferably at least about 20 nt, more preferably at least about 30 nt, even more preferably at least 30- of the reference polynucleotides. Polynucleotides (DNA or RNA) that hybridize to 70 or 80-150 nucleotides. These are used as diagnostic probes and primers described above but not limited to these and described in more detail below. For example, a portion of a polynucleotide that is "at least about 20 nts in length" may comprise a reference polynucleotide (e.g., the sequence of one or both of the deposited cDNAs, the complementary chain of the nucleotide sequence described in Figures 1A and 1B (SEQ ID NO: 1), 5A and 5B (SEQ ID NO: 18) the complementary chain of the nucleotide sequence, the complementary chain of the nucleotide sequence described in SEQ ID NO: 21, the complementary chain of the nucleotide sequence described in SEQ ID NO: 22, the complementary chain of the nucleotide sequence described in SEQ ID NO: 27, and Includes a specifically cited range that is greater than or less than several amino acids (ie, 5, 4, 3, 2, 1 or 0) at one or both ends of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 29 do. Of course, the 3 'terminal poly (A) track of the Neutrokine-alpha cDNA described in poly A sequences (e.g., Figures 1A and 1B (SEQ ID NO: 1), Neutrokine-alphaSV described in Figures 5A and 5B (SEQ ID NO: 18) 3) terminal poly (A) track of cDNA or 3 'terminal poly (A) track of Neutrokine-alphaSV cDNA described in SEQ ID NO: 22) or a polynucleotide that only hydrides to the complementary sequence of T (or U) residues Is not included in the polynucleotides of the invention used to hybridize to a portion of the nucleic acid of the invention. This is because such polynucleotides hybridize to nucleic acid molecules containing poly (A) sequences or to their complementary sequences (e.g., double-stranded cDNA clones generated using oli dT as a primer in practice).

As mentioned, nucleic acid molecules of the invention encoding Neutrokine-alpha polypeptides or Neutrokine-alphaSV polypeptides include, but are not limited to, polynucleotides encoding the amino acid sequences of the individual extracellular domains of the polypeptide and Additional sequences such as the coding sequence for the extracellular domain of the individual polypeptide and the intracellular and transmembrane domain sequence or the coding sequence of the pre-, pro- or pre-pro-protein sequence, ie the additional coding sequence described above And may comprise the coding sequence of an individual extracellular domain of a polypeptide present or absent.

Also encoded by the nucleic acids of the invention are additional non-coding sequences, including but not limited to intron and non-coding 5 'and 3' sequences (e.g. splicing and polya, such as ribosomal binding and stability of mRNA) The protein sequence along with additional coding sequences encoding additional amino acids, such as providing a further action, including transcribed sequences that play a role in mRNA processing and transcription, including denylation signals).

Thus, a sequence encoding a polypeptide can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In a particularly preferred embodiment of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as a tag provided in the pQE vector (QIAGEN, Inc., 91311 Chatsworth Eaton Avenue, 9159), among many available. . See, eg, Gentz et al., Proc. Natl. Acad. Sci. As described in USA 86: 821-824, hexa-histidine provides convenient purification of fusion proteins. Another peptide useful for purification as the "HA" tag corresponds to an epitope derived from influenza hemagglutinin protein described in Wilson et al., Cell 37: 767 (1984). As discussed below, other such fusion proteins include Neutrokine-alpha or Neutrokine-alphaSV polypeptideSV polypeptides fused to Fc at the N- or C-terminus.

The invention also relates to variants of nucleic acid molecules of the invention that encode portions, derivatives or analogs of the Neutrokine-alpha or Neutrokine-alphaSV polypeptides of SEQ ID NO: 2. Variants can occur naturally, such as natural allelic variants. "Allele variants" are one of several different types of genes that occupy a given locus on an organism's chromosome (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). Non-natural variants include, but are not limited to, oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, eg, Carter et al., Nucl. Acids Res. 13: 4331 (1986); And Zoller et al., Nucl.Acids Res. 10: 6487 (1982)), cassette mutagenesis (see Wells et al., Gene 34: 315 (1985)), restriction selection mutagenesis (see Wells et. al., Phiols.Trans.R.Soc.London SerA 317: 415 (1986)), and may be produced using mutagenesis techniques known in the art.

Such variants include those produced by nucleotide substitutions, deletions or additions. Substitutions, deletions or additions may involve one or more nucleotides. The transition can be in a cryptographic area, a non-cryptographic area, or both. Variations in the coding region can lead to conserved or non-conserved amino acid substitutions, deletions or additions. Particularly preferred among these are silent substitutions, additions and deletions that do not alter the properties and activities of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or portions thereof. Also particularly preferred in this connection are conservative substitutions.

Further aspects of the invention include at least one, at most 50, more preferably at most 40, even more preferably at most 30, even more preferably at most 20, 10-20, 5-10, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides having an amino acid sequence containing 1-5, 3-5 or 1-3 conserved amino acid substitutions (e.g., Neutrokine-alpha and And / or polynucleotides encoding the amino acid sequence of the Neutrokine-alphaSV polypeptide fragment). Of course, Neutrokine-alpha and / or Neutrokine- with amino acid sequences containing up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conserved amino acid substitutions in order of increasing preference. Very preferred are polynucleotides encoding the amino acid sequence of an alphaSV polypeptide.

Further embodiments include (a) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having the complete amino acid sequence described in FIGS. 1A and 1B (ie, positions 1 to 285 of SEQ ID NO: 2), (b) N- A nucleotide sequence encoding a Neutrokine-alpha polypeptide having the complete amino acid sequence of SEQ ID NO: 2 (ie positions 2 to 285 of SEQ ID NO: 2), excluding the terminal methionine, (c) Neutrokine-alpha functional activity (e.g., an antigen or A fragment of the polypeptide of (b) having a biological activity, (d) encoding an estimated extracellular domain of a Neutrokine-alpha polypeptide having an amino acid sequence of positions 73 to 285 in FIGS. 1A and 1B (SEQ ID NO: 2) Nucleotide sequence, (e) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in FIGS. 1A and 1B (SEQ ID NO: 2), and (f) a deposit of ATCC Accession No. 97768 A nucleotide sequence encoding a Neutrokine-alpha polypeptide having a complete amino acid sequence encoded by a contained cDNA clone, (g) a Neutrope having an amino acid sequence encoded by a cDNA clone contained in a deposit of ATCC Accession No. 97768 The nucleotide sequence encoding the extracellular domain of a keen-alpha polypeptide and (h) the nucleotide sequence of (a), (b), (c), (d), (e), (f) or (g) A polynucleotide selected from the group consisting of the complementary nucleotide sequences is at least 80%, 85% or 90% identical, more preferably at least 95%, 96%, 97%, 98% or 99% identical Or alternatively include isolated nucleic acid molecules consisting of such polynucleotides. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also included in the present invention.

A very preferred embodiment of the invention is that at least 80%, 85% or 90% identical polynucleotide and nucleotide sequence encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in FIGS. 1A and 1B (SEQ ID NO: 2) And more preferably comprises at least 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotides, or alternatively isolated nucleic acid molecules consisting of such polynucleotides. Preferred embodiments of the invention comprise polynucleotides encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and a polynucleotide having at least 90% identical nucleotide sequence, or alternatively In particular, it relates to nucleic acid molecules consisting of such polynucleotides. More preferred embodiments of the invention comprise polynucleotides encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and a polynucleotide having at least 95% identical nucleotide sequence, or Alternatively it relates to a nucleic acid molecule consisting of such polynucleotides. Even more preferred embodiments of the invention comprise polynucleotides encoding a Neutrokine-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and a polynucleotide having at least 96% identical nucleotide sequence or Alternatively, it relates to a nucleic acid molecule consisting of such polynucleotides.

Further preferred embodiments of the present invention comprise polynucleotides encoding the Neutrokine-alpha polypeptide having an amino acid sequence of positions 134 to 285 in FIGS. 1A and 1B (SEQ ID NO: 2) and a polynucleotide having at least 97% identical nucleotide sequence. Or alternatively relates to nucleic acid molecules consisting of such polynucleotides. In addition, more preferred embodiments of the present invention comprise polynucleotides encoding the Neutrokine-alpha polypeptides having the amino acid sequence of positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and polynucleotides having at least 98% identical nucleotide sequences. Or alternatively relates to nucleic acid molecules consisting of such polynucleotides. In addition, more preferred embodiments of the present invention comprise polynucleotides encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134 to 285 in FIGS. 1A and 1B (SEQ ID NO: 2) and polynucleotides having at least 99% identical nucleotide sequences. Or alternatively relates to nucleic acid molecules consisting of such polynucleotides.

Additional aspects of the invention have an amino acid sequence containing at least one, at most 50, more preferably at most 40, even more preferably at most 30, even more preferably at most 20 conserved amino acid substitutions. It relates to an isolated nucleic acid molecule comprising a polynucleotide encoding an amino acid sequence of a Neutrokine-alphaSV polypeptide (eg, a Neutrokine-alphaSV polypeptide fragment described herein). Of course, in order of increasing preference, 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6, 5, 4, Very preferred are polynucleotides encoding the amino acid sequence of the Neutrokine-alphaSV polypeptide having an amino acid sequence containing up to 3, 2 or 1 conserved amino acid substitutions.

Further embodiments include (a) a nucleotide sequence encoding a Neutrokine-alphaSV polypeptide having the complete amino acid sequence shown in FIGS. 5A and 5B (ie, positions 1 to 266 of SEQ ID NO: 19), (b) an N-terminal methionine Nucleotide sequence encoding a Neutrokine-alphaSV polypeptide having the complete amino acid sequence of SEQ ID NO: 19 (ie, positions 2 to 266 of SEQ ID NO: 2), (c) positions 73 to 285 in FIGS. 5A and 5B (SEQ ID NO: 19) A nucleotide sequence encoding an estimated extracellular domain of a Neutrokine-alphaSV polypeptide having an amino acid sequence of (d) a Neutrope having a complete amino acid sequence encoded by a cDNA clone contained in a deposit of ATCC Accession No. 203518 Nucleotide sequence encoding the Kin-alphaSV polypeptide, (e) amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 203518 Nucleotide sequence encoding the extracellular domain of a Neutrokine-alphaSV polypeptide having a row and (f) a nucleotide complementary to the nucleotide sequence of (a), (b), (c), (d) or (e) above Polynucleotides selected from the group consisting of sequences and nucleotide sequences are at least 80%, 85% or 90% identical, more preferably comprise at least 95%, 96%, 97%, 98% or 99% identical polynucleotides, Alternatively, isolated nucleic acid molecules consisting of such polynucleotides.

In addition, the present invention provides for the nucleotides 1 to 1 except for the nucleotide and nucleotide sequences of 797 to 1082, 810 to 1082 and 346 to 542, measured from the four cDNA clones described above, preferably in FIGS. 1A and 1B (SEQ ID NO: 1). Of at least about 10 contiguous nucleotides, at least about 20 contiguous nucleotides, at least about 25 contiguous nucleotides or at least about 30 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or about 50 contiguous nucleotides of the sequence of nucleotide 1082 At least 90% or at least 95% identical to any portion, or alternatively comprises polynucleotides consisting of such sequences. In addition, the present invention further comprises at least about 10 contiguous nucleotides in the sequence, at least about 20 contiguous nucleotides, about 25, except for the nucleotides measured from the four cDNA clones described above in Figures 5A and 5B (SEQ ID NO: 18). Comprise at least 90% or at least 95% identical sequence to, or alternatively such a sequence of at least contiguous nucleotides or at least about 30 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or at least about 50 contiguous nucleotides It comprises a polynucleotide consisting of. In addition, the present invention further provides at least about 10 consecutive nucleotides, at least about 20 contiguous nucleotides, at least about 25 contiguous nucleotides or about 30 nucleotides in the sequence, except for nucleotides as determined in SEQ ID NO: 21, preferably from the four cDNA clones described above. At least 90 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or at least about 50 or more contiguous nucleotides, at least 90% or at least 95% identical to, or alternatively, polynucleotides consisting of do. In addition, the present invention further provides at least about 10 consecutive nucleotides, at least about 20 contiguous nucleotides, at least about 25 contiguous nucleotides or about 30 nucleotides in the sequence except for nucleotides as determined in SEQ ID NO: 22, preferably from the four cDNA clones described above At least 90 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or at least about 50 or more contiguous nucleotides, at least 90% or at least 95% identical to, or alternatively, polynucleotides consisting of do. In addition, the present invention further provides at least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides or about 30 nucleotides in the sequence except for the nucleotides measured in SEQ ID NO: 27, preferably from the four cDNA clones described above. At least 90 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or at least about 50 or more contiguous nucleotides, at least 90% or at least 95% identical to, or alternatively, polynucleotides consisting of do. In addition, the present invention further provides at least about 10 consecutive nucleotides, at least about 20 contiguous nucleotides, at least about 25 contiguous nucleotides or about 30 nucleotides in the sequence except for nucleotides as determined in SEQ ID NO: 29, preferably from the four cDNA clones described above At least 90 contiguous nucleotides, preferably at least about 40 contiguous nucleotides or at least about 50 or more contiguous nucleotides, at least 90% or at least 95% identical to, or alternatively, polynucleotides consisting of do. Here, "about" includes as large or small as several amino acids (ie, 5, 4, 3, 2 or 1) at one or both ends from a specifically designated range.

Polynucleotides having a reference nucleotide sequence that encodes a Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide and, for example, at least 95% "identical" nucleotide sequence, are those in which the nucleotide sequence of the polynucleotide is Neutrokine-alpha and / or It is meant the same as the reference sequence except that it may include up to 5 mismatches per 100 nucleotides of the reference nucleotide sequence encoding the Neutrokine-alphaSV polypeptide. In other words, up to 5% of the nucleotides of the reference sequence may be deleted, substituted with other nucleotides, or up to 5% of the total nucleotides of the reference sequence to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence. Can be inserted into the reference sequence. These variations of the reference sequence may be at the 5 'or 3' terminal position of the reference nucleotide sequence, at any position between their terminal positions, interspersed individually among the nucleotides in the reference sequence, or interspersed in one or more consecutive groups within the reference sequence May occur. The reference (reference) sequence is a complete nucleotide sequence encoding Neutrokine-alpha or Neutrokine-alphaSV described in FIGS. 1A and 1B (SEQ ID NO: 1) and FIGS. 5A and 5B (SEQ ID NO: 18), respectively, for example SEQ ID NO: 21, All Neutrokine-alpha, such as Neutrokine-alpha polynucleotides described in 22, 27 or 28, or all Neutrokine-alpha or Neutrokine-alphaSV polynucleotide fragments described herein.

Indeed, certain nucleic acid molecules may, for example, have the nucleotide sequence described in FIGS. 1A and 1B, the nucleotide sequence shown in FIGS. 5A and 5B, the nucleotide sequence of the deposited cDNA clone, or the Neutrokine-described in SEQ ID NO: 21, 22, 27 or 28. Best Fit program (Wisconsin 53711 Madison) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to all Neutrokine-alpha polynucleotides or fragments thereof, such as alpha polynucleotides It can be routinely measured using known computer programs such as Science Drive 575 University Research Park, Wisconsin Sequence Analysis Package for Unix, version 8 of the Genetics Computer Group. Bestfit uses Smith and Waterman's local homology algorithms to find optimal homology fragments between two sequences (Advances in Applied Mathematics 2: 482-489 (1981)). When using Best Fit or other sequence alignment programs to determine whether a particular sequence is for example 95% identical to the reference sequence of the present invention, of course the percentage of identity is calculated for the full length of the reference nucleotide sequence and is determined by the nucleotides in the reference sequence. Set the variable to allow gaps in homology below 5% of the total number.

In certain embodiments, the identity between the reference (query) sequence (the sequence of the invention) and the sequence (also called the full sequence sequence) is determined by Brutrag and colleagues' algorithm (Comp. App. Biosic. (1990) 6: 237-245) using a FASTDB computer program. In the sequence sequence both the query and the corresponding sequence are DNA sequences. RNA sequences can be compared by converting U to T. The result of the entire sequence is shown in% identity. Preferred variables used in the FASTDB array of DNA sequences to calculate% identity are as follows: matrix = Unitary, k-tuple = 4, mismatch penalty = 1, binding penalty = 30, random group length = 0, threshold score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the shorter of the corresponding nucleotide sequence. In this embodiment, manual corrections should be made to the results if the sequence is shorter than the query sequence due to the 5 'or 3' deletion, not due to internal deletion. This is because the FASTDB program does not calculate 5 'and 3' cleavage of the sequence when calculating% identity. For those sequences truncated at the 5 'or 3' end to the query sequence,% identity is the percentage of the total base of the query sequence to calculate the number of bases of the query sequence that are 5 'and 3' of that sequence that are not matched / arranged. Correct by doing Whether nucleotides were matched / arranged is determined as a result of FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using a particular variable to obtain a final percent identity score. This corrected score is used for the purposes of this embodiment. For the purpose of manually adjusting the% identity score, only bases other than the 5 'and 3' bases of the sequence indicated by the FASTDB sequence as not matched / arranged with the query sequence are calculated. For example, 90 base corresponding sequences are aligned with 100 base query sequences to determine% identity. Deletion occurs at the 5 'end of the sequence and thus the FASTDB sequence does not show a match / array at the first 10 bases at the 5' end. Ten unpaired bases represent 10% of the sequence (the number of bases present at the unmatched 5 'and 3' ends / total number of bases in the query sequence) and therefore from the% identity score calculated by the FASTDB program. Deduct 10%. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. As another example, 90 base corresponding sequences were compared with 100 base query sequences. At this point, the deletion is an internal deletion, so there is no base at 5 'or 3' of the sequence that is not matched / arranged with the query. In this case,% identity calculated by FASTDB is not corrected manually. Once again, manually correct only the 5 'and 3' bases of the sequence that are not matched / arranged with the query sequence. No other manual calibration is made for the purposes of the present invention.

The present invention relates to a nucleic acid sequence described herein (i.e., a polynucleotide) (e.g., whether or not it encodes a polypeptide having Neutrokine-alpha and / or Neutrokine-alphaSV functional activity (e.g., biological activity)). At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence described in FIGS. 1A and 1B (SEQ ID NO: 1 or the nucleic acid sequence of deposited cDNA) It relates to a nucleic acid molecule. In addition, the present invention relates to the nucleic acid sequence described in FIGS. 5A and 5B (SEQ ID NO: 18) or to the nucleic acid sequence of the deposited cDNA, 80%, 85%, regardless of whether it encodes a polypeptide having Neutrokine-alphaSV activity. At least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical nucleic acid molecules. In addition, the present invention provides 80%, 85%, 90%, 92%, 95% of the nucleic acid sequences set forth in SEQ ID NO: 21, 22, 27 or 28, regardless of whether they encode polypeptides having Neutrokine-alpha activity. At least 96%, 97%, 98% or 99% identical nucleic acid molecules. This means that even if a particular nucleic acid molecule does not encode a polypeptide having Neutrokine-alpha and / or Neutrokine-alphaSV activity, those skilled in the art will use the nucleic acid molecule, for example as a hybridization probe or polymerase chain reaction (PCR) primer. Because I know how to do it. Uses of nucleic acid molecules of the invention that do not encode polypeptides having Neutrokine-alpha and / or Neutrokine-alphaSV activity include, inter alia, (1) Neutrokine-alpha and / or Neutrokine-alphaSV genes in the cDNA library. Or to isolate allelic variants thereof (2) Neutrokine-alpha and / or Neutrokine as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1998). In situ hybridization to mid-term chromosomal dispersion to provide accurate chromosomal location of alphaSV genes and Norson blot analysis to detect Neutrokine-alpha and / or Neutrokine-alphaSV mRNA in certain tissues.

However, preferred is the nucleic acid sequence described herein (e.g., Figures 1A and 1B (SEQ ID NO: 1) that actually encodes a polypeptide having Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide functional activity (e.g., biological activity). A nucleic acid molecule having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence and nucleic acid sequence of a deposited cDNA or fragment thereof) to be. Also preferred is the nucleic acid sequence described in FIGS. 5A and 5B (SEQ ID NO: 18) or deposited, which actually encodes a polypeptide having Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide functional activity (eg, biological activity). The nucleic acid sequence of the cDNA and the sequence is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical. Also preferred are nucleic acid sequences and sequences set forth in SEQ ID NO: 21, 22, 27 or 28 that actually encode polypeptides having Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide functional activity (eg, biological activity). At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical nucleic acid molecules.

"Neutrokine-alpha polypeptide functional activity polypeptides" (e.g. biologically active) and "Neutrokine-alphaSV polypeptide functional activity polypeptides" (e.g. biologically active) specific activity assays (e.g. immune As measured in a biological or biological assay), it refers to a polypeptide that exhibits similar activity, although not necessarily identical to the activity of the extracellular domain or full length Neutrokine-alpha or Neutrokine-alphaSV polypeptide of the invention. For example, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide functional activity may be characterized in that the polypeptide sequences described herein are complete Neutrokine-alpha and / or Neutrokine-alphaSV or Neutrokine-alpha and / or Can be measured by the ability to form multimers (eg, homodimers and homotrimers) with the extracellular domain of Neutrokine-alphaSV and the ability to bind Neutrokine-alpha and / or Neutrokine-alphaSV ligands. have. In addition, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide functional activity may be attributed to the ability of a polypeptide of the invention to induce lymphocyte (eg B cell) proliferation, differentiation or activation and / or to prolong B cell survival. It can measure by measuring. These assays of activity are described herein (see Example 6) and can be routinely performed using techniques known in the art. In addition, the Neutrokine-alpha or Neutrokine-alphaSV polypeptides of the invention modulate cell proliferation, cytotoxicity, cell survival and cell death. In vitro cell proliferation, cytotoxicity, cell survival, and cell death assays for measuring the effect of proteins on specific cells use well known and readily available reagents in the art to detect cell replication and / or death. It can carry out by making it. For example, many such assays for TNF-related protein activity are described in the various references cited herein. In brief, examples of such assays are human or animal (eg mouse) cells harvested and (1) transfected host cell-supernatant or (2) non-transformation containing Neutrokine-alpha protein (or candidate polypeptide). Mixing with the infected host cell-supernatant control and incubating for a specified period of time, followed by measuring the effect on cell number or survival. Cell proliferation and / or survival regulatory activity that can be measured in this type of assay is useful for treating tumors, tumor metastasis, infections, autoimmune diseases, inflammation and other immune-related diseases.

Neutrokine-alpha regulates cell proliferation and differentiation in a dose-dependent manner in this assay. Thus, it is preferred that a "polypeptide with Neutrokine-alpha polypeptide functional activity" (eg, biological activity) comprises a polypeptide that exhibits the same cellular regulatory (particularly immunomodulatory) activity in this assay in a dose-dependent manner. . Although the degree of dose-dependent activity need not be the same as that of the Neutrokine-alpha polypeptide, preferably "polypeptide with Neutrokine-alpha polypeptide functional activity" is given in comparison to Neutrokine-alpha polypeptide. The activity exhibits substantially similar dose-dependence (ie, the candidate polypeptide exhibits greater activity or less activity at about 25-fold or less, preferably about 10-fold or less compared to the reference Neutrokine-alpha polypeptide). Indicates).

In certain preferred embodiments, "polypeptides with Neutrokine-alpha polypeptide functional activity" (eg, biological activity) and "polypeptides with Neutrokine-alphaSV polypeptide functional activity" (eg, biological activity) are shown in FIG. Polypeptides that also exhibit the same B cell (or other cell type) regulatory (particularly immunomodulatory) activity described in 8A, 8B, 9A, 9B, 10, 11, 12A and 12B and Example 6.

Like other members of the TNF family, Neutrokine-alpha is active against leukocytes, including, for example, monocytes, lymphocytes (eg B cells) and neutrophils. For this reason Neutrokine-alpha is active in inducing proliferation, differentiation and migration of their cell types. Such activity is useful for immune boosting or suppression, myeloprotection, stem cell recruitment, acute and chronic inflammation control and leukemia treatment. Assays for measuring this activity are known in the art. See, eg, Peters et al., Immun. Today 17: 273 (1996); Young et al., J. Exp. Med. 182: 1111 (1995); Caux et al., Nature 390: 258 (1992); And Santiago-Schwarz et al., Adv. Exp. Med. Biol. 378: 7 (1995).

Of course, due to the degeneracy of the genetic code, those skilled in the art will appreciate that the nucleic acid sequence contained in the cDNA clone deposited in ATCC Accession No. 97768 or the nucleic acid sequence described in FIGS. 1A and 1B (SEQ ID NO: 1) or fragments and sequences thereof is 80%. Polypeptides having many "nutrokine-alpha polypeptide functional activities" (eg, biological activity) at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical You will immediately recognize that it encrypts your password. Those skilled in the art will also appreciate that the nucleic acid sequences contained in the cDNA clones deposited in ATCC Accession No. 203518 or the nucleic acid sequences and sequences described in FIGS. 5A and 5B (SEQ ID NO: 18) may be 80%, 85%, 90%, 92%, 95%. It will be readily appreciated that many nucleic acid molecules that are at least 96%, 97%, 98% or 99% identical encode a polypeptide having a "Neutrokine-alphaSV polypeptide functional activity" (eg, biological activity). In fact, this is obvious to those skilled in the art, even if the comparative assays described above are not conducted because all of the degenerate variants of those nucleotide sequences encode the same polypeptide. It will also be recognized in the art that even for nucleic acid molecules that are not degenerate variants, a suitable number will also encode Neutrokine-alpha and / or Neutrokine-alphaSV activity. This is because those skilled in the art are fully aware of amino acid substitutions (e.g., substitution of aliphatic amino acids with second aliphatic amino acids) that do not significantly affect or are unlikely to affect protein function as described further below. .

Similarly, a polynucleotide encoding a polypeptide containing all or a portion of regions V-142 to K-160 in SEQ ID NO: 2 detects and / or mutations in the expression of Neutrokine-alpha or Neutrokine-alphaSV polynucleotides. It is likely to be a valuable diagnostic and therapeutic polynucleotide in connection with the application. In addition, a polynucleotide linking amino acid residues T-141 and G-142 of the Neutrokine-alphaSV polypeptide described in SEQ ID NO: 19, between which the amino acid sequences V-142 to K-160 of Neutrokine-alpha are explicitly inserted Seems to be diagnostically and therapeutically useful. Such T-141 / G-142 spanning polynucleotides would represent a much higher possibility of hybridization with Neutrokine-alphaSV polynucleotides than with Neutrokine-alpha polynucleotides. A non-limiting non-exclusive list of Neutrokine-alphaSV polypeptides encoded by the polynucleotides of the invention includes polypeptides comprising or consisting of an amino acid sequence selected from: G of SEQ ID NO: 19 -121 to E-163; E-122 to E-163; G-123 to E-163; N-124 to E-163; S-125 to E-163; S-126 to E-163; Q-127 to E-163; N-128 E163; S-129 to E-163; R-130 to E-163; N131 to E-163; K-132 to E-163; R-133 to E-163; A-134 to E-163; V-135 to E-163; Q-136 to E-163; G-137 to E-163; P-138 to E-163; E-139 to E-163; E-140 to E-163; T-141 to E-163; G-142 to E-163; S-143 to E-163; Y-144 to E-163; T-145 to E-163; F-146 to E-163; V-147 to E-163; P-148 to E-163; W-149 to E-163; L-150 to E-163; L-151 to E-163; S-152 to E-163; F-153 to E-163; K-154 to E-163; R-155 to E-163; G-156 to E-163; S-157 to E-163; A-158 to E-163; L-159 to E-163; E-160 to E-163; E-161 to E-163; K-162 to E-163; G-121 to K-162; G-121 to E-161; G-121 to E-160; G-121 to L-159; G-121 to A-158; G-121 to S-157; G-121 to G-156; G-121 to R-155; G-121 to K-154; G-121 to F-153; G-121 to S-152; G-121 to L-151; G-121 to L-150; G-121 to W-149; G-121 to P-148; G-121 to V-147; G-121 to F-146; G-121 to T-145; G-121 to Y-144; G-121 to S-143; G-121 to G-142; G-121 to T-141; G-121 to E-140; G-121 to E-139; G-121 to P-138; G-121 to G-137; G-121 to Q-136; G-121 to V-135; G-121 A-134; G-121 to R-133; G-121 to K-132; G-121 to N-131; G-121 to R-130; G-121 to S-129; G-121 to N-128; G-121 to Q-127; G-121 to S-126; G-121 to S-125; G-121 to N-124; G-121 to G-123; And G-121 to E-122. Polypeptides encoded by these polynucleotides are also included in the present invention.

Vector and Host Cells

The invention also relates to vectors comprising isolated DNA molecules of the invention, host cells genetically engineered by the recombinant vector or engineered to produce the polypeptides of the invention, and by Neutrogen by recombinant or synthetic techniques. The invention relates to the generation of kin-alpha and / or Neutrokine-alphaSV polypeptides or fragments thereof.

In one embodiment, the polynucleotides of the invention are linked to a vector (eg, cloning or expression vector). The vector can be, for example, a phage, plasmid, virus or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case viral propagation generally occurs only in the complementary host cell. Polynucleotides may be linked to a vector containing a selection marker for propagation in the host. Introduction of vector constructs into host cells is not limited to these, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. And can be accomplished by techniques known in the art. This method is described in many standard experimental guidelines such as Davids et al., Basic Methods in Molecular Biology (1986).

In general, recombinant expression vectors induce the transcription of selection markers (eg, ampicillin resistance genes of E. coli and Saccharomyces cerevisiae TRP1 gene) and downstream structural sequences that allow transformation of the origin of replication and host. Promoters derived from highly-expressed genes. Such promoters can be derived, for example, from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factors, acid phosphatase or heat shock proteins. The heterologous structural sequence is combined with a leader sequence capable of inducing the secretion of the translated initiation and termination sequence and preferably of the translated protein into the periplasmic space or extracellular medium at appropriate stages. Optionally, the heterologous sequence can encode a fusion protein comprising an N-terminal identification peptide that provides a desired feature, such as stabilization or simple purification of the expressed recombinant product.

In one embodiment, the DNA of the invention is a phage lambda PL promoter, E. Operably linked with appropriate heterologous regulatory elements (eg, promoters or enhancers) such as the E. coli lac, trp, phoA and tac promoters, SV40 early and late promoters and promoters of retroviral LTR. Other suitable promoters are known to those skilled in the art.

As mentioned, the expression vector preferably comprises one or more selection markers. Such markers include dihydrofolate reductase, G418 or neomycin resistance genes for eukaryotic cell culture and E. coli. Tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria are included. Representative examples of suitable hosts include, but are not limited to, these. Bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; Yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); Insect cells, such as Drosophila S2 and Spodogerra Sf9 cells; Animal cells such as CHO, COS, 293 and Bow melanoma cells; And plant cells. Suitable culture media and conditions for the host cells described above are known in the art.

The host cell may be a higher eukaryotic cell such as a mammalian cell (eg, a human derived cell) or a lower eukaryotic cell such as a yeast cell or the host cell may be a prokaryotic cell such as a bacterial cell. The host strain may be chosen to control the expression of the inserted gene sequence or to modify and process the gene product in the particular aspect desired. Expression from certain promoters may be elevated in the presence of certain inducers and thus control of expression of the genetically engineered polypeptide. In addition, other host cells have characteristics and specific mechanisms for the translation and post-translational processing and modification of proteins (eg, phosphorylation, cleavage). Appropriate cell lines can be selected to ensure the desired modification and processing of the expressed foreign protein. The selection of appropriate vectors and promoters for expression in host cells is a well known procedure and the techniques necessary for constructing expression vectors, introducing vectors into a host and expression in a host are routine to those skilled in the art.

Expression vectors useful for bacteria are constructed by inserting a structural DNA sequence encoding a protein of interest with an appropriate translational initiation and termination signal onto an operative readout with a functional promoter. The vector includes one or more phenotypic selection markers and origins of replication to ensure maintenance in the host and to provide amplification if necessary. Suitable prokaryotic hosts for transformation, although others may be used as a matter of choice, are E. coli. Several species of the genus E. coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces and Staphylococcus. Expression vectors suitable for use in bacteria can include bacterial marker origins and bacterial markers derived from commercial plasmids comprising the genetic elements of the cloning vector pBR322 (ATCC 37017), which is well known as a representative but not limited to these. . Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotech, Madison, Wisconsin). These pBR322 "skeleton" moieties are combined with a suitable expression vector and the structural sequence to be expressed. Preferred vectors for use in bacteria include pHE4-5 (ATCC Accession No. 209311 and variants thereof), pQE70, pQE60 and pQE-9 available from QIAGEN, Inc; PBS vectors, phagescript vectors, bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from stratazine; And ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Farmersia. Preferred expression vectors for use in the yeast system include, but are not limited to, pYES2, pYD1, pTEE1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K and PAO815 (all available from Invitrogen, Carlsbad, CA). Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from stratazine and pSVK3, pBPV, pMSG and pSVL available from Farmersia. Other suitable vectors will be apparent to those skilled in the art.

After transforming a suitable host strain and growing it to an appropriate cell density, the selected promoter is induced by appropriate means (eg, temperature conversion or chemical induction) and the cells are cultured for an additional period of time. Cells are typically harvested by centrifugation and disrupted by physical or chemical means and the resulting crude extract is maintained for further purification.

Microbial cells used in the expression of proteins can be disrupted by convenient methods including freeze-thaw cycles, sonication, mechanical disruption or the use of cell lysates and such methods are well known to those skilled in the art.

In one embodiment, yeast Pichia pastoris is used to express Neutrokine-alpha proteins in the eukaryotic system. Pichia Pastoris is a methanol-required yeast capable of metabolizing methanol to a unique carbon source. The main step in the methanol metabolic pathway is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by the enzyme alcohol oxidase. To metabolize methanol to its only carbon source, Pichia pastoris must produce high levels of alcohol oxidase due in part to the relatively low affinity of alcohol oxidase for O ₂ . After all, in a growth medium that relies on methanol as its main carbon source, one of the two alcohol oxidase genes (AOX1) promoter region is very active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene accounts for up to about 30% of the total soluble protein of Pichia pastoris (Ellis, SB, et al., Mol. Cell. Biol. 5: 1111-). 21 (1985); Koutz, PJ et al., Yeast 5: 167-77 (1989); Tschopp, JF, et al., Nucl.Acids Res. 15: 3859-76 (1987)). Thus, under transcriptional control of all or part of the AOX1 regulatory sequence, heterologous coding sequences, such as, for example, the Neutrokine-alpha or Neutrokine-alphaSV polynucleotides of the present invention, have a very high level in Pichia yeast grown in the presence of methanol. It is expressed as.

As an example, see "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998] and using the plasmid vector pPIC9K to express DNA encoding Neutrokine-alpha or Neutrokine-alphaSV polypeptides in a Pichia yeast system as described herein. do. This expression vector is a derivative of the Neutrokine-alpha or Neutrokine-alphaSV protein of the invention due to a potent AOX1 promoter linked to a Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (i.e. leader) located upstream of the multiple cloning site. Enable expression and secretion.

As will be readily appreciated by those skilled in the art, many other yeast vectors may be used in place of pPIC9K, examples of which include pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2 , pHIL-S1, pPIC3.5K and PAO815. However, the proposed expression constructs must provide appropriately arranged signals for transcription, translation, secretion (if needed), including the necessary in-frame AUG.

In one embodiment, high level expression of a heterologous coding sequence, such as the Neutrokine-alpha or Neutrokine-alphaSV polynucleotide of the present invention, clones the heterologous polynucleotide of the present invention into an expression vector such as pGAPZ or pGAPZalpha and the absence of methanol By growing the yeast culture under

Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of usually about 10 to 300 bp DNA that act on the promoter to increase transcription. Examples include the SV40 enhancer of bp 100 to 270 located in the second half of the origin of replication, the cytomegalo early promoter enhancer, the polyoma enhancer located in the second half of the origin of replication and the adenovirus enhancer.

Various mammalian cell culture systems can also be used to express recombinant protein. Examples of mammalian expression systems include, but are not limited to, the COS-7 strain of monkey kidney fibroblasts described in Glozman, Cell 23: 175 (1981) and other cell lines capable of expressing a conformity vector (eg, C127, 3T3, CHO, Hella). And BHK cell lines). Mammalian expression vectors include replication origins, suitable promoters and enhancers, and other necessary ribosomal binding sites, polyadenylation sites, splice donor and receptor sites, transcription termination sequences, and 5 'flanking non-transcription sequences. DNA sequences derived from SV40 splices and polyadenylation sites can be used to provide the necessary nontranscribed genetic elements.

In certain embodiments, constructs designed to express some of the extracellular domains of Neutrokine-alpha (eg, amino acid residues Ala-134 to Leu-285) are preferred. Those skilled in the art can design polynucleotide primers using the polynucleotide and polypeptide sequences provided as SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and generate expression constructs therefrom.

In another embodiment, constructs designed to express the complete putative extracellular domain of Neutrokine-alpha (ie, amino acid residues Gln-73 to Leu-285) are preferred. Those skilled in the art can design polynucleotide primers using the polynucleotide and polypeptide sequences provided as SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and generate expression constructs therefrom.

In addition to including host cells containing the vector constructs discussed herein, the present invention also provides for the deletion or substitution of endogenous genetic material (eg, Neutrokine-alpha coding sequences) and / or Neutrokine-alpha polynucleotides of the present invention. Of vertebrate origin, in particular mammalian origin, engineered to contain genetic material (e.g., heterologous polynucleotide sequences) that is operatively linked to and activates, mutates and / or amplifies endogenous Neutrokine-alpha polynucleotides. Primary, secondary and immortalized host cells. For example, using techniques known in the art to homologous recombination regions (eg, promoters and / or enhancers) and endogenous Neutrokine-alpha polynucleotide sequences, homologous recombination (see, eg, June 24, 1997) Patent 5,461,670; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342-435-438 (1989). The entire contents of each of these documents are incorporated herein by reference.

The host cells described below can be used in a conventional manner to produce gene products encoded by recombinant techniques. Alternatively, the cell-free translation system can be used to generate the polypeptide of the invention from RNA derived from the DNA construct of the invention.

Polypeptides of the invention can be expressed or synthesized in a modified form, such as fusion proteins (including polypeptides linked to heterologous protein sequences (of different proteins) via peptide bonds), and can produce additional heterologous regions as well as secretion signals. May include. Such fusion proteins can be obtained by linking the polynucleotides of the invention with nucleic acid sequences encoding the desired amino acid sequences to one another by methods known in the art within an appropriate reading frame and expressing the fusion protein products by methods known in the art. have. Alternatively, such fusion proteins can be made by protein synthesis techniques, for example by the use of peptide synthesizers. Thus, for example, additional amino acids, particularly regions of charged amino acids, may be added to the N-terminus of the polypeptide to enhance stability and persistence in the host cell, during purification, or during subsequent processing and storage. Peptide residues may also be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to allow for secretion or excretion, to improve stability, and to facilitate purification is a familiar and conventional technique in the art.

In one embodiment, polynucleotides encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention are fused to a pelB pectate lyase signal sequence to express and purify such polypeptides in Gram-negative bacteria. Efficiency can be increased (see US Pat. Nos. 5,576,195 and 5,846,818). The entire contents of these patents are incorporated herein by reference.

Preferred fusion proteins include heterologous regions from immunoglobulins useful for stabilizing and purifying proteins. For example, EP-A-O 464 533 (Canadian corresponding patent 2045869) describes fusion proteins comprising several parts of the constant region of an immunoglobulin molecule with other proteins or parts thereof. In many cases, the Fc portion of the fusion protein is very advantageous for use in therapy and diagnosis and thus provides for example improved pharmacokinetic properties (EP-A 032 262). On the other hand, for some uses it may be desirable to remove the Fc moiety after the fusion protein is expressed, detected and purified in the described advantageous manner. This is where the use of the Fc moiety has proven to be an obstacle in the treatment and diagnosis, for example when the fusion protein is used as an antigen for immunization. In drug development, for example, human proteins such as hIL-5 have been fused with the Fc moiety for the purpose of a high-working screening assay to identify antagonists of hIL-5 (see D. Bennet et al., J.). Molecular Recognition 8: 52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995).

Polypeptides of the invention include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plants, insects, and mammalian cells. This includes. Depending on the host used in the recombinant manufacturing procedure, the polypeptides of the present invention may be glycosylated or aglycosylated. In addition, the polypeptides of the present invention may also include methionine residues that are initially modified in some cases as a result of the host-mediated process.

Polypeptides of the invention can be chemically synthesized using techniques known in the art (see, eg, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., NY, and Hunkapiller, M., et al., 1984, Nature 310: 105-111). For example, peptides corresponding to fragments of the complete Neutrokine-alpha or Neutrokine-alphaSV polypeptides of the invention can be synthesized using a peptide synthesizer. Also, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into Neutrokine-alpha or Neutrokine-alphaSV polynucleotide sequences. Uncommon amino acids include, but are not limited to, the D-isomers of common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine acid, t- Butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, design amino acids (e.g., b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids) and common amino acid analogs do. The amino acid may also be D (right turn) or L (left turn).

The present invention is modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, or the like, during or after translation. Neutrokine-alpha or Neutrokine-alphaSV polypeptides that can be distinguished. None of the numerous chemical modifications are limited to these by metabolic synthesis in the presence of cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ , acetylation, formylation, oxidation, reduction, tunicamycin It can be accomplished by known techniques involving specific chemical cleavage.

Further post-translational modifications encompassed by the present invention include, for example, processing at the N-terminus or C-terminus, linking chemical residues to amino acid backbones, chemical modifications of N-linked or O-linked carbohydrate chains and prokaryotic host cells. Addition or deletion of N-terminal methionine residues as a result of expression is included. Polypeptides can also be modified with a detection label such as an enzyme, fluorescence, isotope or affinity label to enable detection and isolation of the protein. In addition, the polypeptides of the invention can be modified by iodide.

In one embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention may also be labeled with biotin. In other related embodiments, the biotinylated Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention can be prepared, for example, by imaging agents or one or more Neutrokine-alpha and / or Neutrokine-alphaSV receptor (s). Or as a means for identifying other co-receptor or co-ligand molecules.

In addition, the present invention provides chemically modified derivatives of Neutrokine-alpha or Neutrokine-alphaSV which may provide additional benefits such as increased solubility, stability and polypeptide in vivo or in vitro circulation time or reduced immunogenicity. (See US Pat. No. 4,179,337). The chemical moiety for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptide may be modified at any position in the molecule and at a predetermined position in the molecule and may comprise one, two, three or more linked chemical moieties.

The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycols, the preferred molecular weight is from about 1 kDa to about 100 kDa for convenient manipulation and preparation and the term "about" refers to the molecular weight in the preparation of polyethylene glycol and some molecules may be more or less weight than the molecular weights described above. have. Different sizes may be used depending on the desired therapeutic profile (eg, desired duration of sustained release, effect on biological activity, ease of manipulation, degree or lack of antigenicity and other known effects of polyethylene glycol on the therapeutic protein or analog). have. For example, polyethylene glycol may contain about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, It may have an average molecular weight of 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

As mentioned above, polyethylene glycol may have a side chain structure. Branched polyethylene glycols are described, for example, in US Pat. No. 5,643,575; Morpurgo, et al., Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18: 2745-2750 (1999); And Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999). The contents of each of these documents are incorporated herein by reference.

Polyethylene glycol molecules (or other chemical moieties) should be linked to the protein taking into account its effect on the functional or antigenic domains of the protein. There are many binding methods available to those skilled in the art (see, eg, EP 0 401 384 (GPE binding to G-CSF) and Malik et al., Exp. Hematol. 20: 1028-1035 (1992)) PEGylation of GM-CSF using sil chloride)]. For example, polyethylene glycol can be covalently linked to an amino acid residue via a reactive group such as a free amino or carboxyl group. Reaction groups are groups to which activated polyethylene glycol molecules can be bound. Amino acid residues having free amino groups may include, for example, lysine residues and N-terminal amino acid residues; Amino acid residues having free carboxyl groups may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Sulhydryl groups can also be used as reaction groups for bonding polyethylene glycol molecules. Binding to amino groups, such as binding to N-terminal or lysine groups, is preferred for therapeutic purposes.

As suggested above, polyethylene glycol can be linked to a protein through linkage with any one of many amino acid residues. For example, polyethylene glycol can be linked to a protein via covalent bonds with lysine, histidine, aspartic acid, glutamic acid or cysteine residues. Polyethylene glycols may be used in one or more reaction chemistries to provide specific amino acid residues of the protein (eg lysine, histidine, aspartic acid, glutamic acid or cysteine) or one or more types of amino acid residues of the protein (eg lysine, histidine, aspartic acid). , Glutamic acid, cysteine and combinations thereof).

Certain chemically modified proteins at the N-terminus may be required. As an example when using polyethylene glycol, the type of polyethylene glycol molecule (by molecular weight, branching, etc.), the ratio of the polyethylene glycol molecule to the protein (or peptide) molecule in the reaction mixture, the type of pegylation reaction to be carried out and the selected N It can be selected from the method of obtaining terminally-peptylated protein. The method of obtaining an N-terminal pegylation agent (i.e., separating this residue from other monopezylation moieties if necessary) can be accomplished by purification of the N-terminal pegylation material from the group of pegylation protein molecules. Certain proteins that are chemically modified at the N-terminus can be achieved by reductive alkylation which exhibits distinct reactivity of other types of primary amino groups (lysine versus N-terminus) that are being used for derivatization at the particular protein. Under appropriate reaction conditions, substantial specific derivatization of the protein at the N-terminus with the carbonyl group containing polymer is achieved.

As described above, the pegylation of proteins according to the invention can be achieved by a number of means. For example, polyethylene glycol can be linked to the protein directly or via an intermediate linker. Linker-free systems for linking polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al., Intern. J. of Hematol. 68: 1-18 (1998); US Patent No. 4,002,531; US Patent No. 5,349,052; WO 95/06058; And WO 98/32466. Each of these documents is incorporated herein by reference.

One system for directly linking polyethylene glycol to amino acid residues of a protein without an intermediate linker is the use of tresylchloride (ClSO ₂ CH ₂ CF ₃ ) to modify the monomethoxy polyethylene glycol (MPEG) resulting in tresylated MPEG. use. When the protein is reacted with tresylated MPEG, polyethylene glycol is directly linked to the amino group of the protein. Accordingly, the present invention includes protein-polyethylene glycol conjugates produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethane sulfonyl group.

Polyethylene glycols can also be linked to proteins using many different intermediate linkers. For example, US Pat. No. 5,612,460 describes urethane linkers for linking polyethylene glycol to proteins. The entire contents of this patent are incorporated herein by reference. Protein-polyethylene glycol conjugates in which polyethylene glycol is linked to the protein by linkers are also known as MPEG-succinimidylsuccinate, MPEG, MPEG-2,4,5-trichloro activated with 1,1'-carbonyldiimidazole. It can be produced by reacting a protein with a compound such as phenylcarbonate, MPEG-p-nitrophenol carbonate and various MPEG-succinate derivatives. Many additional polyethylene glycol derivatives and reaction chemistries for linking polyethylene glycol to proteins are described in WO 98/32466. The entire contents of this patent are incorporated herein by reference. Pegylated protein products produced using the reaction chemistry described herein are included within the scope of the present invention.

The number (ie degree of substitution) of polyethylene glycol residues linked to each protein of the invention may also vary. For example, the fezylated protein of the present invention may, on average, be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. have. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12 per protein molecule. , 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 polyethylene glycol residues. Methods of measuring the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides include, but are not limited to, ammonium sulfate or ethanol sedimentation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, Recovery and purification can be carried out by known methods including chemical chromatography, hydroxyapatite chromatography and lectin chromatography. The most preferred method for purification is high performance liquid chromatography (HPLC).

Neutrokine-alpha polypeptide

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention may be monomers or multimers (ie, dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the present invention, methods for their preparation and compositions containing them (preferably pharmaceutical compositions). . In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further embodiment, the multimers of the invention are at least dimers, at least trimers or at least tetramers.

Multimers belonging to the invention may be homomeric or heteromultimers. The term homomer, as used herein, refers to Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention (Neutrokine-alpha and / or Neutrokine-alphaSV fragments, variants and fusion proteins described herein). It means a multimer containing only). These homomers may contain Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers containing only Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides having the same amino acid sequence. In another specific embodiment, the homomers of the invention are multimers containing Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides having different amino acid sequences. In certain embodiments, multimers of the invention may be homodimers (eg, contain Neutrokine-alpha and / or Neutrokine-alphaSVs having the same or different amino acid sequences) or homotrimers (eg, the same or different amino acid sequences) Containing Neutrokine-alpha and / or Neutrokine-alphaSV). In a preferred embodiment, the multimers of the invention are homotrimers. In a further embodiment, the homodimeric multimers of the invention are at least a homodimer, at least a homotrimer or at least a homo tetramer.

As used herein, the term heteromeric multimer refers to multimers containing heterologous polypeptides (ie, polypeptides of different proteins) in addition to the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention. In certain embodiments, the multimers of the present invention are heterodimers, heterotrimers or hetero tetramers. In a further embodiment, the multimers of the present invention are at least heterodimers, at least heterotrimers or at least hetero tetramers. In further conventional embodiments, the heteromultimers of the invention contain CD40 ligand polypeptide sequence (s) or biologically active fragment (s) or variant (s) thereof.

Multimers of the present invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent bonds and / or may be indirectly linked, for example by liposome formation. Thus, in one embodiment, multimers of the invention, such as homodimers or homotrimers, are formed when a polypeptide of the invention is contacted with one other polypeptide in solution. In another embodiment, a heterodimer of the present invention, such as a heterodimer or a heterotrimer, is characterized in that the polypeptide of the invention is directed to an antibody against the polypeptide of the invention (in a heterologous polypeptide sequence of the fusion protein of the invention) in solution. Antibody). In another embodiment, multimers of the invention are formed by covalent bonds to and / or covalent bonds between the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention. Such covalent bonds include one or more amino acid residues contained in a polypeptide sequence (eg, residues set forth in SEQ ID NO: 2 or SEQ ID NO: 19 or residues contained in a polypeptide encoded by a clone deposited in connection with the present application). As one example, a covalent bond is a bridge between cysteine residues located within a polypeptide sequence that interacts in a native polypeptide. As another example, covalent bonds are the result of chemical or recombinant manipulation. Alternatively, the covalent linkage may involve one or more amino acid residues contained in the heterologous polypeptide sequence in the Neutrokine-alpha and / or Neutrokine-alphaSV fusion protein. As an example, covalent linkages are made between heterologous sequences contained in the fusion proteins of the invention (see, eg, US Pat. No. 5,478,925). As a specific example, covalent linkages are made between heterologous sequences contained in the Neutrokine-alpha-Fc and / or Neutrokine-alphaSV-Fc fusion proteins described herein. As another specific example, the covalent linkage of a fusion protein according to the invention is between heterologous polypeptide sequences from other TNF class ligand / receptor members capable of forming covalently linked multimers, such as osteoprotegerin. International Publication No. WO 98/49305. The entire contents of this patent are incorporated herein by reference. As another specific example, the covalent linkage of a fusion protein according to the invention is between heterologous polypeptide sequences from CD40L or soluble fragments thereof. In another embodiment, two or more Neutrokine-alpha and / or Neutrokine-alpha polypeptides according to the invention are linked via synthetic linkers (eg, peptides, carbohydrates or soluble polymer linkers). Examples include peptide linkers described in US Pat. No. 5,073,627. The contents of this patent are incorporated herein by reference. Proteins comprising multiple Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides separated by a peptide linker can be produced by conventional recombinant DNA techniques.

The method for preparing multimeric Neutrokine-alpha and / or Neutrokine-alpha SV polypeptides according to the present invention comprises Neutrokine-alpha and / or Neutrokine-alpha fused to a leucine zipper or isoleucine zipper polypeptide sequence. The use of SV polypeptides. Leucine zipper or isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were first identified in several DNA binding proteins (Landschulz et al., Science 240: 1759, (1988)) and have since been found in several other proteins. Among the known leucine zippers or isoleucine zippers include natural peptides and derivatives thereof which dimer or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric Neutrokine-alpha and / or Neutrokine-alphaSV proteins include those described in PCT Patent Publication No. WO 94/10308. The contents of this patent are incorporated herein by reference. Recombinant fusion proteins comprising soluble Neutrokine-alpha and / or Neutrokine-alphaSV fused to a dimer or trimerized peptide in solution are expressed in a suitable host cell using techniques known in the art and from the culture supernatant. The resulting soluble multimeric Neutrokine-alpha and / or Neutrokine-alphaSV is recovered.

It is believed that certain members of the TNF class of proteins exist in trimeric form (Beutler and Huffel, Science 264: 667, 1994; Banner et al., Cell 73: 431, 1993). Thus, trimer Neutrokine-alpha and / or Neutrokine-alphaSV may provide the benefit of increased biological activity. Preferred leucine zipper residues are those which preferentially form trimers. One example is a leucine zipper derived from lung supernatant protein D (SPD) as described in Hoppe et al., FEBS Letters 344: 191, (1994) and US patent application 08 / 446,922. The contents of these documents are incorporated herein by reference. Other peptides derived from natural trimer proteins can be used to prepare trimer Neutrokine-alpha and / or Neutrokine-alphaSV.

As another example, the protein of the invention is bound by interaction between flag polypeptide sequences contained in the flag-Neutrokine alpha or flag-Neutrokine-alphaSV fusion protein of the invention. In a further aspect, the protein of the invention is bound by the interaction between the heterologous polypeptide sequence contained in the flag-Neutrokine alpha or flag-Neutrokine-alphaSV fusion protein of the invention and an anti-flag antibody.

Multimers of the present invention can be produced using chemical techniques known in the art. For example, polypeptides that need to be contained in the multimers of the present invention can be chemically crosslinked using linker molecule length optimization techniques and linker molecules known in the art (see, eg, US Pat. No. 5,478,925). ). The entire contents of this patent are incorporated herein by reference. In addition, the multimers of the present invention may be produced using techniques known in the art to form one or more intermolecular crosslinks between cysteine residues located within the sequence of polypeptides that need to be contained in the multimer (see Eg US Pat. No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In addition, techniques known in the art can be applied to form liposomes containing polypeptide components that need to be contained in the multimers of the present invention (see, eg, US Pat. No. 5,478,925). The contents of this patent are incorporated herein by reference.

Alternatively, the multimers of the present invention can be formed using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in the multimers of the present invention are recombinantly produced using fusion protein techniques described herein or known in the art (see, eg, US Pat. No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In certain embodiments, the polynucleotides encoding homodimers of the invention link a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further from the original C-terminus to the N-terminus. Produced by linking the translation product of the polypeptide (lacking the leader sequence) in the opposite direction to a synthetic polynucleotide encoding (see, eg, US Pat. No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In another embodiment, recombinant techniques described herein or known in the art are applied to produce recombinant polypeptides according to the invention which contain transmembrane domains and which can be introduced into liposomes by membrane reconstitution techniques (see examples). : US Pat. No. 5,478,925). The entire contents of this patent are incorporated herein by reference.

In one embodiment, the invention provides an isolated Neutrokine-alpha polypeptide or portion of a polypeptide thereof having an amino acid sequence encoded by a cDNA clone contained in ATCC 97768 or an amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2). Provided are polypeptides comprising (ie, fragments). In another embodiment, the invention provides an isolated Neutrokine-alphaSV polypeptide or polypeptide thereof having an amino acid sequence encoded by a cDNA clone contained in ATCC 203518 or an amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19). A polypeptide is provided that includes a portion (ie, a fragment).

Polypeptide fragments of the invention are encoded by cDNA contained in SEQ ID NO: 2, or contained in the plasmid of ATCC Accession No. 97768, or are described in the nucleotide sequence contained in the deposited clone or described in Figure 1A-B (SEQ ID NO: 1). A polypeptide comprising an amino acid sequence encoded by a nucleic acid that hybridizes (eg, under stringent hybridization conditions) to the complementary chain of the nucleotide sequence, or alternatively comprises a polypeptide consisting of such sequence.

In addition, the polypeptide fragment of the present invention is encoded by cDNA contained in SEQ ID NO: 19, or contained in the plasmid of ATCC Accession No. 203518 or contained in the deposited clone or FIG. 5A-B (SEQ ID NO: 18). A polypeptide comprises an amino acid sequence encoded by a nucleic acid that hybridizes (eg, under stringent hybridization conditions) to the complementary chain of the nucleotide sequence described herein, or alternatively comprises a polypeptide consisting of such sequence.

In addition, polypeptide fragments of the invention comprise, or alternatively comprise, an amino acid sequence encoded by a nucleic acid that hybridizes (eg, under the hybridization conditions described herein) to the complementary chain of the nucleotide sequence described in SEQ ID NO: 21. Includes polypeptides consisting of such sequences.

In addition, the polypeptide fragments of the invention comprise an amino acid sequence encoded by a nucleic acid contained in SEQ ID NO: 23 or hybridizing with the complementary chain of the nucleotide sequence described in SEQ ID NO: 22 (eg, under the hybridization conditions described herein). Or alternatively comprises polypeptides consisting of such sequences.

In addition, the polypeptide fragments of the present invention comprise an amino acid sequence encoded by a nucleic acid contained in SEQ ID NO: 28 or hybridizing with the complementary chain of the nucleotide sequence described in SEQ ID NO: 27 (eg, under the hybridization conditions described herein). Or alternatively comprises polypeptides consisting of such sequences.

In addition, the polypeptide fragments of the invention comprise an amino acid sequence encoded by a nucleic acid contained in SEQ ID NO: 30 or hybridizing with the complementary chain of the nucleotide sequence shown in SEQ ID NO: 29 (eg, under the hybridization conditions described herein) or Alternatively, polypeptides consisting of such sequences are included.

Polypeptide fragments of the present invention are either nucleotide sequences contained in SEQ ID NO: 2, encoded by cDNA contained in deposited clones, or contained in the deposited clones, or the nucleotide sequences described in FIGS. 1A and 1B (SEQ ID NO: 1) or A polypeptide comprises an amino acid sequence encoded by a nucleic acid that hybridizes (eg under stringent hybridization conditions) to its complementary chain, or alternatively comprises a polypeptide consisting of such sequence. Protein fragments may be present independently or may be present in fragments within larger polypeptides that occupy some or a region most preferably as a single continuous region. Representative examples of polypeptide fragments according to the invention include, or alternatively include, about 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, and / or 251 to 285 amino acid residues of SEQ ID NO: 2. Fragments consisting of these residues are included. In addition, polypeptide fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 or more amino acids in length.

In certain embodiments, polypeptide fragments of the invention comprise amino acid residues 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148- as shown in FIGS. 1A and 1B (SEQ ID NO: 2). 152, 166-181, 185-209, 210-221, 226-237, 244-249, 253-265 and / or 277-284, or alternatively consist of these residues.

Neutrokine of the invention comprising a 19 amino acid residue insertion not found in the Neutrokine-alphaSV polypeptide sequence (ie amino acid residues Val-142 to Lys-160 in the sequences described in FIGS. 1A and 1B and SEQ ID NO: 2) It will be appreciated by those skilled in the art that mutations targeting regions of alpha polypeptides can affect the observed biological activity of Neutrokine-alpha polypeptides. More specifically, the list of partial, non-limiting, non-exclusive residues in Neutrokine-alpha polypeptide sequences that may be targeted by mutations includes the following amino acid residues in the Neutrokine-alpha polypeptide sequence described in SEQ ID NO: 2. V-142; T-143; Q-144; D-145; C-146; L-147; Q-148, L-149; I-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157; I-158; Q-159; And K-160. Polynucleotides encoding Neutrokine-alpha polypeptides having one or more mutations in regions V-142 to K-160 of SEQ ID NO: 2 are contemplated. Polypeptides encoded by these polynucleotides are also included in the present invention.

Polypeptide fragments may be present independently or may be present in fragments within larger polypeptides that occupy some or a region most preferably as a single continuous region. Representative examples of polypeptide fragments according to the invention include about 1 to 15, 16 to 30, 31 to 46, 47 to 55, 56 to 72, 73 to 104, 105 to 163, 163 to 188, 186 amino acid residues of SEQ ID NO: 2. To 210 and 210 to 284, or alternatively fragments consisting of such moieties. Further representative examples of polypeptide fragments according to the invention include about 1 to 143, 1 to 150, 47 to 143, 47 to 150, 73 to 143, 73 to 150, 100 to 150, 140 to 145, amino acid residues of SEQ ID NO: 19; 142 to 148, 140 to 150, 140 to 200, 140 to 225 and 140 to 266, or alternatively fragments consisting of these residues. In addition, polypeptide fragments may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 or more amino acids in length. Here, "about" means a range that is larger or smaller by several, ie 5, 4, 3, 2 or 1 amino acid residues at the amino and carboxy terminus in the specified range. Polynucleotides encoding these polypeptide fragments are also included in the present invention.

Further preferred embodiments include the putative intracellular domain of Neutrokine-alpha (amino acid residues 1-46 of SEQ ID NO: 2), the putative transmembrane domain of Neutrokine-alpha (amino acid residues 47-72 of SEQ ID NO: 2), Neutrokine- Polypeptide fragments comprising, or consisting of, the putative extracellular domain of alpha (amino acid residues 73 to 285 of SEQ ID NO: 2), and the putative TNF conserved domain of Neutrokine-alpha (amino acids 191 to 284 of SEQ ID NO: 2) Polypeptides comprising or consisting of an putative intracellular domain fused to an putative extracellular domain of keen-alpha (amino acid residues 1 to 46 fused to amino acid residues 73 to 285 of SEQ ID NO: 2). Polynucleotides encoding these polypeptides are also included in the present invention.

Still further preferred embodiments include the putative intracellular domain of Neutrokine-alphaSV (amino acid residues 1 to 46 of SEQ ID NO: 19), and the putative transmembrane domain of Neutrokine-alphaSV (amino acid residues 47 to 72 of SEQ ID NO: 19). Include, or include, an estimated extracellular domain of Neutrokine-alphaSV (amino acid residues 73 to 266 of SEQ ID NO: 19), and an estimated TNF conserved domain of Neutrokine-alphaSV (amino acids 172 to 265 of SEQ ID NO: 19). A polypeptide comprising or consisting of an estimated intracellular domain fused to an estimated extracellular domain of Neutrokine-alphaSV (amino acid residues 1 to 46 fused to amino acid residues 73 to 266 of SEQ ID NO: 19) Peptides. Polynucleotides encoding these polypeptides are also included in the present invention.

Certain further embodiments of the invention include polypeptide fragments comprising or consisting of the putative beta-fleet sheet regions described in FIG. 7A. These polypeptide fragments of the invention are amino acid residues Gln-144 to Ala-151, Phe-172 to Lys-173, Ala-177 to Glu-179, Asn-183 to Ile-185, Gly-191 to Lys of SEQ ID NO: 2 -204, His-210 to Val-219, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to Ile-263 and / or Val-276 to Leu-284, or alternatively Consisting of these residues. In another non-limiting embodiment, these polypeptide fragments of the invention comprise amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160, Asn-164 to Ile-166, Gly-172 to Lys of SEQ ID NO: 19. -185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile-244 and / or Vla-257 to Leu-265 and amino acid residues of SEQ ID NO: 23 Phe-42 to Lys-43, Ala-47 to Glu-49, Asn-53 to Ile-55, Gly-61 to Pro-74, His-80 to Val-89, Leu-96 to Pro-107, Asn- 112 to Ala-121, Gly-126 to Ile-133 and / or Asp-146 to Leu-154. In a further non-limiting embodiment, those polypeptide fragments of the invention also include amino acid residues Gln-78 to Ala-85, Phe-106 to Lys-107, Ala-111 to Glu-113, Asn-117 to Ile-119, Gly-125 to Lys-138, His-144 to Val-153, Leu-160 to Pro-171, Asn-176 to Ala-185, Gly-190 to Ile-197 and / or Val-210 to Leu-218 and amino acid residues Gln-78 to Ala-85, Phe-106 to Lys-107, Ala-111 to Glu-113, Asn-117 to Ile-119, Gly-125 to Lys-138, His of SEQ ID NO: 30 -144 to Val-153, Leu-160 to Pro-171, Asn-176 to Ala-185, Gly-190 to Ile-197 and / or Val-210 to Leu-218. Polynucleotide fragments encoding these polypeptides are also provided by the present invention.

Examples of non-limiting examples of polypeptides according to the invention comprising, or alternatively consisting of, combinations of amino acid sequences according to the invention include fused to [Gly-250 to Leu-285] of SEQ ID NO: 2. [Met- fused to [Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to Val-199 to Ala-248] 1 to Lys-113; Fusion to [Ile-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-250 to Leu-285] of SEQ ID NO: 2 [Met-1 to Lys-113]; Or [Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly-250 to Leu-285] of SEQ ID NO: 2 Fused [Met-1 to Lys-113] are included. Other combinations may include polypeptide fragments combined in any order other than those described above (eg, fused to [Gly-250 to Leu-285] fused to [Ile-142 to Lys-160] of SEQ ID NO: 2). [Leu-114 to Thr-141] fused to [Val-199 to Ala-248]. Other combinations may also include the heterologous polypeptide fragments described herein and / or other polypeptides and polypeptide fragments of the invention (eg, in [Gly-250 to Leu-285] of SEQ ID NO: 2 fused to FLAG tags). [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to fused [Gly-161 to Gln-198]. Polynucleotides encoding these polypeptides are also included in the present invention.

Further non-limiting examples of polypeptides according to the invention comprising a combination of amino acid sequences or alternatively consisting of such combinations [Val-180 to fused to [Gly-230 to Leu-266] of SEQ ID NO: 19 [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to Ala-229]; [Met-1 to Lys-113] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO: 19; Or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO: 19 Included. Other combinations may include polypeptide fragments combined in other orders than those described above (eg, fused to [Gly-230 to Leu-266] fused to [Gly-142 to Gln-179] of SEQ ID NO: 19). [Leu-114 to Thr-141] fused to [Val-180 to Ala-229]. Other combinations may also include the heterologous polypeptide fragments described herein and / or other polypeptides and polypeptide fragments of the invention (eg, Gly-230 to Leu-266 of SEQ ID NO: 19 fused to a FLAG tag). [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179]. Polynucleotides encoding these polypeptides are also included in the present invention.

Further non-limiting examples of polypeptides according to the invention comprising a combination of amino acid sequences or alternatively consisting of such combinations [Val-225 to fused to [Gly-273 to Leu-309] of SEQ ID NO: 23 [Met-1 to Lys- fused to [Leu-107 to Thr-134] fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to Ala-272] 106; Fusion to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to [Val-225 to Ala-272] fused to [Gly-273 to Leu-309] of SEQ ID NO: 23 [Met-1 to Lys-106] fused to [Glu-135 to Asn-165]; Or [Glu-135 to Asn-165] fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to [Gly-273 to Leu-309] of SEQ ID NO: 23 [Met-1 to Lys-106] fused to fused [Leu-107 to Thr-134] are included. Other combinations may include polypeptide fragments combined in other orders than those described above (eg, fused to [Leu-107 to Thr-134] fused to [Gly-273 to Leu-309] of SEQ ID NO: 23). [Met-1 to Lys-106] fused to [Gly-185 to Gln-224] fused to [Ile-167 to Lys-184] fused to [Val-225 to Ala-272]. Other combinations may also include the heterologous polypeptide fragments described herein and / or other polypeptides and polypeptide fragments of the invention (eg, in [Gly-273 to Leu-309] of SEQ ID NO: 23 fused to FLAG tags). Fused to [Glu-135 to Asn-165] fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to fused [Val-225 to Ala-272] [Met-1 to Lys-106]. Polynucleotides encoding these polypeptides are also included in the present invention.

Further non-limiting examples of polypeptides according to the invention comprising a combination of amino acid sequences or alternatively consisting of such combinations [Val-133 to fused to [Gly-183 to Ala-219] of SEQ ID NO: 28 [Tyr-1 to Lys- fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to Ala-182] 47; [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] of SEQ ID NO: 28; Or [Tyr-1 to Lys-47] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] fused to [Gly-183 to Ala-219] of SEQ ID NO: 28 Included. Other combinations may include polypeptide fragments combined in other orders than those described above (eg, fused to [Val-133 to Ala-182] fused to [Leu-48 to Thr-75] of SEQ ID NO: 28). [Tyr-1 to Lys-47] fused to [Gly-183 to Ala-219]). Other combinations may also include the heterologous polypeptide fragments described herein and / or other polypeptides and polypeptide fragments of the invention (eg, [Val-133 to Ala-182] of SEQ ID NO: 28 fused to an Fc receptor tag). [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132]. Polynucleotides encoding these polypeptides are also included in the present invention.

[Val-133 to fused to [Gly-183 to Ala-219] of SEQ ID NO: 30 as examples of further non-limiting examples of polypeptides according to the invention comprising, or alternatively consisting of, amino acid sequences; [Tyr-1 to Lys- fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to Ala-182] 47; [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] of SEQ ID NO: 30; Or [Tyr-1 to Lys-47] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] fused to [Gly-183 to Ala-219] of SEQ ID NO: 30 Included. Other combinations may include polypeptide fragments combined in other orders than those described above (eg, fused to [Val-133 to Ala-182] fused to [Leu-48 to Thr-75] of SEQ ID NO: 30). [Tyr-1 to Lys-47] fused to [Gly-183 to Ala-219]). Other combinations may also include heterologous polypeptide fragments described herein and / or other polypeptides and polypeptide fragments of the invention (eg, in [Val-133 to Ala-182] of SEQ ID NO: 30 fused to an Fc tag). [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132]. Polynucleotides encoding these polypeptides are also included in the present invention.

Additional aspects of the invention are described in FIGS. 3 and 6 and Table 1 and the Garnier-Robson alpha-regions, beta-regions, turn-regions and coil-regions, Chow-Pasman alpha-described as described herein. Jameson of domains, beta- and coil-domains, kite-dulit hydrophilic and hydrophobic domains, Eisenberg alpha- and beta-bilateral domains, Carplus-Shultz flexion zones, Emini surface-forming regions and high antigenic index A Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide fragment that comprises or alternatively consists of a functional region of a polypeptide of the invention, such as a wolf region. In a preferred embodiment, the polypeptide fragments of the invention are antigenic. The data described in columns VIII, IX, XIII and XIV in Table 1 can be used routinely to determine regions of Neutrokine-alpha exhibiting high levels of antigenic potential. Highly antigenic regions are assigned to columns VIII, IX, XIII and / or IV by selecting values representing regions of the polypeptide that are likely to be exposed to the surface of the polypeptide in an environment where antigen recognition may occur during the initiation of an immune response. Measure from the data described. Very preferred fragments of the invention include regions of Neutrokine-alpha and / or Neutrokine-alphaSV that combine several structural features such as several (eg, 1, 2, 3 or 4) among the features described above. Included fragments are included. Polynucleotides encoding these polypeptides are also included in the present invention.

In another aspect, the invention provides a polypeptide comprising or alternatively consisting of an epitope-containing portion of a polypeptide according to the invention. Polynucleotides encoding these polypeptides are also included in the present invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of the polypeptide according to the invention. An "immunogenic epitope" is defined as part of a protein that elicits an antibody response when the complete protein is an immunogen. On the other hand, the region of the protein molecule to which the 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, eg, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)).

Regarding the selection of polypeptides containing antigenic epitopes (ie, containing regions of protein molecules to which antibodies can bind), relatively short synthetic peptides that mimic part of the protein sequence react with partially simulated proteins. It is well known that it can conventionally induce antiserum (see, eg, Sutcliffe, JG, Shinnick, TM, Green, N. and Learner, RA (1983) "Antibodies that react with predetermined sites on proteins", Science, 219 (660-666). Peptides capable of eliciting protein-reactive serum are commonly indicated in the primary sequence of a protein and can be characterized by a set of simple chemical rules and complete the immunodominant region (ie, immunogenic epitope) or amino or carboxyl of a complete protein. It is not limited to the terminal anywhere. Thus, antigenic epitope-containing peptides and polypeptides according to the present invention are useful for eliciting antibodies (including monoclonal antibodies) that specifically bind to the polypeptides of the present invention (Wilson et al., Cell 37: 767-778 (1984) at 777).

Antigenic epitope-containing peptides and polypeptides of the invention preferably contain at least 4, at least 5, at least 6, at least 7 amino acid sequences contained within the amino acid sequences of the polypeptides of the invention, more preferably More than 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, It contains at least 50 amino acid sequences, most preferably from about 15 to about 30 amino acid sequences. Preferred polypeptides comprising immunogenic or antigenic epitopes are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 in length. Or 100 amino acid residues. Additional conventional preferred antigenic epitopes include the antigenic epitopes described herein as well as portions thereof.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Neutrokine-alpha- and / or Neutrokine-alphaSV-specific antibodies include the following: FIGS. 1A and 1B (SEQ ID NO: 2) A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Phe-115 to about Leu-147 shown; Polypeptides comprising, or alternatively consisting of, amino acid residues of about Ile-150 to about Tyr-163 shown in FIGS. 1A and 1B (SEQ ID NO: 2); A polypeptide comprising or alternatively consisting of the amino acid residues of about Ser-171 to about Phe-194 shown in FIGS. 1A and 1B (SEQ ID NO: 2); Polypeptides comprising, or alternatively consisting of, amino acid residues of about Glu-223 to about Tyr-246 shown in FIGS. 1A and 1B (SEQ ID NO: 2); And a polypeptide comprising, or alternatively consisting of, amino acid residues of about Ser-271 to about Phe-278 shown in FIGS. 1A and 1B (SEQ ID NO: 2). Here, "about" means a range that is greater or less than a few, ie 5, 4, 3, 2 or 1 amino acid residues in either or both of the amino- and carboxy-terminus in the specified range. These polypeptide fragments were determined to contain the antigenic epitopes of Neutrokine-alpha polypeptides by analysis of the Jameson-Wolf Index, as described in FIG. 3 and Table 1.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate Neutrokine-alpha- and / or Neutrokine-alphaSV-specific antibodies include the following: Figures 5A and 5B (SEQ ID NO: 19). A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Pro-32 to about Leu-47 shown; A polypeptide comprising or alternatively consisting of the amino acid residues of about Glu-116 to about Ser-143 shown in FIGS. 5A and 5B (SEQ ID NO: 19); Polypeptides comprising or alternatively consisting of amino acid residues of about Phe-153 to about Tyr-173 shown in FIGS. 5A and 5B (SEQ ID NO: 19); A polypeptide comprising or alternatively consisting of the amino acid residues of about Pro-218 to about Tyr-227 shown in FIGS. 5A and 5B (SEQ ID NO: 19); Polypeptides comprising, or alternatively consisting of, amino acid residues of about Ala-232 to about Gln-241 shown in FIGS. 5A and 5B (SEQ ID NO: 19); A polypeptide comprising or alternatively consisting of the amino acid residues of about Ile-244 to about Ala-249 shown in FIGS. 5A and 5B (SEQ ID NO: 19); And a polypeptide comprising, or alternatively consisting of, amino acid residues of about Ser-252 to about Val-257 shown in FIGS. 5A and 5B (SEQ ID NO: 19). Here, "about" means a range that is greater or less than a few, ie 5, 4, 3, 2 or 1 amino acid residues in either or both of the amino- and carboxy-terminus in the specified range. Polynucleotides encoding these polypeptides or are included in the present invention. These polypeptide fragments were prepared by the analysis of the Jameson-Wolf antigen index described in FIG. 6 above and tabulation of the data of FIG. 6 obtained by the Protean component of the DNA * STAR computer program. It was determined to contain antigenic epitopes.

Epitope-containing peptides and polypeptides of the invention can be produced by conventional means (see Houghten, RA (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody). interaction at the level of individual amino acids.Proc.Natl.Acad.Sci.USA 82: 5131-5135). This "simultaneous multiple peptide synthesis (SMPS)" method is also described in US Pat. No. 4,631,211 (1986) to Houghten et al.

Epitope-containing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art, including but not limited to (Sutcliffe et al .; Wilson et al .; Chow , M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985)). Immunogenic epitope-containing peptides of the present invention, ie, portions of proteins that elicit an antibody response when the complete protein is an immunogen, are identified according to methods known in the art (see, eg, Geysen et al., Supra). In addition, Geysen, U.S. Pat.No. 5,194,392 (1990) also describes a sequence of monomers (amino acids or other compounds) that are topological equivalents (ie, "mimotops") of epitopes complementary to specific paratopes (antigen binding sites) of the antibody. The general method of detecting or measuring) is described. More generally, U.S. Patent No. 4,433,092 (1989) to Geysen describes a method for detecting or measuring the sequence of monomers that are topological equivalents of ligands complementary to the ligand binding sites of that particular receptor. Similarly, U.S. Patent No. 5,480,971 to Houghten, RA et al. (1996) on peralkylated oligopeptide mixtures preferentially binds to the corresponding receptor molecules as well as to linear C1-C7-alkyl peralkylated oligopeptides and sets and libraries of such peptides. Methods of using the oligopeptide sets and libraries to determine the sequence of peralkylated oligopeptides are described. Thus, by these methods, non-peptide analogues of the epitope-containing peptides of the present invention may also be conventionally prepared.

The present invention hybridizes with an epitope of a polypeptide having an amino acid sequence of SEQ ID NO: 2 or a polynucleotide sequence contained in ATCC Accession No. 97768 or a complementary sequence of a sequence of SEQ ID NO: 1 or a cDNA sequence contained in ATCC Accession No. 97768 Epitopes of polypeptide sequences encoded by polynucleotides (eg, under the hybridization conditions described herein), or alternatively include polypeptides consisting of such epitopes. In addition, the present invention includes a polynucleotide sequence encoding an epitope of a polypeptide sequence of the invention (e.g., the sequence described in SEQ ID NO: 1), or alternatively consisting of such a sequence, an epitope of the invention. Polynucleotide sequences of the complementary chain of the polynucleotide sequence and polynucleotide sequences that hybridize (eg, under the hybridization conditions described herein) to the complementary chain.

In addition, the present invention is the epitope of the polypeptide having a amino acid sequence of SEQ ID NO: 19 is encoded by the cDNA sequence contained in ATCC Accession No. 203518 or the cDNA contained in the complementary sequence of the sequence of SEQ ID NO: 18 or ATCC Accession No. 203518 Epitopes of polypeptide sequences encoded by polynucleotides that hybridize with a sequence (eg, under the hybridization conditions described herein), or alternatively include polypeptides consisting of such epitopes. In addition, the present invention includes a polynucleotide sequence encoding an epitope of a polypeptide sequence of the invention (e.g., the sequence set forth in SEQ ID NO: 18), or alternatively consists of such a sequence, an epitope of the invention. Polynucleotide sequences of the complementary chain of the polynucleotide sequence and polynucleotide sequences that hybridize (eg, under the hybridization conditions described herein) to the complementary chain.

The term "epitope" as used herein refers to a portion of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, most preferably a human. In a preferred embodiment, the present invention includes a polypeptide containing an epitope as well as a polynucleotide encoding the polypeptide. As used herein, an "immunogenic epitope" is defined as part of a protein that elicits an antibody response in an animal as measured by methods known in the art, such as the methods for producing antibodies described in the literature, below. Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983). As used herein, an “antigenic epitope” is a portion of a protein that is capable of immunospecific binding to its antigen as determined by methods well known in the art, such as the immunoassay described herein. Defined. Immunospecific binding excludes nonspecific binding but does not necessarily exclude cross reactivity with other antigens. Antigenic epitopes do not necessarily need to be immunogenic.

Fragments that act as epitopes can be prepared by conventional means (see, eg, Houghten, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985) and US Pat. No. 4,631,211).

In the present invention, the antigenic epitope is preferably 4 or more, 5 or more, 6 or more, 7 or more, more preferably 8 or more, 9 or more, 10 or more, 11 or more, 12 or more , At least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, most preferably from about 15 to about 30 amino acids. . Preferred polypeptides containing immunogenic or antigenic epitopes are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 in length. Or 100 or more amino acids. Additional conventional preferred antigenic epitopes include the antigenic epitopes described herein as well as portions thereof. Antigenic epitopes are useful for, for example, inducing antibodies (including monoclonal antibodies) that specifically bind to epitopes. Preferred antigenic epitopes include the antigenic epitopes described herein, as well as combinations of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (see, for example, Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 (1983)).

Similarly, immunogenic epitopes can be used to induce antibodies, for example, according to methods well known in the art (see, eg, Sutcliffe et al .; Wilson et al .; Chow et al., Proc. Natl.Acad. Sci. USA 82: 910-914; and Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985)). Preferred antigenic epitopes include the combination of two, three, four, five or more of these immunogenic epitopes as well as the immunogenic epitopes described herein. Polypeptides comprising one or more immunogenic epitopes are provided in the animal kingdom (e.g. rabbit or mouse) together with a carrier protein such as albumin to induce an antibody response or if the polypeptide is of sufficient length (about 25 amino acids or more) The polypeptide can be provided without a carrier. However, immunogenic epitopes comprising 8 to 10 fewer amino acids have been shown to be sufficient to induce antibodies that can bind to minimally straight chain epitopes in denatured polypeptides (eg in Western blotting).

Epitope-containing polypeptides of the invention can be used to induce antibodies according to methods well known in the art, including but not limited to in vivo immunization, in vitro immunization and phage display methods (see, for example, Sutcliffe et al .; Wilson et al .; and Bittle et al., J. Gen. Virol., 66: 2347-2345 (1985). If in vivo immunization is used, the animal can be immunized with free peptides. However, anti-peptide antibody titers can be elevated by linking the peptide to a macromolecular carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxin. For example, peptides containing cysteine residues can be linked to the carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) while other peptides are more common linking agents such as glutaraldehyde. It can be connected to the carrier using. Animals such as rabbits, rats, and mice are for example by intraperitoneal and / or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's excipients or other excipients known to stimulate the immune response. Immunized with free or carrier-bound peptide. Several booster injections may be required, for example at intervals of about 2 weeks, to provide useful titers of anti-peptide antibodies that can be assayed by, for example, ELISA assays using free peptides adsorbed on solid surfaces. The titer of anti-peptide antibodies in serum from immunized animals can be increased by selection of anti-peptide antibodies, for example by adsorption of peptides on solid supports and elution of selected antibodies according to methods well known in the art. have.

As will be appreciated by those skilled in the art and discussed above, polypeptides of the invention comprising immunogenic or antigenic epitopes may be fused to other polypeptide sequences. For example, a polypeptide of the invention can be fused with a constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3 or combinations and portions thereof) to produce chimeric polypeptides. . Such fusion proteins can facilitate purification and increase half-life in vivo. This has been demonstrated for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and several domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (see eg EP394,827; Traunecker et al., Nature, 331: 84-86 (1988). Increased delivery of antigens to the immune system along the epithelial barrier has been demonstrated for antigens bound to FcRn binding partners such as IgG or Fc fragments (see, eg, PCT Publication Nos. WO 96/22024 and WO 99/04813). IgG fusion proteins with disulfide-linked dimer structures due to IgG partial disulfide binding have also been found to be more efficient at binding and neutralizing other molecules than monomeric polypeptides alone or fragments thereof (see, eg, Fountoulakis et al., J.). Biochem., 270: 3958-3964 (1995) The nucleic acid encoding the epitope is also an epitope tag (eg, hemagglutinin (“HA”) tag or flag to aid in the detection and purification of the expressed polypeptide. Tag), for example, the system described by Janknecht et al. Allows for the convenient purification of undenatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9872-897) In this system, the gene is linked to an amino-terminal tag in which the open reading frame of the gene consists of six histidine residues. Such as fusion dokjeok is cloned into a vaccinia recombination plasmid ah tag matrix for a fusion protein - the loaded agarose column - acts as a binding domain to extract acetic acid Ni ²⁺ nitrile from cells infected with a recombinant vaccinia virus And histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and epitope-containing fragments thereof are fused with heterologous antigens (eg, polypeptides, carbohydrates, phospholipids or nucleic acids). In certain embodiments, the heterologous antigen is an immunogen.

In more particular embodiments, the heterologous antigen is a gp120 protein of HIV or a fragment thereof. Polynucleotides encoding these polypeptides are also included in the present invention.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and epitope-containing fragments thereof are fused with polypeptide sequences (or biologically active fragments or variants thereof) of other TNF ligand family members. . In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention are fused with a CD40L polypeptide sequence. In a preferred embodiment, the CD40L polypeptide sequence is soluble.

Of techniques of Neutrokine-alpha and / or Neutrokine-alphaSV using techniques of gene-shuffling, motif-shuffling, exon-shuffling and / or codon-shuffling (collectively referred to as "DNA shuffling"). It can modulate activity and thereby effectively produce agonists and antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV (see, eg, US Pat. Nos. 5,605,793, 5,605,793, 5,811,238, 5,830,721). , 5,834,252 and 5,837,458 and Patten, PA, et al., Curr.Opinion Biotechnol. 8: 724-33 (1997); Harayama, S. Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, LO, et al., J. Mol. Biol. 287: 265-76 (1999); and Lorenzo, MM and Blasco, R. Biotechniques 24 (2): 308-13 (1998). Each of these patents and documents is incorporated herein by reference. In one embodiment, variations of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and corresponding polypeptides can be achieved by DNA shuffling. DNA shuffling involves the insertion of two or more DNA fragments into a desired Neutrokine-alpha and / or Neutrokine-alphaSV molecule by homologous recombination or site-specific recombination. In another embodiment, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and corresponding polypeptides are subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Can be applied and mutated. In another embodiment, one or more components, motifs, regions, parts, domains, fragments, etc., of Neutrokine-alpha and / or Neutrokine-alphaSVs are incorporated into one or more components, motifs, regions, parts, domains, of one or more heterologous molecules, Fragments and the like. In a preferred embodiment, the heterologous molecule is for example TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta) , OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (WO 96/14328), AIM-I (WO 97/34911), APRIL (J.Exp Med. 188 (6): 1185-1190), endocine-alpha (International Publication No. WO 98/07880), OPG, OX40 and Nerve Growth Factor (NGF) and Fas, CD30, CD27, CD40 and 4-IBB Soluble forms, TR2 (WO 96/34095), DR3 (WO 97/33904), DR4 (WO 98/32856), TR4 (WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication Number WO 98/41629), TRANK, TR9 (International Publication Number WO 98/56892), TR10 (International Publication Number WO 98/54202), 312C2 (International Publication Number WO 98). / 06842), TR12, CAD and v-FLIP. In further embodiments, the heterologous molecule includes all members of the TNF family.

In a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (including biologically active fragments or variants thereof) of the invention are fused with soluble CD40L polypeptides or biologically active fragments or variants thereof.

Protein engineering can be used to enhance or modify the characteristics of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides. Recombinant DNA techniques known to those skilled in the art can be used to generate new mutant proteins or muteins including one or more amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or increased stability. In addition, they can be purified in higher yields and exhibit better solubility than the corresponding natural polypeptides, at least under certain purification and storage conditions. For example, it is known in the art that for many proteins, including extracellular domains or mature forms of secreted proteins, one or more amino acids can be deleted from the N- or C-terminus without substantial loss of biological function. have. See, eg, Ron et al., J. Biol. Chem., 268: 2984-2988 (1993) describe modified KGF proteins that exhibit heparin binding activity, although 3, 8 or 27 amino-terminal amino acid residues are deleted.

In the present case, since the protein of the present invention is a member of the TNF polypeptide family, the deletion of the N-terminal amino acid from Figures 1A and 1B (SEQ ID NO: 2) to the Gly (G) residue at position 191, for example lymphocytes (eg , B cells) may maintain some biological activity, such as the ability to stimulate proliferation, differentiation and / or activation and cytotoxicity to appropriate target cells. Polypeptides with additional N-terminal deletions, including Gly (G) residues, maintain biological activity because these residues in TNF-related polypeptides are known to be within the starting point of a conserved domain necessary for biological activity. It is not expected. However, although one or more biological functions of the protein are modified or lost when one or more amino acids are deleted from the N-terminus of the protein, other functional activity can still be maintained. Thus, the ability of shortened proteins to induce and / or bind to antibodies that recognize the complete or extracellular domain of the protein is maintained when sub-majority among the residues of the complete or extracellular domain of the protein is removed from the N-terminus. Will be. Whether a particular polypeptide lacking an N-terminal residue of a complete protein retains such immunological activity can be readily determined by conventional methods described herein and also known in the art.

Accordingly, the present invention also provides a polypeptide in which one or more residues are deleted from the amino terminus of the amino acid sequence of Neutrokine-alpha shown in FIGS. And polynucleotides encoding such polypeptides. In particular, the invention residues n ¹ -285 in SEQ ID NO: 2 comprises the amino acid sequence of (where, n ¹ is from SEQ ID NO: 2 is an integer in the range of from amino acid position of amino acid residues 2-190 of the amino acid sequence), or alternatively Provides polypeptides consisting of such sequences. Polynucleotides encoding these polypeptides are also included in the present invention. More particularly, the present invention relates to residues 2-285 of SEQ ID NO: 2,

And an amino acid sequence selected from the group consisting of 190-285, or alternatively, encoding a polypeptide consisting of such sequence. Polypeptides encoded by these polynucleotides are also included in the present invention. The invention also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, A nucleic acid molecule comprising or alternatively consisting of at least 98% or 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Furthermore, since the putative extracellular domain of Neutrokine-alpha according to the invention can maintain its own biological activity, it is possible to obtain from the putative extracellular region of the polypeptide (positions Gln-73 to Leu-285 of SEQ ID NO: 2). Deletion of N- and C-terminal amino acid residues results in some biological activity such as ligand binding, stimulation of lymphocyte (eg B cell) proliferation, differentiation and / or activation and regulation of cell replication or regulation of target cell activity. I can keep it. However, even if one or more biological functions of the polypeptide are modified or lost when one or more amino acids are deleted from the N-terminus of the putative extracellular domain of the Neutrokine-alpha polypeptide, other functional activities will still be maintained. Can be. Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete or mature or extracellular domain of the polypeptide is less than or equal to N- less than one of the residues of the complete or mature or extracellular domain of the polypeptide. Will be retained when removed from the end. Whether a particular polypeptide lacking an N-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and also known in the art.

Thus, the present invention also provides polypeptides and polynucleotides encoding one or more residues deleted from the amino terminus of the amino acid sequence of Neutrokine-alpha described in SEQ ID NO: 2 to the glycine residue at position 280. In particular, the present invention relates to residues n ² -285 in SEQ ID NO: 2, wherein n ² is an integer falling within the range of amino acid positions of amino acid residues 73-280 in SEQ ID NO: 2 and 73 is a molecule of the Neutrokine-alpha polypeptide set forth in SEQ ID NO: 2 Polypeptides comprising the amino acid sequence of the putative extracellular domain, which is the position of the first residue from the N-terminus), or alternatively consisting of such sequence. Polynucleotides encoding these polypeptides are also included in the present invention. More particularly, in certain embodiments, the present invention relates to residues Q-73 to L-285 of SEQ ID NO: 2; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; A-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; L-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 through L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 through L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; N-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; Provided are polynucleotides comprising amino acid sequences selected from the group consisting of F-279 to L-285 and G-280 to L-285, or alternatively encoding polypeptides consisting of such sequences. Polypeptides encoded by these polynucleotides are also included in the present invention. The invention also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, A nucleic acid molecule comprising or alternatively consisting of at least 98% or 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Very preferred embodiments of the invention are at least 80%, 85%, 90% identical, more than 80%, 85%, 90% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2) Preferably it relates to a nucleic acid molecule comprising or alternatively consisting of polynucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% identical nucleotide sequences. Preferred embodiments of the invention include polynucleotides having a nucleotide sequence that is at least 90% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). Or alternatively, to a nucleic acid molecule consisting of such polynucleotides. More preferred embodiments of the invention include polynucleotides having a nucleotide sequence that is at least 95% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). Or alternatively, to a nucleic acid molecule consisting of such polynucleotides. More preferred embodiments of the invention include polynucleotides having a nucleotide sequence that is at least 96% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). Or alternatively, to a nucleic acid molecule consisting of such polynucleotides. In addition, a more preferred aspect of the invention is a poly having a nucleotide sequence that is at least 97% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising or alternatively consisting of such polynucleotides. In addition, a more preferred aspect of the invention is a poly having a nucleotide sequence that is at least 98% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising or alternatively consisting of such polynucleotides. In addition, a more preferred embodiment of the invention is a poly having a nucleotide sequence that is at least 99% identical to the polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence of positions 134-285 in FIGS. 1A and 1B (SEQ ID NO: 2). It relates to a nucleic acid molecule comprising or alternatively consisting of such polynucleotides.

In certain embodiments, a polypeptide comprising or alternatively consisting of Neutrokine-alpha and / or one of the following N-terminal deleted polypeptide fragments is preferred: amino acid residues Ala-71 to SEQ ID NO: 2 to Leu-285, amino acid residues Ala-81 to Leu-285, amino acid residues Leu-112 to Leu-285, amino acid residues Ala-134 to Leu-285, amino acid residues Leu-147 to Leu-285 and amino acid residues Gly-161 to Leu-285. Polynucleotides encoding these polypeptides are also included in the present invention.

Similarly, many examples of biologically functional C-terminal deletion muteins are known. For example, interferon gamma shows up to 10 times greater activity by deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7: 199-2 (1998)). Since the protein is a member of the TNF polypeptide family, deletions of C-terminal amino acids up to the leucine residue at position 284 may, but are not the case for all biological activities, for example ligand binding, lymphocyte (eg B cell) proliferation, differentiation. And / or maintain most of the activity, such as the ability to stimulate activation and the regulation of cell replication. Polypeptides having a deletion of up to about 10 additional C-terminal residues (ie, up to the glycine residue at position 274) also include a conserved TNF domain of which the polypeptide extends to about Leu-284 in SEQ ID NO: 2. Although it is deleted, it can maintain some activity such as receptor binding. However, even if the deletion of one or more amino acids from the C-terminus of the protein leads to modification or loss in one or more biological functions of the protein, other functional activity can still be maintained. Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete or mature protein will be maintained when less than the major portion of the residues of the complete or mature protein is removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of a complete protein maintains such immunological activity can be readily determined by conventional methods described herein and known in the art.

Thus, the present invention also provides for polypeptides encoding one or more residues deleted from the carboxy terminus of the amino acid sequence of the Neutrokine-alpha polypeptide shown in FIGS. 1A and 1B (SEQ ID NO: 2) to the glycine residue at position 274. Polynucleotides are provided. In particular, the present invention includes the amino acid sequence of residues 1-m ¹ in SEQ ID NO: 2, wherein m ¹ is an integer falling within the range of amino acid positions of amino acid residues 274-284 in SEQ ID NO: 2, or alternatively such sequences It provides a polypeptide consisting of. Polynucleotides encoding these polypeptides are also included in the present invention. More particularly, in certain embodiments, the present invention provides residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, Provided are polynucleotides comprising amino acid sequences selected from the group consisting of 1-283 and 1-284, or alternatively encoding polypeptides consisting of such sequences. Polypeptides encoded by these polynucleotides are also included in the present invention. The invention also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, A nucleic acid molecule comprising or alternatively consisting of at least 98% or 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Also, one or more amino acids deleted from both amino and carboxyl termini, which may be generally described as having residues n ¹ -m ¹ of SEQ ID NO: 2, wherein n ¹ and m ¹ are integers as defined above Polypeptides comprising or consisting of such amino acids are provided. Also, a portion of the complete Neutrokine-alpha amino acid sequence encoded by the deposited cDNA clone contained in ATCC Accession No. 97768, wherein the portion is from the amino terminus of the complete amino acid sequence encoded by the cDNA clone in the deposited plasmid. Poly excluding 1 to 190 amino acids or excluding 1 to 11 amino acids from the C-terminus or pointing to a combined deletion from these N-terminus and C-terminus) or alternatively consisting of Nucleotide sequences encoding peptides are included. Polynucleotides encoding all of the deletion polypeptides are also included in the present invention.

Similarly, the deletion of the C-terminal amino acid residue of the putative extracellular domain of Neutrokine-alpha from SEQ ID NO: 2 to the leucine residue at position 79 may include, for example, ligand binding, lymphocyte (eg B cell) proliferation, differentiation and And / or maintain some biological activity such as stimulation of activation and regulation of cell replication or regulation of target cell activation. Polypeptides with additional C-terminal deletions comprising Leu-79 of SEQ ID NO: 2 are not expected to maintain biological activity.

However, even if the deletion of one or more amino acids from the C-terminus of the protein leads to modification or loss in one or more biological functions of the protein, other functional activity can still be maintained. Thus, the ability of a shortened protein to induce and / or bind to an antibody that recognizes a complete, mature or extracellular form of polypeptide removes less than a major portion of the residues of a complete, mature or extracellular protein from the C-terminus. Will be maintained when Whether a particular polypeptide lacking the C-terminal residue of a complete protein maintains such immunological activity can be readily determined by conventional methods described herein and known in the art.

Accordingly, the invention also relates to polypeptides and polypeptides wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the putative extracellular domain of the Neutrokine-alpha polypeptide described in SEQ ID NO: 2 to the glycine residue in position 79 in SEQ ID NO: 2. Provided are polynucleotides that encode a peptide. In particular, the present invention SEQ ID NO: 2, residues 73-m ² in (where, m is an integer from ² SEQ ID NO: 2 within the range of the amino acid position of amino acid residues 79-285 and residues 78 neutroavidin Kin-alpha polypeptide (described in SEQ ID NO: 2 A polypeptide consisting of, or alternatively consisting of, the amino acid sequence of the C-terminal end of the putative extracellular domain). Polynucleotides encoding these polypeptides are also included in the present invention. More particularly, in certain embodiments, the present invention relates to residues Q-73 to Leu-285 of SEQ ID NO: 2; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to S-247; Q-73 to Y-246; Q-73 to K-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to K-232; Q-73 to R-231; Q-73 to F-230; Q- 73 to L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-®; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-73 to I-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q- 73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Provided are polynucleotides comprising, or alternatively encoding, an amino acid sequence selected from the group consisting of Q-73 to R-80 and Q-73 to L-79. Polypeptides encoded by these polynucleotides are also included in the present invention. The invention also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, A nucleic acid molecule comprising or alternatively consisting of at least 98% or 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

The invention also relates to the amino of the putative extracellular domain of Neutrokine-alpha, which may be generally described as having residues n ² -m ² of SEQ ID NO: 2, wherein n ² and m ² are integers defined above. And polypeptides having one or more amino acids deleted from both carboxyl termini.

In another embodiment, a portion of an extracellular domain of a Neutrokine-alpha amino acid sequence encoded by a cDNA plasmid contained in a deposit of ATCC Accession No. 97768, wherein the portion is a cDNA contained in a deposit of ATCC Accession No. 97768 C- of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid, excluding 1 to about 206 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by the plasmid or contained in the deposit of ATCC Accession No. 97768 Refers to the combined deletion from the N-terminus and C-terminus of the complete extracellular domain of the amino acid sequence, which is excluded by 1 to about 206 amino acids from the terminal or encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768 Nucleotide encoding a polypeptide consisting of Sequences are included.

As mentioned above, although a deletion of one or more amino acids from the N-terminus of a polypeptide induces modification or loss in one or more functional activities (eg, biological activities) of that polypeptide, other functional or biological activities still remain. Can be maintained. Thus, the ability of a shortened Neutrokine-alpha to induce and / or bind an antibody that recognizes the full-length or mature form or extracellular domain of the polypeptide is dependent on the residues of the full-length or mature form or extracellular domain of the polypeptide. It will remain when the middle major part is removed from the N-terminus. Whether a particular polypeptide lacking an N-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art. Neutrokine-alpha muteins with large numbers of deleted N-terminal amino acid residues are unlikely to be able to maintain some functional (eg biological or immunogenic) activity. In fact, peptides consisting of up to six Neutrokine-alpha amino acid residues can often elicit an immune response.

Thus, the present invention also encodes polypeptides having one or more residues deleted from the amino terminus of the putative full-length amino acid sequence of Neutrokine-alpha described in SEQ ID NO: 2, to the glycine residue at position 280 described in SEQ ID NO: 2, and such polypeptides. To provide a polynucleotide. In particular, the amino acid sequence of the invention residues n ³ -285 of the sequence described in SEQ ID NO: 2 (where, n ³ is an integer in the range of the amino acid residues 1 to 280 in the amino acid sequence of SEQ ID NO: 2, amino acid positions) To provide a polypeptide.

More particularly, the present invention provides residues D-2 to L-285 of SEQ ID NO: 2; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K-43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; C-60 to L-285; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 through L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 through L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; Provided are polynucleotides comprising, or alternatively encoding, amino acid sequences selected from the group consisting of F-279 to L-285 and G-280 to L-285. The present application is directed to a polynucleotide sequence encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

As mentioned above, although a deletion of one or more amino acids from the C-terminus of a protein induces modification or loss in one or more functional activities (eg, biological activities) of that protein, other functional or biological activities are still maintained. Can be. Thus, the ability of a shortened Neutrokine-alpha mutein to induce and / or bind an antibody that recognizes a complete or mature form or extracellular domain of the polypeptide may result in a full or mature form or extracellular domain of the polypeptide. Less than the major part of the residues of will be retained when removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art. Neutrokine-alpha muteins with large numbers of deleted C-terminal amino acid residues are unlikely to be able to maintain some functional (eg biological or immunogenic) activity. In fact, peptides consisting of up to six Neutrokine-alpha amino acid residues can often elicit an immune response.

Thus, in another embodiment, the present invention also provides a polypeptide and one or more polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of Neutrokine-alpha described in SEQ ID NO: 2 to the glutamic acid residue at position 6 described in SEQ ID NO: 2. It provides a polynucleotide encoding a. In particular, the present invention comprises an amino acid sequence of residues 1-m ³ of the sequence set forth in SEQ ID NO: 2, wherein m ³ is an integer falling within the range of amino acid positions of amino acid residues 6 to 284 in the amino acid sequence of SEQ ID NO: 2 Provide a polypeptide.

More particularly, the present invention provides residues M-1 to L-284 of SEQ ID NO: 2; M-1 to K-283; M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to K-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1 to K-215; M-1 to R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183; M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-®; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49 M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; Provided are polynucleotides comprising, or alternatively encoding, amino acid sequences selected from the group consisting of M-1 to R-7 and M-1 to E-6. The present application is directed to a polynucleotide sequence encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, the present invention provides amino and carboxyl termini of Neutrokine-alpha polypeptides which may be generally described as having residues n ³ -m ³ of SEQ ID NO: 2, wherein n ³ and m ³ are integers as defined above. Provided are polypeptides from which one or more amino acids are deleted from all.

In addition, the putative extracellular domain of the Neutrokine-alphaSV polypeptide according to the invention may itself exhibit functional activity (e.g., biological activity), although the polynucleotide of positions Gln-73 to Leu-266 in SEQ ID NO: 19 may Deletion of the N- and C-terminal amino acid residues of the putative extracellular domain of the peptide may be, for example, ligand binding, lymphocyte (eg B cell) proliferation, stimulation of differentiation and / or activation and regulation of cell replication or target cells. Some biological activity, such as regulation of activation, can be maintained. However, although the deletion of one or more amino acids from the N-terminus of the putative extracellular domain of the Neutrokine-alphaSV polypeptide induces modification or loss in one or more biological functions of the protein, other functional activity will still be maintained. Can be. Thus, the ability of shortened proteins to induce and / or bind antibodies that recognize a complete or mature or extracellular domain of a polypeptide is such that less than or equal to the major portion of the residues of the complete or mature or extracellular domain of the polypeptide is N-. Will be retained when removed from the end. Whether a particular polypeptide lacking an N-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art.

Accordingly, the present invention also provides polypeptides and polynucleotides encoding one or more residues deleted from the amino terminus of the amino acid sequence of Neutrokine-alphaSV described in SEQ ID NO: 19 to the glycine residue at position 261. In particular, the present invention relates to residues n ⁴ -266 in SEQ ID NO: 19, wherein n ⁴ is an integer falling within the range of amino acid positions of amino acid residues 73-261 in the amino acid sequence of SEQ ID NO: 19 and 261 is the putative extracellular described in SEQ ID NO: 19. Polypeptides comprising the amino acid sequence of the domain Neutrokine-alphaSV polypeptide, which is the position of the first residue from the N-terminus), or alternatively consisting of such sequence.

More particularly, in certain embodiments, the present invention provides residues Q-73 to L-266 of SEQ ID NO: 19; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; Provided are polynucleotides comprising or alternatively encoding amino acid sequences selected from the group consisting of F-260 to L-266 and G-261 to L-266. The present application is directed to a polynucleotide sequence encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Similarly, deletion of the C-terminal amino acid residue of the putative extracellular domain of Neutrokine-alphaSV from SEQ ID NO. And / or maintain some functional activity such as the ability to stimulate activation, regulation of cell replication, regulation of target cell activity and / or immunogenicity. Polypeptides with additional C-terminal deletions comprising Leu-79 of SEQ ID NO: 19 are not expected to maintain biological activity.

However, although the deletion of one or more amino acids from the C-terminus of the polypeptide induces modification or loss in one or more functional activities (eg, biological activity) of the polypeptide, other functional activity can still be maintained. Thus, the ability of shortened proteins to induce and / or bind antibodies that recognize a complete or mature or extracellular domain of a polypeptide is such that C- or less than a major portion of the residues of a complete or mature or extracellular domain of a polypeptide Will be retained when removed from the end. Whether a particular polypeptide lacking the C-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art.

Accordingly, the present invention also relates to polypeptides and polypeptides wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the putative extracellular domain of Neutrokine-alphaSV described in SEQ ID NO: 19 to the leucine residue in SEQ ID NO: 19. Provided are polynucleotides that encode peptides. In particular, the present invention provides a polypeptide having an amino acid sequence of residues 73-m ⁴ of the amino acid sequence of SEQ ID NO: 19, wherein m ⁴ is an integer falling within the range of amino acid positions of amino acid residues 79-266 in the amino acid sequence of SEQ ID NO: 19; To provide.

More particularly, in certain embodiments, the present invention provides residues Q-73 to L-265 of SEQ ID NO: 19; Q- 73 to K-264; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; Q-73 to Q-189; Q-73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q- 73 to F-®; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q- 73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Provided are polynucleotides comprising or alternatively encoding amino acid sequences selected from the group consisting of Q-73 to L-79 and Q-73 to S-78. The present application also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98 A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, the present invention provides for the presumed extracellular domain of Neutrokine-alphaSV, which may be generally described as having residues n ⁴ -m ⁴ of SEQ ID NO: 19, wherein n ⁴ and m ⁴ Provided are polypeptides wherein one or more amino acids are deleted from both amino and carboxyl termini.

In another embodiment, a portion of the extracellular domain of the Neutrokine-alphaSV amino acid sequence encoded by a cDNA clone contained in a deposit of ATCC Accession No. 203518, wherein the portion is in a deposit of ATCC Accession No. 203518. Extracellular domain of amino acid sequence encoded by cDNA clone that excludes 1 to about 260 amino acids from the amino terminus of the extracellular domain of amino acid sequence encoded by the contained cDNA clone or is contained in a deposit of ATCC Accession No. 203518 The combined deletion from said amino terminus and the carboxy terminus of the complete extracellular domain of the amino acid sequence, excluding 1 to about 187 amino acids from the carboxy terminus of or encoded by a cDNA clone contained in a deposit of ATCC Accession No. 203518. Nucleotide encoding a polypeptide consisting of It includes sequences de.

As mentioned above, although the deletion of one or more amino acids from the N-terminus of the polypeptide leads to modification or loss in one or more functional activities (eg, biological activities) of the polypeptide, other functional activities are still maintained. Can be. Thus, the ability of shortened Neutrokine-alphaSVs to induce and / or bind to antibodies that recognize the full-length or mature form or extracellular domain of a polypeptide is dependent on the full-length or mature form or extracellular domain of the polypeptide. Less than the major part of the residues will be retained when removed from the N-terminus. Whether a particular polypeptide lacking an N-terminal residue of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art. Neutrokine-alphaSV muteins with multiple deleted N-terminal amino acid residues are unlikely to be able to maintain some functional (eg immunogenic) activity. In fact, peptides consisting of as few as six Neutrokine-alphaSV amino acid residues can often trigger an immune response.

Accordingly, the present invention also relates to polypeptides having one or more residues deleted from the amino terminus of the putative full-length amino acid sequence of Neutrokine-alphaSV described in SEQ ID NO: 19 up to the glycine residue at position 261 described in SEQ ID NO: 19 and such polypeptides. Provided are polynucleotides that encode. In particular, the present invention comprises the amino acid sequence of residues n ⁵ -266 of the sequence set forth in SEQ ID NO: 19, wherein n ⁵ is an integer falling within the range of amino acid positions 1 to 261 of the amino acid sequence in the amino acid sequence of SEQ ID NO: 19; Provide a polypeptide.

More particularly, the present invention provides residues D-2 to L-266 of SEQ ID NO: 19; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; Provided are polynucleotides comprising or alternatively encoding amino acid sequences selected from the group consisting of F-260 to L-266 and G-261 to L-266. The present application also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98 A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

As mentioned above, although the deletion of one or more amino acids from the C-terminus of a protein leads to modification or loss in one or more functional activities (eg biological activity) of that protein, other functional activity can still be maintained. have. Thus, the ability of the shortened Neutrokine-alphaSV muteins to induce and / or bind to antibodies that recognize a complete or mature form or extracellular domain of the polypeptide may be a complete or mature form or extracellular form of the polypeptide. Less than the major part of the residues of the domain will be retained when removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residues of a complete polypeptide maintains such immunological activity can be readily determined by conventional methods described herein and known in the art. Neutrokine-alpha muteins with multiple deleted C-terminal amino acid residues are unlikely to be able to maintain some functional (eg immunogenic) activity. In fact, peptides consisting of as few as six Neutrokine-alphaSV amino acid residues can often trigger an immune response.

Accordingly, the present invention also relates to polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of Neutrokine-alphaSV as described in SEQ ID NO: 19 to the glutamic acid residue at position 6 as described in SEQ ID NO: 19 and to the polypeptides encoding such polypeptides. Provide nucleotides. In particular, the present invention comprises an amino acid sequence of residues 1-m ⁵ of the sequence set forth in SEQ ID NO: 19, wherein m ⁵ is an integer falling within the range of amino acid positions 6 to 265 of amino acid residues in the amino acid sequence of SEQ ID NO: 19; Provide a polypeptide.

More particularly, the present invention provides residues M-1 to L-265 of SEQ ID NO: 19; M-1 to K-264; M-1 to L-263; M-1 to A-262; M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234; M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-215; M-1 to I-214; M-1 to C-213; M-1 to R-212; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-1 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-®; M-1 to Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to I-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; Provided are polynucleotides comprising, or alternatively encoding, amino acid sequences selected from the group consisting of M-1 to R-7 and M-1 to E-6. The present application is directed to a polynucleotide sequence encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or A nucleic acid molecule comprising or alternatively consisting of at least 99% identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, the present invention relates to both the amino and carboxyl termini of the Neutrokine-alpha polypeptide which may be generally described as having residues n ⁵ -m ⁵ of SEQ ID NO: 19, wherein n ⁵ and m ⁵ are integers defined above. Providing a polypeptide from which one or more amino acids are deleted.

In a further aspect, the invention provides a polypeptide comprising an amino acid sequence of residues 134-m ⁶ in SEQ ID NO: 2, wherein m ⁶ is an integer from 140 to 285 corresponding to the position of the amino acid residues in SEQ ID NO: 2. . For example, the present invention provides residues A-134 to Leu-285 of SEQ ID NO: 2; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-®; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; Provided are polynucleotides comprising or alternatively encoding amino acid sequences selected from the group consisting of A-134 to V-142 and A-134 to T-141. The present application also provides polynucleotide sequences encoding the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides described above and 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98 A nucleic acid molecule comprising or alternatively consisting of at least 99% identical or identical polynucleotide sequences. In addition, the present invention includes such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also included in the present invention. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. Polypeptides consisting of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Further preferred polypeptide fragments of the invention include residues M-1 to C-15 of SEQ ID NO: 2; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; K-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; A-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; L-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R-®; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-171 to I-185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to I-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G-249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274; L-261 to D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to L-282; Q-269 to K-283; Amino acid residues selected from the group consisting of I-270 to L-284 and S-271 to L-285, or alternatively consist of such residues. Preferably, these polypeptide fragments have at least one functional activity (eg, biological activity, antigenicity and immunogenicity) of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention, for example It can be used to generate or select antibodies as described. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. It relates to a polypeptide consisting of a sequence. In addition, the present invention includes such amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also included in the present invention.

Further preferred polypeptide fragments of the invention include residues M-1 to C-15 of SEQ ID NO: 19; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; A-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; L-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to G-142; S-129 to S-143; R-130 to Y-144; N-131 to D-145; K-132 to F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-®; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; I-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-215; G-202 to N-216; D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-248; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to K-264; Amino acid residues selected from the group consisting of I-251 to L-265 and S-252 to L-266, or alternatively consist of such residues. Preferably, these polypeptide fragments have at least one functional activity (eg, biological activity, antigenicity and immunogenicity) of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention, for example It can be used to generate or select antibodies as described. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. It relates to a polypeptide consisting of a sequence. In addition, the present invention includes such amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also included in the present invention.

Further preferred polypeptide fragments of the invention include residues M-1 to F-15 of SEQ ID NO: 38; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q-74; S-61 to A-75; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109; A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to N-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-®; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-168 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to V-190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-240 to I-254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to E-258; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-256 to E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to L-285 A-272; L-286; Q-273 to K-287; Amino acid residues selected from the group consisting of I-274 to L-288 and S-275 to L-289, or alternatively consist of such residues. Preferably, these polypeptide fragments have at least one functional activity (eg, biological activity, antigenicity and immunogenicity) of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention, for example It can be used to generate or select antibodies as described. In addition, the present invention includes or alternatively comprises an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. It relates to a polypeptide consisting of a sequence. In addition, the present invention includes such amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also included in the present invention.

Those skilled in the art will appreciate that some amino acid sequences of the Neutrokine-alpha and Neutrokine-alpha SV polypeptides may be modified without significantly affecting the structure or function of the polypeptide. If such differences in sequence are considered, it should be borne in mind that there are important regions in the polypeptide that measure activity.

Accordingly, the present invention is directed to variations of Neutrokine-alpha polypeptides that exhibit Neutrokine-alpha polypeptide functional activity (eg, biological activity) or include regions of Neutrokine-alpha polypeptides, such as the polypeptide fragments described herein. It includes. The invention also relates to Neutrokine-alphaSV polynucleotides that exhibit a Neutrokine-alphaSV polypeptide functional activity (eg, biological activity) or that include a region of Neutrokine-alphaSV polypeptides, such as the polypeptide fragments described herein. Variations of the peptides. Such variants include deletions, insertions, inversions, repetitions and type substitutions selected according to general rules known in the art to have little effect on activity. For example, instructions on how to make expressionally silent amino acid substitutions are described in Bowie. J. U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247: 1306-1310 (1990). Here, the authors suggest that there are two main ways to study resistance to variations in amino acid sequences. The first depends on the evolutionary process in which mutations are allowed or rejected by natural selection. The second method uses screening or screening to identify sequences that maintain genetic engineering and functionality that introduce amino acid mutations at specific positions of the cloned genes.

As the authors stated, these studies have shown that proteins are surprisingly resistant to amino acid substitutions. The authors also indicate which amino acid variations can be accepted at specific positions of the protein. For example, most buried amino acid residues require nonpolar side chains, while surface side chain characteristics are generally rarely preserved. Other phenotypic silent substitutions are described in Biwie, J. U. et al., Supra, and references cited therein. Typical conservative substitutions include the substitution of one of the aliphatic amino acids Ala, Val, Leu and Ile to another, the exchange of the acidic residues Asp and Glu, the substitution of the amide residues Asn and Gln, and the exchange of the basic residues Lys and Arg. And substitution between the aromatic residues Phe, Thr.

Thus, fragments, derivatives or analogues of those encoded by the polypeptides or deposited cDNA plasmids shown in FIGS. 1A and 1B (SEQ ID NO: 2) are those in which (i) at least one amino acid residue is nonconserved Amino acid residues), which may or may not be encoded by the genetic code, (ii) at least one of the amino acid residues comprises a substituent group, (iii) extracellular of the polypeptide The domain is fused with another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol) or (iv) the additional amino acid is an extracellular domain of the polypeptide, e.g., an IgG Fc fusion region peptide or leader, or To the sequence or proprotein sequence used to purify the secretory sequence or extracellular domain of the polypeptide. It may be connected. Such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein.

Moreover, fragments, derivatives or analogues of those encoded by the polypeptides or deposited cDNA plasmids shown in FIGS. 5A and 5B (SEQ ID NO: 19) may comprise (i) amino acid residues (preferably conserved Amino acid residues), which may or may not be encoded by the genetic code, (ii) at least one of the amino acid residues comprises a substituent group, (iii) extracellular of the polypeptide The domain is fused with another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol) or (iv) the additional amino acid is a member of an extracellular domain of the polypeptide, e.g., another TNF ligand family member (e.g., Soluble biologically active fragments of the CD40 ligand), IgG Fc fusion region peptides or leader or secretory sequences or polypeptides More Id cell may be connected to the pro sequence or protein sequence that is used to purify the domain. Such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein.

Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention may comprise one or more amino acid substitutions, deletions or additions by natural mutations or by manipulation of humans. As mentioned, small natural variations are preferred, such as auxiliary amino acid substitutions where the variation does not significantly affect the folding or activity of the protein (see Table 2).

Conserved amino acid substitutions Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine polarity Glutamine Asparagine Basic Arginine Lysine Histidine acid Aspartic Acid Glutamic Acid small type Alanine Serine Threonine Methionine Glycine

In one embodiment of the invention, the polypeptide is an amino acid containing at least one, at most 50, preferably at most 40, more preferably at most 30, even more preferably at most 20 conserved amino acid substitutions. It comprises or alternatively consists of the amino acid sequence of Neutrokine-alpha or Neutrokine-alphaSV polypeptide having a sequence. Of course, the amino acid sequence of the Neutrokine-alpha polypeptide containing one or more 10, 9, 8, 7, 6, 5, 4, 3, 2 or less conservative amino acid substitutions in increasing order of preference. Very preferred are peptides or polypeptides having an amino acid sequence.

For example, site directed variations at the amino acid level of Neutrokine-alpha can be made by substituting specific amino acids for conservative substitutions. Preferred conservative substitution mutations in the Neutrokine-alpha amino acid sequence provided in SEQ ID NO: 2 include the substitution of M1 with A, G, I, L, S, T or V; D2 is substituted by E; D3 is substituted by E; S 4 is substituted with A, G, I, L, T, M or V; T5 is substituted with A, I, L, S, M or V; E6 is substituted by D; R 7 is substituted with H or K; E8 is substituted by D; Q9 is substituted by N; S10 is substituted with A, G, I, L, T, M or V; R 11 is substituted by H or K; L12 is substituted with A, G, I, S, T, M or V; T13 is substituted by A; Substitution with G, I, L, S, M or V; S14 is substituted with A, G, I, L, T, M or V; L 16 is substituted with A, G, I, S, T, M or V; K17 is substituted with H or R; K18 is substituted with H or R; R19 is substituted by H or K; E20 is substituted by D; E21 is substituted by D; M 22 is substituted with A, G, I, L, S, T or V; K23 is substituted by H or R; L24 is substituted with A, G, I, S, T, M or V; K25 is substituted with H or R; E26 is substituted by D; V28 is substituted with A, G, I, L, S, T or M; S29 is substituted with A, G, I, L, T, M or V; I30 is substituted with A, G, L, S, T, M or V; L31 is substituted with A, G, I, S, T, M or V; R33 is substituted by H or K; K34 is substituted by H or R; E35 is substituted by D; S36 is substituted by A, G, I, L, T, M or V; S38 is substituted with A, G, I, L, T, M or V; V39 is substituted by A, G, I, L, S, T or M; R40 is substituted with H or K; S41 is substituted with A, G, I, L, T, M or V; S42 is substituted with A, G, I, L, T, M or V; K43 is substituted by H or R; D44 is substituted by E; G45 is substituted with A, I, L, S, T, M or V; K46 is substituted by H or R; L47 is substituted with A, G, I, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; A49 is substituted with G, I, L, S, T, M or V; A 50 is substituted with G, I, L, S, T, M or V; T51 is substituted with A, G, I, L, S, M or V; L52 is substituted with A, G, I, S, T, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; A55 is substituted with G, I, L, S, T, M or V; L56 is substituted by A, G, I, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; S58 is substituted with A, G, I, S, T, M or V; L61 is substituted with A, G, I, S, T, M or V; T62 is substituted with A, G, I, S, T, M or V; V63 is substituted with A, G, I, L, S, T or V; V64 is substituted with A, G, I, S, T, M or V; S65 is substituted with A, G, I, L, T, M or V; F66 is substituted by W or Y; Y67 is substituted by F or W; Q68 is substituted by N; V69 is substituted with A, G, I, L, S, T or M; A70 is substituted with G, I, L, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; L72 is substituted with A, G, I, S, T, M or V; Q73 is substituted by N; G74 is substituted with A, I, L, S, T, M or V; D75 is substituted by E; L76 is substituted with A, G, I, S, T, M or V; A77 is substituted with G, I, L, S, T, M or V; S78 is substituted with A, G, I, L, T, M or V; L79 is substituted with A, G, I, S, T, M or V; R 80 is substituted with H or K; A81 is substituted with G, I, L, S, T, M or V; E82 is substituted by D; L83 is substituted with A, G, I, S, T, M or V; Q84 is substituted by N; G85 is substituted with A, I, L, S, T, M or V; H86 is substituted by K or R; H87 is substituted by K or R; A88 is substituted with G, I, L, S, T, M or V; E89 is substituted by D; K90 is substituted by H or R; L91 is substituted with A, G, I, S, T, M or V; A93 is substituted with G, I, L, S, T, M or V; G94 is substituted with A, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; G96 is substituted with A, I, L, S, T, M or V; A97 is substituted with G, I, L, S, T, M or V; K99 is substituted by H or R; A100 is substituted with G, I, L, S, T, M or V; G101 is substituted with A, I, L, S, T, M or V; L102 is substituted with A, G, I, S, T, M or V; E103 is substituted by D; E104 is substituted by D; A105 is substituted with G, I, L, S, T, M or V; A107 is substituted with G, I, L, S, T, M or V; V108 is substituted with A, G, I, S, T, M or V; T109 is substituted with A, G, I, S, T, M or V; A110 is substituted with G, I, L, S, T or M; G111 is substituted with A, I, L, S, T, M or V; L112 is substituted with A, G, I, S, T, M or V; K113 is substituted with H or R; I114 is substituted by A, G, L, S, T, M or V; F115 is substituted by W or Y; E116 is substituted by D; A119 is substituted with G, I, L, S, T, M or V; G121 is substituted with A, I, L, S, T, M or V; E122 is substituted by D; G123 is substituted with A, I, L, S, T, M or V; N124 is substituted by Q; S125 is substituted with A, G, I, L, T, M or V; S126 is substituted with A, G, I, L, T, M or V; Q127 is substituted by N; N128 is substituted by Q; S129 is substituted with A, G, I, L, T, M or V; R 130 is substituted with H or K; N131 is substituted by Q; K132 is substituted by H or R; R133 is substituted by H or K; A134 is substituted with G, I, L, S, T, M or V; V135 is substituted with A, G, I, L, T or M; Q136 is substituted by N; G137 is substituted with A, I, L, S, T, M or V; E139 is substituted by D; E140 is substituted by D; T141 is substituted with A, G, I, L, S, M or V; V142 is substituted with A, G, I, L, S, T or M; T143 is substituted with A, G, I, L, S, M or V; Q144 is substituted by N; D145 is substituted with E; L147 is substituted with A, G, I, S, T, M or V; Q148 is substituted by N; L149 is substituted with A, G, I, S, T, M or V; I150 is substituted with A, G, L, S, T, M or V; A151 is substituted with G, I, L, S, T, M or V; D152 is substituted by E; S153 is substituted with A, G, I, L, T, M or V; E154 is substituted by D; T155 is substituted with A, G, I, L, S, M or V; T157 is substituted with A, G, I, L, S, M or V; I158 is substituted by A, G, L, S, T, M or V; Q159 is substituted by N; K160 is substituted with H or R; G161 is substituted with A, I, L, S, T, M or V; S162 is substituted with A, G, I, L, T, M or V; Y163 is substituted with F or W; T164 is substituted with A, G, I, L, S, M or V; F165 is substituted by W or Y; V166 is substituted with A, G, I, L, S, T or M; L169 is substituted with A, G, I, S, T, M or V; L170 is substituted with A, G, I, S, T, M or V; S171 is substituted with A, G, I, L, T, M or V; F172 is substituted with W or Y; K173 is substituted with H or R; R174 is substituted with H or K; G175 is substituted with A, I, L, S, T, M or V; S176 is substituted with A, G, I, L, T, M or V; A177 is substituted with G, I, L, S, T, M or V; L178 is substituted with A, G, I, S, T, M or V; E179 is substituted by D; E180 is substituted by D; K181 is substituted with H or R; E182 is substituted by D; N183 is substituted by Q; K184 is substituted by H or R; I185 is substituted with A, G, L, S, T, M or V; L186 is substituted with A, G, I, S, T, M or V; V187 is substituted with A, G, I, L, S, T or M; K188 is substituted by H or R; E189 is substituted by D; T190 is substituted with A, G, I, L, S, M or V; G191 is substituted with A, I, L, S, T, M or V; Y192 is substituted with F or W; F193 is substituted with W or Y; F194 is substituted with W or Y; I195 is substituted with A, G, L, S, T, M or V; Y196 is substituted with F or W; G197 is substituted with A, I, L, S, T, M or V; Q198 is substituted by N; V199 is substituted with A, G, I, L, S, T or M; L200 is substituted with A, G, I, S, T, M or V; Y201 is substituted with F or W; T202 is substituted with A, G, I, L, S, M or V; D203 substituted by E; K204 is substituted by H or R; T205 is substituted with A, G, I, L, S, M or V; Y206 is substituted with F or W; A207 is substituted with G, I, L, S, T, M or V; M208 is substituted with A, G, I, L, S, T or V; G209 is substituted with A, I, L, S, T, M or V; H210 is substituted by K or R; L211 is substituted with A, G, I, S, T, M or V; I212 is substituted by A, G, L, S, T, M or V; Q213 is substituted with N; R214 is substituted with H or K; K215 is substituted by H or R; K216 is substituted by H or R; V217 is substituted with A, G, I, L, S, T or M; H218 is substituted with K or R; V219 is substituted with A, G, I, L, S, T or M; F220 is substituted by W or Y; G221 is substituted with A, I, L, S, T, M or V; D222 is substituted with E; E223 is substituted by D; L224 is substituted with A, G, I, S, T, M or V; S225 is substituted with A, G, I, L, T, M or V; L226 is substituted with A, G, I, S, T, M or V; V227 is substituted with A, G, I, L, S, T or M; T228 is substituted with A, G, I, L, S, M or V; L229 is substituted with A, G, I, S, T, M or V; F230 is substituted by W or Y; R231 is substituted by H or K; I233 substituted with A, G, L, S, T, M or V; Q234 is substituted by N; N235 is substituted by Q; M236 is substituted with A, G, I, L, S, T or V; E238 is substituted with D; T239 is substituted with A, G, I, L, S, M or V; E238 is substituted with D; T239 is substituted with A, G, I, L, S, M or V; L240 is substituted with A, G, I, S, T, M or V; N242 is substituted by Q; N243 is substituted by Q; S244 is substituted with A, G, I, L, T, M or V; Y246 is substituted by F or W; S247 is substituted with A, G, I, L, T, M or V; A248 is substituted with G, I, L, S, T, M or V; G249 is substituted with A, I, L, S, T, M or V; I250 is substituted with A, G, L, S, T, M or V; A251 is substituted with G, I, L, S, T, M or V; K252 is substituted by H or R; L253 is substituted with A, G, I, S, T, M or V; E254 is substituted by D; E255 is substituted by D; G256 is substituted with A, I, L, S, T, M or V; D257 substituted by E; E258 is substituted with D; L259 is substituted with A, G, I, S, T, M or V; Q260 substituted by N; L261 is substituted with A, G, I, S, T, M or V; A262 is substituted with G, I, L, S, T, M or V; I263 substituted with A, G, L, S, T, M or V; R265 is substituted with H or K; E266 is substituted by D; N267 is substituted by Q; A268 is substituted with G, I, L, S, T, M or V; Q269 is substituted by N; I270 substituted by A, G, L, S, T, M or V; S271 is substituted with A, G, I, L, T, M or V; L272 is substituted by A, G, I, S, T, M or V; D273 is substituted by E; G274 is substituted with A, I, L, S, T, M or V; D275 is substituted by E; V276 is substituted by A, G, I, L, S, T or M; T277 is substituted with A, G, I, L, S, M or V; F278 is substituted by W or Y; F279 is substituted by W or Y; G280 is substituted with A, I, L, S, T, M or V; A281 is substituted with G, I, L, S, T, M or V; L282 is substituted with A, G, I, S, T, M or V; K283 is substituted with H or R; L284 is substituted with A, G, I, S, T, M or V and / or L285 is substituted with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention may be conventionally screened for Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (eg, increased or decreased stability and / or solubility). . Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In another embodiment, site directed variations at the amino acid level of Neutrokine-alphaSV can be made by substituting specific amino acids for conservative substitutions. Preferred conservative substitution mutations in the Neutrokine-alphaSV amino acid sequence provided in SEQ ID NO: 19 include the substitution of M1 with A, G, I, L, S, T or V; D2 is substituted by E; D3 is substituted by E; S 4 is substituted with A, G, I, L, T, M or V; T5 is substituted with A, I, L, S, M or V; E6 is substituted by D; R 7 is substituted with H or K; E8 is substituted by D; Q9 is substituted by N; S10 is substituted with A, G, I, L, T, M or V; R 11 is substituted by H or K; L12 is substituted with A, G, I, S, T, M or V; T13 is substituted with A, G, I, L, S, M or V; S14 is substituted with A, G, I, L, T, M or V; L 16 is substituted with A, G, I, S, T, M or V; K17 is substituted with H or R; K18 is substituted with H or R; R19 is substituted by H or K; E20 is substituted by D; E21 is substituted by D; M 22 is substituted with A, G, I, L, S, T or V; K23 is substituted by H or R; L24 is substituted with A, G, I, S, T, M or V; K25 is substituted with H or R; E26 is substituted by D; V28 is substituted with A, G, I, L, S, T or M; S29 is substituted with A, G, I, L, T, M or V; I30 is substituted with A, G, L, S, T, M or V; L31 is substituted with A, G, I, S, T, M or V; R33 is substituted by H or K; K34 is substituted by H or R; E35 is substituted by D; S36 is substituted by A, G, I, L, T, M or V; S38 is substituted with A, G, I, L, T, M or V; V39 is substituted by A, G, I, L, S, T or M; R40 is substituted with H or K; S41 is substituted with A, G, I, L, T, M or V; S42 is substituted with A, G, I, L, T, M or V; K43 is substituted by H or R; D44 is substituted by E; G45 is substituted with A, I, L, S, T, M or V; K46 is substituted by H or R; L47 is substituted with A, G, I, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; A49 is substituted with G, I, L, S, T, M or V; A 50 is substituted with G, I, L, S, T, M or V; T51 is substituted with A, G, I, L, S, M or V; L52 is substituted with A, G, I, S, T, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; A55 is substituted with G, I, L, S, T, M or V; L56 is substituted by A, G, I, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; S58 is substituted with A, G, I, S, T, M or V; L61 is substituted with A, G, I, S, T, M or V; T62 is substituted with A, G, I, S, T, M or V; V63 is substituted with A, G, I, L, S, T or V; V64 is substituted with A, G, I, S, T, M or V; S65 is substituted with A, G, I, L, T, M or V; F66 is substituted by W or Y; Y67 is substituted by F or W; Q68 is substituted by N; V69 is substituted with A, G, I, L, S, T or M; A70 is substituted with G, I, L, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; L72 is substituted with A, G, I, S, T, M or V; Q73 is substituted by N; G74 is substituted with A, I, L, S, T, M or V; D75 is substituted by E; L76 is substituted with A, G, I, S, T, M or V; A77 is substituted with G, I, L, S, T, M or V; S78 is substituted with A, G, I, L, T, M or V; L79 is substituted with A, G, I, S, T, M or V; R 80 is substituted with H or K; A81 is substituted with G, I, L, S, T, M or V; E82 is substituted by D; L83 is substituted with A, G, I, S, T, M or V; Q84 is substituted by N; G85 is substituted with A, I, L, S, T, M or V; H86 is substituted by K or R; H87 is substituted by K or R; A88 is substituted with G, I, L, S, T, M or V; E89 is substituted by D; K90 is substituted by H or R; L91 is substituted with A, G, I, S, T, M or V; A93 is substituted with G, I, L, S, T, M or V; G94 is substituted with A, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; G96 is substituted with A, I, L, S, T, M or V; A97 is substituted with G, I, L, S, T, M or V; K99 is substituted by H or R; A100 is substituted with G, I, L, S, T, M or V; G101 is substituted with A, I, L, S, T, M or V; L102 is substituted with A, G, I, S, T, M or V; E103 is substituted by D; E104 is substituted by D; A105 is substituted with G, I, L, S, T, M or V; A107 is substituted with G, I, L, S, T, M or V; V108 is substituted with A, G, I, L, S, T or M; T109 is substituted with A, G, I, S, T, M or V; A110 is substituted with G, I, L, S, T, M or V; G111 is substituted with A, I, L, S, T, M or V; L112 is substituted with A, G, I, S, T, M or V; K113 is substituted with H or R; I114 is substituted by A, G, L, S, T, M or V; F115 is substituted by W or Y; E116 is substituted by D; A119 is substituted with G, I, L, S, T, M or V; G121 is substituted with A, I, L, S, T, M or V; E122 is substituted by D; G123 is substituted with A, I, L, S, T, M or V; N124 is substituted by Q; S125 is substituted with A, G, I, L, T, M or V; S126 is substituted with A, G, I, L, T, M or V; Q127 is substituted by N; N128 is substituted by Q; S129 is substituted with A, G, I, L, T, M or V; R 130 is substituted with H or K; N131 is substituted by Q; K132 is substituted by H or R; R133 is substituted by H or K; A134 is substituted with G, I, L, S, T, M or V; V135 is substituted with A, G, I, L, T, M or V; Q136 is substituted by N; G137 is substituted with A, I, L, S, T, M or V; E139 is substituted by D; E140 is substituted by D; T141 is substituted with A, G, I, L, S, M or V; G142 is substituted with A, I, L, S, T, M or V; S143 is substituted with A, G, I, L, T, M or V; Y144 is substituted by F or W; T145 is substituted with A, G, I, L, S, M or V; F146 is substituted by W or Y; V147 is substituted with A, G, I, L, S, T or M; W149 is substituted by F or Y; L150 is substituted with A, G, I, S, T, M or V; L151 is substituted with A, G, I, S, T, M or V; S152 is substituted with A, G, I, L, T, M or V; F153 is substituted by W or Y; K154 is substituted by H or R; R155 is substituted by H or K; G156 is substituted with A, I, L, S, T, M or V; S157 is substituted with A, G, I, L, T, M or V; A158 is substituted with G, I, L, S, T, M or V; L159 is substituted with A, G, I, S, T, M or V; E160 is substituted by D; E161 is substituted by D; K162 is substituted with H or R; E163 is substituted by D; N164 is substituted by Q; K165 is substituted with H or R; I166 is substituted with A, G, L, S, T, M or V; L167 is substituted with A, G, I, S, T, M or V; V168 is substituted with A, G, I, L, S, T or M; K169 is substituted with H or R; E170 is substituted by D; T171 is substituted with A, G, I, L, S, M or V; G172 is substituted with A, I, L, S, T, M or V; Y173 is substituted with F or W; F174 is substituted with W or Y; F175 substituted by W or Y; I176 is substituted with A, G, L, S, T, M or V; Y177 is substituted by F or W; G178 is substituted with A, I, L, S, T, M or V; Q179 is substituted by N; V180 is substituted with A, G, I, L, S, T or M; L181 is substituted with A, G, I, S, T, M or V; Y182 is substituted with F or W; T183 is substituted with A, G, I, L, S, M or V; D184 is substituted with E; K185 is substituted with H or R; T186 is substituted with A, G, I, L, S, M or V; Y187 is substituted with F or W; A188 is substituted with G, I, L, S, T, M or V; M 189 is substituted with A, G, I, L, S, T or V; G190 is substituted with A, I, L, S, T, M or V; H191 is substituted with K or R; L192 is substituted with A, G, I, S, T, M or V; I193 is substituted with A, G, L, S, T, M or V; Q194 is substituted by N; R 195 is substituted with H or K; K196 is substituted by H or R; K197 is substituted with H or R; V198 is substituted with A, G, I, L, S, T or M; H199 is substituted by K or R; V200 is substituted with A, G, I, L, S, T or M; F201 is substituted with W or Y; G202 is substituted with A, I, L, S, T, M or V; D203 substituted by E; E204 is substituted by D; L205 is substituted with A, G, I, S, T, M or V; S206 is substituted with A, G, I, L, T, M or V; L207 is substituted with A, G, I, S, T, M or V; V208 is substituted with A, G, I, L, S, T or M; T209 is substituted with A, G, I, L, S, M or V; L210 is substituted with A, G, I, S, T, M or V; F211 is substituted by W or Y; R212 is substituted with H or K; I214 is substituted with A, G, L, S, T, M or V; Q215 is substituted by N; N216 substituted by Q; M217 is substituted with A, G, I, L, S, T or V; E219 is substituted by D; T220 is substituted with A, G, I, L, S, M or V; L221 is substituted with A, G, I, S, T, M or V; N223 is substituted by Q; N224 is substituted by Q; S225 is substituted with A, G, I, L, T, M or V; Y227 is substituted with F or W; S228 is substituted with A, G, I, L, T, M or V; A229 is substituted with G, I, L, S, T, M or V; G230 is substituted with A, I, L, S, T, M or V; I231 substituted by A, G, L, S, T, M or V; A232 is substituted with G, I, L, S, T, M or V; K233 is substituted with H or R; L234 is substituted with A, G, I, S, T, M or V; E235 is substituted by D; E236 is substituted by D; G237 is substituted with A, I, L, S, T, M or V; D238 is substituted with E; E239 is substituted by D; L240 is substituted with A, G, I, S, T, M or V; Q241 is substituted by N; L242 is substituted with A, G, I, S, T, M or V; A243 is substituted by G, I, L, S, T, M or V; I244 is substituted with A, G, L, S, T, M or V; R246 is substituted by H or K; E247 is substituted by D; N248 is substituted with Q; A249 is substituted with G, I, L, S, T, M or V; Q250 is substituted by N; I251 substituted with A, G, L, S, T, M or V; S252 is substituted with A, G, I, L, T, M or V; L253 is substituted with A, G, I, S, T, M or V; D254 is substituted with E; G255 is substituted with A, I, L, S, T, M or V; D256 is substituted by E; V257 substituted with A, G, I, L, S, T or M; T258 is substituted with A, G, I, L, S, M or V; F259 is substituted by W or Y; F260 is substituted with W or Y; G261 is substituted with A, I, L, S, T, M or V; A262 is substituted with G, I, L, S, T, M or V; L263 is substituted with A, G, I, S, T, M or V; K264 is substituted by H or R; L265 is substituted with A, G, I, S, T, M or V and / or L266 is substituted with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention may be conventionally screened for Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (eg, increased or decreased stability and / or solubility). . Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In another embodiment, site directed variations at the amino acid level of Neutrokine-alpha can be made by substituting specific amino acids with conservative substitutions. Preferred conservative substitution mutations in the Neutrokine-alpha amino acid sequence provided in SEQ ID NO: 23 include R1 substituted with H or K; V2 is substituted with A, G, I, L, S, T or M; V3 is substituted with A, G, I, L, S, T or M; D4 is substituted by E; L 5 is substituted with A, G, I, S, T, M or V; S6 is substituted with A, G, I, L, T, M or V; A7 is substituted with G, I, L, S, T, M or V; A 10 is substituted with G, I, L, S, T, M or V; L 13 is substituted with A, G, I, S, T, M or V; G15 is substituted with A, I, L, S, T, M or V; R17 is substituted by H or K; H18 is substituted by K or R; S19 is substituted by A, G, I, L, T, M or V; Q20 is substituted by N; H21 is substituted by K or R; D22 is substituted by E; D23 is substituted by E; N24 is substituted by Q; G25 is substituted with A, I, L, S, T, M or V; M 26 is substituted with A, G, I, L, S, T or V; N27 is substituted by Q; L28 is substituted with A, G, I, S, T, M or V; R29 is substituted by H or K; N30 is substituted by Q; R31 is substituted by H or K; T32 is substituted with A, G, I, L, S, M or V; Y33 is substituted by F or W; T34 is substituted with A, G, I, L, S, M or V; F35 is substituted by W or Y; V36 is substituted by A, G, I, L, S, T or M; W38 is substituted by F or Y; L39 is substituted with A, G, I, S, T, M or V; L40 is substituted with A, G, I, S, T, M or V; S41 is substituted with A, G, I, L, T, M or V; F42 is substituted by W or Y; K43 is substituted by H or R; R44 is substituted by H or K; G45 is substituted with A, I, L, S, T, M or V; N46 is substituted by Q; A47 is substituted with G, I, L, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; E49 is substituted by D; E50 is substituted by D; K51 is substituted with H or R; E52 is substituted by D; N53 is substituted by Q; K54 is substituted by H or R; I55 is substituted with A, G, L, S, T, M or V; V56 is substituted by A, G, I, L, S, T or M; V57 is substituted with A, G, I, L, S, T or M; R58 is substituted by H or K; Q59 is substituted by N; T 60 is substituted with A, G, I, L, S, M or V; G61 is substituted with A, G, I, L, S, M or V; Y62 is substituted by F or W; F63 is substituted by W or Y; F64 is substituted by W or Y; I65 is substituted by A, G, L, S, T, M or V; Y66 is substituted by F or W; S67 is substituted with A, G, I, L, T, M or V; Q68 is substituted by N; V69 is substituted with A, G, I, L, S, T or M; L70 is substituted with A, G, I, S, T, M or V; Y71 is substituted with F or W; T72 is substituted with A, G, I, L, S, M or V; D73 is substituted by E; I75 is substituted with A, G, L, S, T, M or V; F76 is substituted by W or Y; A77 is substituted with G, I, L, S, T, M or V; M78 is substituted with A, G, I, L, S, T or V; G79 is substituted with A, I, L, S, T, M or V; H80 is substituted with K or R; V81 is substituted with A, G, I, L, S, T or M; I82 is substituted with A, G, L, S, T, M or V; Q83 is substituted by N; R 84 is substituted with H or K; K85 is substituted by H or R; K86 is replaced by H or R; V87 is substituted with A, G, I, L, S, T or M; H88 is substituted by K or R; V89 is substituted with A, G, I, L, S, T or M; F90 is substituted by W or Y; G91 is substituted with A, I, L, S, T, M or V; D92 is substituted by E; E93 is substituted by D; L94 is substituted with A, G, I, S, T, M or V; S95 is substituted with A, G, I, L, T, M or V; L96 is substituted with A, G, I, S, T, M or V; V97 is substituted with A, G, I, L, S, T or M; T98 is substituted with A, G, I, L, S, M or V; L99 is substituted with A, G, I, S, T, M or V; F100 is substituted by W or Y; R101 is substituted by H or K; I103 is substituted with A, G, L, S, T, M or V; Q104 is substituted by N; N105 is substituted by Q; M 106 is substituted with A, G, I, L, S, T or V; K108 is substituted by H or R; T109 is substituted with A, G, I, L, S, M or V; L110 is substituted with A, G, I, S, T, M or V; N112 is substituted by Q; N113 is substituted by Q; S114 is substituted by A, G, I, L, T, M or V; Y116 is substituted by F or W; S117 is substituted by A, G, I, L, T, M or V; A118 is substituted with G, I, L, S, T, M or V; G119 is substituted with A, I, L, S, T, M or V; I120 is substituted with A, G, L, S, T, M or V; A121 is substituted with G, I, L, S, T, M or V; R122 is substituted by H or K; L123 is substituted with A, G, I, S, T, M or V; E124 is substituted by D; E125 is substituted by D; G126 is substituted with A, I, L, S, T, M or V; D127 is substituted by E; E128 is substituted by D; I129 is substituted with A, G, L, S, T, M or V; Q130 is substituted by N; L131 is substituted with A, G, I, S, T, M or V; A132 is substituted with G, I, L, S, T, M or V; I133 is substituted with A, G, L, S, T, M or V; R135 is substituted by H or K; E136 is substituted by D; N137 is substituted by Q; A138 is substituted with G, I, L, S, T, M or V; Q139 is substituted by N; I140 is substituted with A, G, L, S, T, M or V; S141 is substituted with A, G, I, L, T, M or V; R142 is substituted by H or K; N143 is substituted by Q; G144 is substituted with A, I, L, S, T, M or V; D145 is substituted with E; D146 is substituted with E; T147 is substituted with A, G, I, L, S, M or V; F148 is substituted by W or Y; F149 is substituted by W or Y; G150 is substituted with A, I, L, S, T, M or V; A151 is substituted with G, I, L, S, T, M or V; L152 is substituted with A, G, I, S, T, M or V; K153 is substituted by H or R; L154 is substituted with A, G, I, S, T, M or V and / or L155 is substituted with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention may be conventionally screened for Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (eg, increased or decreased stability and / or solubility). . Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In another embodiment, site directed variations at the amino acid level of Neutrokine-alpha can be made by substituting specific amino acids with conservative substitutions. Preferred conservative substitution mutations in the Neutrokine-alpha amino acid sequence provided in SEQ ID NO: 38 include the substitution of M1 with A, G, I, L, S, T or V; D2 is substituted by E; E3 is substituted by D; S 4 is substituted with A, G, I, L, T, M or V; A5 is substituted with G, I, L, S, T, M or V; K 6 is substituted with H or R; T7 is substituted with A, G, I, L, S, M or V; L 8 is substituted with A, G, I, S, T, M or V; L 13 is substituted with A, G, I, S, T, M or V; F15 is substituted by W or Y; S17 is substituted with A, G, I, L, T, M or V; E18 is substituted by D; K19 is substituted by H or R; G20 is substituted with A, I, L, S, T, M or V; E21 is substituted by D; D22 is substituted by E; M 23 is substituted with A, G, I, L, S, T or V; K24 is substituted with H or R; V25 is substituted with A, G, I, L, S, T or M; G26 is substituted with A, I, L, S, T, M or V; Y27 is substituted by F or W; D28 is substituted by E; I30 is substituted with A, G, L, S, T, M or V; T31 is substituted with A, G, I, L, S, M or V; Q33 is substituted by N; K34 is substituted by H or R; E35 is substituted by D; E36 is substituted by D; G37 is substituted with A, I, L, S, T, M or V; A38 is substituted with G, I, L, S, T, M or V; W39 is substituted by F or Y; F40 is substituted by W or Y; G41 is substituted with A, I, L, S, T, M or V; I42 is substituted with A, G, L, S, T, M or V; R44 is substituted by H or K; D45 is substituted by E; G46 is substituted with A, I, L, S, T, M or V; R47 is substituted by H or K; L48 is substituted with A, G, I, S, T, M or V; L49 is substituted with A, G, I, S, T, M or V; A 50 is substituted with G, I, L, S, T, M or V; A51 is substituted with G, I, L, S, T, M or V; T52 is substituted with A, G, I, L, S, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; L55 is substituted with A, G, I, S, T, M or V; A56 is substituted with G, I, L, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; L58 is substituted with A, G, I, S, T, M or V; S59 is substituted with A, G, I, L, T, M or V; S60 is substituted with A, G, I, L, T, M or V; S61 is substituted with A, G, I, L, T, M or V; F62 is substituted by W or Y; T63 is substituted with A, G, I, L, S, M or V; A64 is substituted with G, I, L, S, T, M or V; M65 is substituted by A, G, I, L, S, T or V; S66 is substituted with A, G, I, L, T, M or V; L67 is substituted with A, G, I, S, T, M or V; Y68 is substituted by F or W; Q69 is substituted by N; L70 is substituted with A, G, I, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; A72 is substituted with G, I, L, S, T, M or V; L73 is substituted with A, G, I, S, T, M or V; Q74 is substituted by N; A75 is substituted with G, I, L, S, T, M or V; D76 is substituted by E; L77 is substituted with A, G, I, S, T, M or V; M78 is substituted with A, G, I, L, S, T or V; N79 is substituted by Q; L80 is substituted with A, G, I, S, T, M or V; R81 is substituted by H or K; M82 is substituted with A, G, I, L, S, T or V; E83 is substituted by D; L84 is substituted with A, G, I, S, T, M or V; Q85 is substituted by N; S86 is substituted with A, G, I, L, T, M or V; Y87 is substituted by F or W; R88 is substituted by H or K; G89 is substituted with A, I, L, S, T, M or V; S90 is substituted with A, G, I, L, T, M or V; A91 is substituted with G, I, L, S, T, M or V; T92 is substituted with A, G, I, L, S, M or V; A94 is substituted with G, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; A96 is substituted with G, I, L, S, T, M or V; G97 is substituted with A, I, L, S, T, M or V; A98 is substituted with G, I, L, S, T, M or V; E100 is substituted by D; L101 is substituted with A, G, I, S, T, M or V; T102 is substituted with A, G, I, L, S, M or V; A103 is substituted with G, I, L, S, T, M or V; G104 is substituted with A, I, L, S, T, M or V; V105 is substituted with A, G, I, L, S, T or M; K106 is substituted by H or R; L107 is substituted with A, G, I, S, T, M or V; L108 is substituted with A, G, I, S, T, M or V; T109 is substituted with A, G, I, L, S, M or V; A 111 is substituted with G, I, L, S, T, M or V; A112 is substituted with G, I, L, S, T, M or V; R114 is substituted by H or K; H116 is substituted with K or R; N117 substituted by Q; S118 is substituted by A, G, I, L, T, M or V; S119 is substituted with A, G, I, L, T, M or V; R 120 is substituted with H or K; G121 is substituted with A, I, L, S, T, M or V; H122 is substituted by K or R; R123 is substituted with H or K; N124 is substituted by Q; R125 is substituted by H or K; R126 is substituted by H or K; A127 is substituted with G, I, L, S, T, M or V; F128 is substituted by W or Y; Q129 is substituted by N; G130 is substituted with A, I, L, S, T, M or V; E132 is substituted by D; E133 is substituted by D; T134 is substituted with A, G, I, L, S, M or V; E135 is substituted by D; Q136 is substituted by N; D137 is substituted by E; V138 is substituted with A, G, I, L, S, T or M; D139 is substituted by E; L140 is substituted with A, G, I, S, T, M or V; S141 is substituted with A, G, I, L, T, M or V; A142 is substituted with G, I, L, S, T, M or V; A145 is substituted with G, I, L, S, T, M or V; L148 is substituted with A, G, I, S, T, M or V; G150 is substituted with A, I, L, S, T, M or V; R152 is substituted by H or K; H153 is substituted by K or R; S154 is substituted with A, G, I, L, T, M or V; Q155 is substituted by N; H156 is substituted with K or R; D157 is substituted by E; D158 is substituted with E; N159 is substituted by Q; G160 is substituted with A, I, L, S, T, M or V; M161 is substituted with A, G, I, L, S, T or V; N162 is substituted by Q; L163 is substituted with A, G, I, S, T, M or V; R164 is substituted with H or K; N165 is substituted by Q; I166 is substituted with A, G, L, S, T, M or V; I167 substituted with A, G, L, S, T, M or V; Q168 is substituted with N; D169 is substituted with E; L171 is substituted with A, G, I, S, T, M or V; Q172 is substituted by N; L 173 is substituted with A, G, I, S, T, M or V; I174 is substituted with A, G, L, S, T, M or V; A175 is substituted with G, I, L, S, T, M or V; D176 is substituted by E; S177 is substituted with A, G, I, L, T, M or V; D178 is substituted with E; T179 is substituted with A, G, I, L, S, M or V; A181 is substituted with G, I, L, S, T, M or V; L182 is substituted with A, G, I, S, T, M or V; E183 is substituted with D; E184 is substituted by D; K185 is substituted with H or R; E186 is substituted by D; N187 substituted by Q; K188 is substituted by H or R; I188 is substituted by A, G, L, S, T, M or V; V190 is substituted with A, G, I, L, S, T or M; V191 is substituted with A, G, I, L, S, T or M; R192 is substituted with H or K; Q193 is substituted with N; T194 is substituted with A, G, I, L, S, M or V; G195 is substituted with A, I, L, S, T, M or V; Y196 is substituted with F or W; F197 is substituted with W or Y; F198 is substituted with W or Y; I199 is substituted with A, G, L, S, T, M or V; Y200 is substituted with F or W; S201 is substituted with A, G, I, L, T, M or V; Q202 is substituted by N; V203 substituted with A, G, I, L, S, T or M; L204 is substituted with A, G, I, S, T, M or V; Y205 is substituted by F or W; T206 is substituted with A, G, I, L, S, M or V; D207 substituted by E; I209 is substituted with A, G, L, S, T, M or V; F210 is substituted by W or Y; A211 is substituted with G, I, L, S, T, M or V; M212 is substituted with A, G, I, L, S, T or V; G213 is substituted with A, I, L, S, T, M or V; H214 is substituted by K or R; V215 is substituted with A, G, I, L, S, T or M; I216 substituted with A, G, L, S, T, M or V; Q217 is substituted by N; R218 is substituted by H or K; K219 is substituted with H or R; K220 is substituted by H or R; V221 is substituted with A, G, I, L, S, T or M; H222 is substituted with K or R; V223 is substituted with A, G, I, L, S, T or M; F224 is substituted with W or Y; G225 is substituted with A, I, L, S, T, M or V; D226 is substituted by E; E227 is substituted with D; L228 is substituted with A, G, I, S, T, M or V; S229 is substituted with A, G, I, L, T, M or V; L230 is substituted with A, G, I, S, T, M or V; V231 is substituted with A, G, I, L, S, T or M; T232 is substituted with A, G, I, L, S, M or V; L233 is substituted with A, G, I, S, T, M or V; F234 is substituted with W or Y; R235 is substituted with H or K; I237 is substituted with A, G, L, S, T, M or V; Q238 is substituted with N; N239 is substituted by Q; M240 is substituted with A, G, I, L, S, T or V; K242 is substituted by H or R; T243 is substituted by A, G, I, L, S, M or V; L244 is substituted with A, G, I, S, T, M or V; N246 is substituted by Q; N247 is substituted with Q; S248 is substituted with A, G, I, L, T, M or V; Y250 is substituted with F or W; S251 is substituted with A, G, I, L, T, M or V; A252 is substituted with G, I, L, S, T, M or V; G253 is substituted with A, I, L, S, T, M or V; I254 is substituted with A, G, L, S, T, M or V; A255 is substituted with G, I, L, S, T, M or V; R256 is substituted by H or K; L257 is substituted with A, G, I, S, T, M or V; E258 is substituted with D; E259 is substituted by D; G260 is substituted with A, I, L, S, T, M or V; D261 is substituted by E; E262 substituted by D; I263 substituted with A, G, L, S, T, M or V; Q264 is substituted by N; L265 is substituted with A, G, I, S, T, M or V; A266 is substituted with G, I, L, S, T, M or V; I267 is substituted with A, G, L, S, T, M or V; R 269 is substituted with H or K; E270 substituted by D; N271 is substituted by Q; A272 is substituted with G, I, L, S, T, M or V; Q273 is substituted by N; I274 is substituted with A, G, L, S, T, M or V; S275 is substituted with A, G, I, L, T, M or V; R276 is substituted with H or K; N277 is substituted by Q; G278 is substituted with A, I, L, S, T, M or V; D279 is substituted by E; D280 is substituted with E; T281 is substituted with A, G, I, L, S, M or V; F282 is substituted with W or Y; F283 is substituted by W or Y; G284 is substituted with A, I, L, S, T, M or V; A285 is substituted with G, I, L, S, T, M or V; L286 is substituted with A, G, I, S, T, M or V; K287 is substituted with H or R; L288 is substituted with A, G, I, S, T, M or V and / or L289 is substituted with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention may be conventionally screened for Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (eg, increased or decreased stability and / or solubility). . Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

The amino acids of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention essential for function can be identified by methods known in the art such as site directed mutagenesis or alanine-scanning mutagenesis [ See Cunningham and Wells, Science 244: 1081-1085 (1989). The latter procedure introduces one alanine mutation at every residue of the molecule. The resulting mutant molecules are then tested for functional activity such as ligand binding and the ability to stimulate lymphocyte (eg B cell) proliferation, differentiation and / or activation.

Particularly advantageous is the substitution of charged amino acids with other charged or neutral amino acids, which can provide a protein which is highly desirable in characterization such as reduced aggregation. Aggregation can cause problems as well as reduce activity in the manufacture of a medicament because the aggregate can be immunogenic [Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabets 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993).

In another aspect, the invention provides a polypeptide having an amino acid sequence that contains a non-conservative substitution of the amino acid sequence provided in SEQ ID NO: 2. For example, non-conservative substitutions of the Neutrokine-alpha protein sequence provided in SEQ ID NO: 2 include M1 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D2 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D3 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S 4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E6 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R7 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E8 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R 11 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L 12 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T 13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S14 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; C15 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 16 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K17 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K18 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R 19 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E20 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E21 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M 22 is substituted with D, E, H, K, R, F, W, Y, P or C; K23 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L24 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K25 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E26 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C27 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; V28 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S29 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; I30 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L31 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R33 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K34 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S36 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V39 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R40 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S42 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K43 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D44 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K46 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L47 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A49 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A 50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L52 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L56 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S58 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C59 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C 60 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T62 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V64 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S65 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F66 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y67 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L72 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q73 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G74 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D75 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L76 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S78 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L79 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R80 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A81 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E82 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L83 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q84 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G85 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; H86 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H87 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A88 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E89 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K90 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P92 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A93 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P98 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K99 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A100 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G101 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L102 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; E103 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E104 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P106 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G 111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L112 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; K113 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I114 is D, E, H, K, R, N, Q, F, W, Y, P or C; F115 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E116 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P117 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P118 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P120 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E122 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S125 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S126 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q127 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N128 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S129 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R 130 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K132 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R133 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V135 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G137 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P138 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E139 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E140 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V142 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; T143 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q144 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D145 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C146 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L147 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q148 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L149 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; I150 is D, E, H, K, R, N, Q, F, W, Y, P or C; A151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D152 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S153 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; E154 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T155 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P156 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; T157 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I158 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q159 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K160 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G161 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S162 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y163 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T164 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F165 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V166 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P167 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W168 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L169 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L170 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F172 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K173 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R174 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G175 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S176 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A177 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L178 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E179 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E180 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K181 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E182 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N183 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K184 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I185 is D, E, H, K, R, N, Q, F, W, Y, P or C; L186 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; V187 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K188 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E189 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T190 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G191 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y192 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F193 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F194 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I195 is D, E, H, K, R, N, Q, F, W, Y, P or C; Y196 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G197 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q198 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V199 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L200 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y201 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T202 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D203 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K204 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T205 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y206 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A207 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M208 is substituted with D, E, H, K, R, F, W, Y, P or C; G209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H210 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L211 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L212 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q213 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R214 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K215 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K216 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V217 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H218 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V219 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F220 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D222 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E223 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L224 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L226 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V227 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L229 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F230 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R231 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C232 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I233 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q234 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N235 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M236 is substituted with D, E, H, K, R, F, W, Y, P or C; P237 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E238 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T239 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L240 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P241 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N242 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N243 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S244 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C245 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y246 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S247 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A248 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G249 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I250 is D, E, H, K, R, N, Q, F, W, Y, P or C; A251 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K252 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E254 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E255 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G256 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D257 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E258 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L259 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q260 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L261 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A262 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I263 is D, E, H, K, R, N, Q, F, W, Y, P or C; P264 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R265 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E266 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N267 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A268 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q269 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I270 is D, E, H, K, R, N, Q, F, W, Y, P or C; S271 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L272 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D273 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G274 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D275 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V276 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; T277 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F278 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F279 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G280 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A281 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L282 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K283 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L284 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L285 is D, E, H, K, R, N, Q, F, W, Substitutions with Y, P or C are included. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention are described herein and known in the art Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility). Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In a further embodiment, the Neutrokine-alpha polypeptides of the invention comprise one or more amino acids (eg, 2, 3, 4, 5, 6, 7) substituted with substituted amino acids (preserved or non-conserved) as described above. , 8, 9, 10, 15, 20, 30 and 50) or alternatively consist of these amino acids.

In another embodiment of the invention, non-conservative substitutions of the Neutrokine-alphaSV protein sequence provided in SEQ ID NO: 19 include M1 being D, E, H, K, R, N, Q, F, W, Y, P or C Substituted by; D2 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D3 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S 4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E6 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R7 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E8 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R 11 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L 12 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T 13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S14 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; C15 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 16 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K17 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K18 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R 19 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E20 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E21 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M 22 is substituted with D, E, H, K, R, F, W, Y, P or C; K23 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L24 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K25 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E26 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C27 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; V28 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S29 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; I30 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L31 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R33 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K34 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S36 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V39 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R40 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S42 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K43 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D44 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K46 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L47 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A49 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A 50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L52 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L56 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S58 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C59 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C 60 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T62 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V64 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S65 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F66 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y67 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L72 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q73 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G74 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D75 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L76 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S78 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L79 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R80 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A81 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E82 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L83 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q84 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G85 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; H86 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H87 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A88 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E89 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K90 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P92 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A93 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P98 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K99 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A100 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G101 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L102 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; E103 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E104 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P106 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G 111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L112 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; K113 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I114 is D, E, H, K, R, N, Q, F, W, Y, P or C; F115 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E116 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P117 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P118 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P120 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E122 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S125 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S126 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q127 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N128 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S129 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R 130 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K132 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R133 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V135 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G137 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P138 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E139 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E140 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G142 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S143 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Y144 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T145 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F146 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V147 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P148 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W149 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S152 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F153 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K154 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R155 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G156 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S157 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A158 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L159 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; E160 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E161 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K162 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E163 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N164 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K165 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I166 is D, E, H, K, R, N, Q, F, W, Y, P or C; L167 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V168 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K169 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E170 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G172 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y173 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F174 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F175 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I176 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y177 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G178 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q179 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V180 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L181 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y182 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T183 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D184 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K185 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T186 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y187 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A188 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M 189 is substituted with D, E, H, K, R, F, W, Y, P or C; G190 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H191 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L192 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I193 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q194 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R 195 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K196 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K197 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V198 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H199 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V200 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F201 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G202 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D203 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E204 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L205 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S206 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L207 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; V208 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L210 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F211 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R212 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C213 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I214 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q215 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N216 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M217 is substituted with D, E, H, K, R, F, W, Y, P or C; P218 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E219 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T220 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P222 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N223 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N224 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C226 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y227 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A229 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G230 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I231 is D, E, H, K, R, N, Q, F, W, Y, P or C; A232 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K233 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L234 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E235 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E236 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G237 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D238 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E239 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L240 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q241 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L242 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A243 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I244 is D, E, H, K, R, N, Q, F, W, Y, P or C; P245 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R246 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E247 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N248 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A249 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q250 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I251 is D, E, H, K, R, N, Q, F, W, Y, P or C; S252 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D254 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G255 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D256 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V257 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T258 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F259 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F260 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G261 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A262 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L263 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K264 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L265 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L266 is D, E, H, K, R, N, Q, F, W, Substitutions with Y, P or C are included. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention are described herein and known in the art Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (eg, increased or decreased stability and / or Solubility). Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In a further embodiment, the Neutrokine-alpha polypeptides of the invention comprise one or more amino acids (eg, 2, 3, 4, 5, 6, 7) substituted with substituted amino acids (preserved or non-conserved) as described above. , 8, 9, 10, 15, 20, 30 and 50) or alternatively consist of these amino acids.

For example, preferred non-conservative substitutions of the Neutrokine-alpha protein sequence provided in SEQ ID NO: 23 include those where R1 is D, E, A, G, I, L, S, T, M, V, N, Q, F, W , Y, P or C; V2 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V3 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D4 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L 5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S6 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A7 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P8 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A 10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P11 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C12 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P14 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G15 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; C16 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R17 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H18 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S19 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q20 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H21 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D22 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D23 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N24 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G25 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M 26 is substituted with D, E, H, K, R, F, W, Y, P or C; N27 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L28 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R29 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N30 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R31 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T32 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Y33 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T34 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F35 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V36 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W38 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L39 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L40 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F42 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K43 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R44 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N46 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A47 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; E49 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E50 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K51 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E52 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N53 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K54 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I55 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; V56 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; V57 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R58 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q59 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T 60 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y62 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F63 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F64 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I65 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Y66 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S67 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y71 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T72 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D73 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P74 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I75 is D, E, H, K, R, N, Q, F, W, Y, P or C; F76 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M78 is substituted with D, E, H, K, R, F, W, Y, P or C; G79 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H80 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V81 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; I82 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q83 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R84 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K85 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K86 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V87 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; H88 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V89 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F90 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D92 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E93 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L 94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T98 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L99 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; F100 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R101 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C102 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I103 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q104 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N105 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M 106 is substituted with D, E, H, K, R, F, W, Y, P or C; P107 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K108 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P111 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N112 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N113 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S114 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; C115 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y116 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S117 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A118 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I120 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R122 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E124 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E125 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G126 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D127 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E128 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I129 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q130 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L131 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A132 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I133 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P134 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R135 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E136 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N137 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A138 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q139 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I140 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S141 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; R142 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N143 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G144 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D145 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D146 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T147 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F148 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F149 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L152 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; K153 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L154 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L155 is D, E, H, K, R, N, Q, F, W, Substitutions with Y, P or C are included. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention are described herein and known in the art Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility). Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In a further embodiment, the Neutrokine-alpha polypeptides of the invention comprise one or more amino acids (eg, 2, 3, 4, 5, 6, 7) substituted with substituted amino acids (preserved or non-conserved) as described above. , 8, 9, 10, 15, 20, 30 and 50) or alternatively consist of these amino acids.

For example, preferred non-conservative substitutions of the Neutrokine-alpha protein sequence provided in SEQ ID NO: 38 include M1 being substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D2 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E3 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S 4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K6 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T7 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L 8 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P10 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P11 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C12 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L 13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C14 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; F15 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; C16 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; S17 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E18 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K19 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G20 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E21 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D22 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M 23 is substituted with D, E, H, K, R, F, W, Y, P or C; K24 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V25 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; G26 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y27 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D28 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P29 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I30 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; T31 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; Q33 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K34 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E36 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G37 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; W39 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F40 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I42 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; C43 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R44 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D45 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G46 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R 47 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L48 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L49 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A 50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T52 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L55 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A56 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L58 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S59 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S60 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F62 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A64 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M65 is substituted by D, E, H, K, R, F, W, Y, P or C; S66 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L67 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Y68 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q69 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A72 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L73 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q74 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A75 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D76 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L77 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; M78 is substituted with D, E, H, K, R, F, W, Y, P or C; N79 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L80 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R 81 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M82 is substituted with D, E, H, K, R, F, W, Y, P or C; E83 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L84 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q85 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S86 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y87 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R88 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G89 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S90 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T92 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P93 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A98 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P99 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E100 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L101 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; T102 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A103 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G104 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K106 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P110 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A 111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A112 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P113 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R114 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P115 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; H116 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N117 substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S118 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R 120 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H122 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R123 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R 125 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R126 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A127 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F128 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q129 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G130 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E132 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E133 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E135 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D137 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V138 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D139 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L140 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S141 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A142 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P143 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P144 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A145 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P146 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C147 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L148 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P149 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C151 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R152 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H153 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S154 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q155 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H156 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D157 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D158 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N159 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G160 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M161 is substituted with D, E, H, K, R, F, W, Y, P or C; N162 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L163 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R164 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N165 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I166 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I167 substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; Q168 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D169 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C170 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q172 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L 173 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I174 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A175 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D176 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S177 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D178 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T179 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P180 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A181 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L182 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E183 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E184 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K185 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E186 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N187 substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K188 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I189 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V190 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V191 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R192 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q193 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T194 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G195 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y196 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F197 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F198 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I199 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y200 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S201 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q202 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V203 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L204 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y205 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T206 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D207 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P208 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F210 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A211 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M212 is substituted with D, E, H, K, R, F, W, Y, P or C; G213 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H214 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V215 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; I216 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q217 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R218 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K219 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K220 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H222 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V223 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F224 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D226 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E227 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S229 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L230 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V231 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T232 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L233 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F234 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R235 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C236 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I237 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q238 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N239 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M240 is substituted with D, E, H, K, R, F, W, Y, P or C; P241 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K242 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T243 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; L244 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; P245 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N246 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N247 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S248 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C249 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y250 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S251 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A252 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I254 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A255 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R256 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L257 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E258 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E259 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G260 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D261 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E262 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I263 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q264 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L265 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A266 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I267 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P268 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R 269 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E270 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N271 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A272 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q273 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I274 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S275 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R276 is substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N277 is substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G278 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D279 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D280 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T281 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F282 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F283 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G284 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; A285 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L286 is substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; K287 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L288 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L289 is D, E, H, K, R, N, Q, F, W, Substitutions with Y, P or C are included. Polynucleotides encoding these polypeptides are also included in the present invention. Neutrokine-alpha protein products of the invention are described herein and known in the art Neutrokine-alpha and / or Neutrokine-alphaSV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility). Preferably, the resulting proteins of the invention have increased and / or decreased Neutrokine-alpha and / or Neutrokine-alphaSV functional activity. More preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV protein products of the present invention have one or more increased and / or decreased Neutrokine-alpha and / or Neutrokine-alpha SV functional activity and / or physical properties. Has

In a further embodiment, the Neutrokine-alpha polypeptides of the invention comprise one or more amino acids (eg, 2, 3, 4, 5, 6, 7) substituted with substituted amino acids (preserved or non-conserved) as described above. , 8, 9, 10, 15, 20, 30 and 50) or alternatively consist of these amino acids.

Substitution of amino acids can also change the selectivity of binding of ligands to cell surface receptors. For example, Ostade et al., Nature 361: 266-268 (1993) describe specific mutations that cause selective binding of TNF-alpha to only one type of two known types of TNF receptors. Because Neutrokine-alpha and Neutrokine-alphaSV are members of the TNF polypeptide family, mutations similar to those of TNF-alpha are likely to show similar effects as in Neutrokine-alpha and / or Neutrokine-alphaSV.

Sites important for ligand-receptor binding can also be determined by structural analysis such as measurement, nuclear magnetic resonance or photoaffinity labels (Smith et al., J. Mol. Biol. 224: 899-904 (1992) and de Vos et al. Science 255: 306-312 (1992).

Since Neutrokine-alpha is a member of the TNF-associated protein lineage, in order to modulate, rather than completely eliminate, the functional activity (eg, biological activity) of Neutrokine-alpha, the mutations in the sequence encoding the amino acid in the TNF conserved domain Ie at positions Gly-191 to Leu-284 in FIGS. 1A and 1B (SEQ ID NO: 2), more preferably TNF family (e.g., TNF-alpha, TNF-beta, LT-beta, and Fas ligand) (see, eg, FIG. At residues in this region that are not conserved in all, most or several members of 2A-B). By making specific mutations of Neutrokine-alpha at locations where such conserved amino acids are typically found in related TNFs, Neutrokine-alpha muteins act as antagonists and thus lymphocyte (eg B cell) proliferation, differentiation and / or Or retains activity that inhibits activation. Thus, the polypeptides of the present invention include Neutrokine-alpha mutants. Such Neutrokine-alpha mutants comprise or alternatively consist of fragments, variants or derivatives of the full length or preferably extracellular domain of the Neutrokine-alpha amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2). . Polynucleotides encoding such Neutrokine-alpha mutants are also included in the present invention.

Because Neutrokine-alphaSV is a member of the TNF-associated protein lineage, the mutation encodes an amino acid in the TNF conserved domain to modulate rather than completely eliminate the functional activity (eg, biological activity) of Neutrokine-alphaSV. In the sequence, ie at positions Gly-172 to Leu-265 in FIGS. 5A and 5B (SEQ ID NO: 19), more preferably TNF family (e.g. TNF-alpha, TNF-beta, LT-beta and Fas ligand) (Reference Examples , Residues in this region that are not conserved in all, most or several members of FIGS. 2A-B). By making specific mutations of Neutrokine-alphaSV at positions where such conserved amino acids are typically found in related TNFs, the Neutrokine-alphaSV muteins act as antagonists, thus proliferating lymphocytes (eg, B cells), Retain activity that inhibits differentiation and / or activation. Thus, the polypeptides of the present invention include Neutrokine-alphaSV mutants. Such Neutrokine-alphaSV mutants comprise or alternatively include fragments, variants or derivatives of the full length or preferably extracellular domain of the Neutrokine-alpha amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19). Is done. Polynucleotides encoding such Neutrokine-alphaSV mutants are also included in the present invention.

Those skilled in the art also include the insertion of 19 amino acid residues that are not found in Neutrokine-alphaSV polypeptide sequences (ie, amino acid residues Val-142 to Lys-160 of the sequences described in Figures 1A and 1B and SEQ ID NO: 2). It will be appreciated that mutations targeting regions of the Neutrokine-alpha polypeptides of the invention may affect the observed functional activity (eg, biological activity) of the Neutrokine-alpha polypeptides. More specifically, some, non-limiting, non-exclusive lists of residues of Neutrokine-alpha polypeptide sequences that may be targeted by mutations include the following amino acid residues of the Neutrokine-alpha polypeptide sequences described in SEQ ID NO: 2. It includes: V-142; T-143; Q-144; D-145; C-146; L-147; Q-148; L-149; I-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157; I-158; Q-159 and K-160.

Recombinant DNA techniques known to those of skill in the art (see, eg, DNA shuffling) can be used to generate novel mutant proteins or muteins, including one or more amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or increased stability. In addition, they can be purified in higher yields and exhibit better solubility, at least under certain purification and storage conditions compared to corresponding natural polypeptides.

Accordingly, the present invention also provides for the deletion, addition or substitution of one or more amino acid residues so that Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides can be produced, which result in better expression, mass production, etc. in selected host cells. Neutrokine-alpha and / or Neutrokine-alphaSV derivatives and analogs. For example, cysteine residues may be deleted or substituted with other amino acid residues to remove disulfide bridges; For example, the N-linked glycosylation sites can be modified or eliminated to achieve expression of homogeneous products that are more readily recovered and purified from yeast hosts known to hyperglycosylate N-linked sites. Consequently, several amino acid substitutions at one or both of the first or third amino acid positions at any one or more of the glycosylation recognition sequences in the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention and / or Amino acid deletion at the second position of any one or more of the recognition sequences will prevent glycosylation of Neutrokine-alpha and / or Neutrokine-alphaSV in the modified tripeptide sequence (see, for example, Miyajimo et al., EMBO). J 5 (6): 1193-1197).

In addition, one or more of the amino acid residues (eg, arginine and lysine residues) of the polypeptide according to the invention may be deleted or substituted with other amino acids to eliminate undesirable processing by proteases such as for example purine or hexine. Can be. One possible consequence of this mutation is that the Neutrokine-alpha polypeptides of the invention are not cleaved and released from the cell surface.

In certain embodiments, Lys-132 and / or Arg-133 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 are mutated or deleted together with other amino acid residues to form Neutrokine-alpha in a soluble form from a cell expressing Neutrokine-alpha. Is prevented or reduced from being released. In a more particular embodiment, Lys-132 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 is mutated to Ala-132. In another particular specific embodiment, Arg-133 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 is mutated to Ala-133. These mutated proteins and / or polynucleotides encoding these proteins manipulate cells expressing Neutrokine-alpha polypeptides so that the polypeptides remain on the surface of the engineered cells, for example in vitro therapy or gene therapy. Can be used for

In certain embodiments, Cys-146 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 is mutated or deleted together with another amino acid residue to, for example, a mutation when expressed in an expression system (essentially as described in Example 1) Plays a role in preventing or reducing oligomerization of Neutrokine-alpha polypeptides. In certain embodiments, Cys-146 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also included in the present invention.

In another specific embodiment, Cys-232 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 is mutated or deleted together with another amino acid residue, such as when expressed in an expression system (essentially as described in Example 1). ) Help to prevent or reduce oligomerization of mutated Neutrokine-alpha polypeptides. In certain embodiments, Cys-232 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also included in the present invention.

In another specific embodiment, Cys-245 of the Neutrokine-alpha sequence described in SEQ ID NO: 2 is mutated to another amino acid residue or deleted together, for example when expressed in an expression system (essentially as described in Example 1). ) Help to prevent or reduce oligomerization of mutated Neutrokine-alpha polypeptides. In certain embodiments, Cys-245 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also included in the present invention.

The polypeptides of the invention are preferably provided in isolated form and are preferably substantially purified. Recombinantly produced forms of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are substantially purified by a one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). can do.

Polypeptides of the invention are complete polypeptides, deposited cDNA, encoded by deposited cDNA comprising intracellular, transmembrane and extracellular domains of the polypeptide encoded by deposited cDNA (ATCC Accession No. 97768). The mature soluble polypeptide encoded by the extracellular domain, excluding the intracellular and transmembrane domains of the protein, the complete polypeptide of FIGS. 1A and 1B (amino acid residues 1-285 of SEQ ID NO: 2), and the mature soluble poly of FIGS. 1A and 1B. The peptides (amino acid residues 134-285 of SEQ ID NO: 2), the extracellular domains of FIGS. 1A and 1B (amino acid residues 73-285 of SEQ ID NO: 2) excluding the intracellular and transmembrane domains, as well as 80%, 85%, Polypeptides having at least 90% similarity, more preferably at least 95% similarity, even more preferably at least 96%, 97%, 98% or 99% similarity. Polynucleotides encoding these polypeptides are also included in the present invention.

Polypeptides of the invention are complete polypeptides, deposited cDNA, encoded by deposited cDNA comprising intracellular, transmembrane and extracellular domains of the polypeptide encoded by deposited cDNA (ATCC Accession No. 203518). Mature soluble polypeptide encoded by the extracellular domain, excluding the intracellular and transmembrane domains of the protein, the complete polypeptide of FIGS. 5A and 5B (amino acid residues 1-266 of SEQ ID NO: 19), excluding the intracellular and transmembrane domains As well as the extracellular domains of FIGS. 5A and 5B (amino acid residues 73-266 of SEQ ID NO: 19) as well as those described above at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, even more preferably 96 Polypeptides having at least%, 97%, 98% or 99% similarity. Polynucleotides encoding these polypeptides are also included in the present invention.

In addition, the polypeptide of the present invention is at least 80% or at least 85%, more preferably at least 80% with the polypeptide encoded by the deposited cDNA (ATCC Accession No. 97768) or the polypeptides of FIGS. 1A and 1B (SEQ ID NO: 2). Said polypeptide comprising at least 90% or at least 95%, even more preferably at least 96%, 97%, 98% or 99% identical polypeptide and consisting of at least 30 amino acids, more preferably at least 50 amino acids Include part of. Polynucleotides encoding these polypeptides are also included in the present invention.

Moreover, the polypeptide of the present invention is at least 80% or at least 85%, more preferably at least 80% with the polypeptide encoded by the deposited cDNA (ATCC Accession No. 203518) or the polypeptides of FIGS. 5A and 5B (SEQ ID NO: 19). Said polypeptide comprising at least 90% or at least 95%, even more preferably at least 96%, 97%, 98% or 99% identical polypeptide and consisting of at least 30 amino acids, more preferably at least 50 amino acids Include part of. Polynucleotides encoding these polypeptides are also included in the present invention.

The% similarity of the two polypeptides is based on the best fit program (Wisconsin 53711 Madison Science Drive 575 University Research Park Genetics Computer Group, Wisconsin Sequencing Package, Version 8 for Unix) and the default setting to measure similarity. Similarity score obtained by comparing amino acid sequences of peptides. Bestfit uses Smith and Waterman's local homology algorithms (Advances in Applied Mathematics 2: 482-489) to find the best fragments of similarity between the two sequences.

Polypeptides having an amino acid sequence "equivalent" of, for example, 95% or more of the reference amino acid sequence of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides have an amino acid sequence of this polypeptide of Neutrokine-alpha and / or Neutrope. Meaning the same as the reference sequence, except that up to five amino acid variations per 100 amino acids of the reference amino acids of the Kin-alphaSV polypeptide may be included. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or 5% of the total amino acid residues in the reference sequence. Amino acids up to can be inserted into the reference sequence. These variations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or may occur anywhere between residues of the reference sequence or between terminal positions interspersed in one or more adjacent groups in the reference sequence.

As a practical matter, certain polypeptides are described, for example, in the amino acid sequences described in Figures 1A and 1B and (SEQ ID NO: 2), deposited cDNA clone HNEDU15 (ATCC Accession No. 97768) or fragments thereof, for example in Figures 5A and Amino acid sequence as shown in 5B (SEQ ID NO: 19), amino acid sequence encoded by deposited cDNA clone HDPMC52 (ATCC Accession No. 203518) and 80%, 85%, 90%, 95%, 96%, 97%, 98% Alternatively, 99% or more of the same may be measured using a known computer program, such as a BestFit program (Wisconsin sequencing package, version 8 for Unix, of Genetics Computer Group, University of Wisconsin 53711 Madison Science Drive 575). When using best-fit or other sequence alignment programs to determine whether a particular sequence is for example 95% identical to the reference sequence of the invention, the percentage of identity is of course calculated over the full length of the reference amino acid sequence and the amino acid residues in the reference sequence. Set the variable to allow gaps in homology below 5% of the total number of.

In certain embodiments, the identity between a reference (query) sequence (a sequence of the present invention) and the sequence in question is based on a FRATTDB computer program based on Brutrag's algorithm (Comp. App. Biosic. 6: 237-245 (1990)). Measure by Preferred variables used for the FASTDB amino acid sequence are: matrix = PAM 0, k-tuple = 2, mismatch penalty = 1, binding penalty = 20, random group length = 0, threshold score = 1, window size = sequence length Gap penalty = 5, gap size penalty = 0.05, window size = 500 or the shorter of the corresponding amino acid sequence. According to this embodiment, manual corrections should be made to the results if the sequence is shorter than the query sequence due to N- or C-terminal deletions, not due to internal deletions. This is because the FASTDB program does not calculate the N- and C-terminal truncations of the sequence when calculating the percent total identity. For those sequences that are truncated at the N- and C-terminus relative to the query sequence, the percent identity is the percentage of the total base of the query sequence to calculate the number of bases of the query sequence that are the N- and C-terminus of that sequence that are not matched / arranged. Correct by doing Whether residues were matched / arranged is determined as a result of FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using a particular variable to obtain a final percent identity score. This final percent identity is what is used for the purposes of the present invention. Only residues at the N- and C-terminal ends of the sequence that are not matched / arranged with the query sequence are considered for the purpose of manually controlling the% identity score. That is, only query residue positions other than the furthest N- and C-terminal residues of the sequence are considered. For example, a 90 amino acid corresponding sequence is aligned with a 100 residue query sequence to determine% identity. Deletion occurs at the N-terminus of the sequence so that the FASTDB sequence does not represent a match / array of the first 10 residues at the N-terminus. Ten unpaired residues represent 10% of the sequence (the number of residues present at the unmatched N- and C-terminus / total number of residues in the query sequence), so the percent identity score calculated by the FASTDB program Deduct 10% from If the remaining 90 residues were perfectly matched the final percent identity would be 90%. As another example, 90 residue corresponding sequences were compared with 100 residue query sequences. At this point the deletion is an internal deletion, so there are no residues at the N- or C-terminus of the sequence that are not matched / arranged with the query. In this case,% identity calculated by FASTDB is not corrected manually. Once again, manually correct only the residue positions outside the N- and C-terminus of the sequence as shown in the FASTDB sequence as not matched / arranged with the query sequence. No other manual calibration is made for the purposes of the present invention.

Polypeptides of the invention are used as molecular weight markers on SDS-PAGE gels or molecular sieve gel filtration columns using methods known to those skilled in the art, but not limited to these. In addition, as described in detail below, the polypeptides of the invention are useful in assays for detecting, but are not limited to, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or Neutrokine-alpha and / or Or polyclonal and monoclonal antibodies useful as agonists and antagonists capable of enhancing or inhibiting Neutrokine-alphaSV action. In addition, the polypeptides of the invention have therapeutic uses as described herein. Such polypeptides can also be used in yeast pair-hybrid systems for "capturing" Neutrokine-alpha and / or Neutrokine-alphaSV, which are also candidate agonists and antagonists according to the present invention. Yeast pair hybrid systems are described in Fields and Song, Nature 340: 245-246 (1989).

Transgenic and "Knockout" Animals

Polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, piglets, goats, sheep, cattle and non-human primates (e.g. baboons, monkeys and chimpanzees) It can be used to generate metastasized animals. In certain embodiments, the techniques described herein or techniques known in the art are used to express the polypeptides of the invention in the human body as part of a gene therapy protocol.

Any technique known in the art can be used to introduce transgenes (ie, polynucleotides of the present invention) into an animal to generate the helped lineage of the transgenic animal. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993) Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., USP 4,873,191 (1989); Retroviral mediated gene delivery into germ cells (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)), embryonic cells or embryos; Gene tracking in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); Electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); Introduction of polynucleotides of the invention using gene guns (Ulmer et al., Science 259: 1745 (1993)); Introduction of nucleic acid constructs into embryonic multipotent stem cells and delivery of stem cells into embryonic cells; And sperm-mediated gene delivery (Lavitrano et al., Cell 57: 717-723 (1989)). The contents of this document are incorporated herein by reference. Such techniques are described in Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115: 171-229 (1989). The entire contents of this document are incorporated herein by reference. See also, US Pat. No. 5,464,764 to Capecchi, et al., Positive-Negative Selection Methods and Vectors; U.S. Patent 5,631,153 to Capecchi, et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-Negative Selection Methods and Vectors for Making Same; US Pat. No. 4,736,866 to Leder et al., Transgenic Non-Human Animals; And US Pat. No. 4,873,191 to Wangner, et al., Genetic Transformation of Zygotes. The entire contents of each of these patents are incorporated herein by reference.

Any technique known in the art produces transgenic clones containing the polynucleotides of the invention (eg, nuclear transfer from resting embryonic cultured germ cells, mammary cells or mature cells to nucleated oocytes). (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)).

The present invention provides transgenic animals containing transgenes in all cells as well as animals containing transgenes in some cells but not in all cells (ie, mosaic animals or chimeric animals). Transgenes can be integrated as single transgenes or as multiple copies, such as in concatemers (eg, head-to-head or head-to-tail). Transgenes are described, for example, in Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992) may optionally be introduced and activated into specific types of cells. The regulatory sequences required for such cell-type specific activation will depend upon the particular cell type in question and will be apparent to those skilled in the art. If it is necessary to integrate the polynucleotide transgene into the chromosomal region of the endogenous gene, the tracking of the gene is preferred. Briefly, when this technique is used, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating through homologous recombination with the chromosomal sequence and disrupting the function of the nucleotide sequence of the endogenous gene. In addition, the teachings described in Gu et al., Science 265: 103-106 (1994) may optionally introduce transgenes into specific types of cells to inactivate endogenous genes only in those types of cells. . The regulatory sequences required for such cell-type specific inactivation depend on the particular cell type in question and are apparent to those skilled in the art. In addition to expressing a polypeptide of the invention in a transgenic or tissue specific manner in a transgenic animal, one of ordinary skill in the art will typically regulate the expression of the polypeptide by a number of different means (eg, developmentally or chemically regulated expression). Constructs can be formed.

Once transgenic animals have been generated, standard techniques can be used to assay expression of recombinant genes. Initial selection can be accomplished by performing an analysis on animal tissues demonstrating that integration of the transgene has been achieved by Southern blotting analysis or PCR techniques. In addition, tissues of transgenic animals using techniques including, but not limited to, Northern blotting analysis, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) and TaqMan PCR of tissue samples obtained from animals MRNA expression level of the transgene can be assessed. Samples of transgenic gene-expressing tissue can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the primitive animals have been produced, they can be breeded, allogened, crossbred or hybridized to produce a colony of specific animals. Examples of such breeding include, but are not limited to, gene transfer that expresses higher levels of the transgene because of the cross-breeding of an animal with one or more integration sites and the effect of further expression of each transgene to establish a separate lineage. Generation of an animal homozygous for a given integration site by crossing heterozygous transgenic animals to increase the breeding and expression of separate strains to create a group of animals and to eliminate the need for selection of the animal by DNA analysis By crossing the homozygous strains, the generation of heterozygous or homozygous strain groups and breeding to establish the transgene on a distinct background suitable for the experimental model in question.

The transgenic and "knockout" animals of the present invention study the biological action of, but are not limited to, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides, and have abnormal Neutrokine-alpha and / or Neutrokine- It is used as an animal model system to study the symptoms and / or diseases associated with alphaSV expression and to select compounds effective for alleviating such symptoms and / or diseases.

In a further aspect of the invention, cells genetically engineered to express the polypeptide of the invention or cells engineered to express the polypeptide of the invention (knockout) are administered in vivo to the patient. Such cells may be obtained from patients (ie, animals including humans) or MHC-compliant donors, including but not limited to fibroblasts, bone marrow cells, blood cells (ie, lymphocytes), adipocytes, myocytes, endothelial cells, and the like. May be included. Transfection or transduction (using viral vectors, preferably vectors incorporating transgenes into the cell genome), including but not limited to plasmids, cosmids, YACs, extruded DNA, electroporation, liposomes, etc. These cells are genetically engineered according to recombinant DNA techniques to introduce the coding sequences of the polypeptides according to the invention into cells or disrupt the coding sequences and / or endogenous regulatory sequences associated with the polypeptides of the invention. The coding sequence of the polypeptide according to the invention can be set under the control of a potent constitutive or inducible promoter or promoter / enhancer such that the expression and preferably secretion of the polypeptide according to the invention is achieved. Engineered cells expressing and preferably secreting the polypeptides of the invention can be introduced systemically into the patient (eg, circulating or intraperitoneally).

Alternatively, cells can be incorporated into the matrix or inserted into the body. For example, genetically engineered fibroblasts can be implanted as part of skin graft tissue and genetically engineered endothelial cells can be transplanted as part of lymphatic or vascular graft tissue. See, eg, US Pat. 5,399,349 and US Pat. No. 5,460,959 to Mulligan & Wilson. The entire contents of these documents are incorporated herein by reference.

If the cells to be administered are non-autologous or non-MHC compatible cells, these cells can be administered using well known techniques that inhibit the development of a host immune response against the introduced cells. For example, cells can be introduced into a capsule form, which allows components to be exchanged into an adjacent extracellular environment while preventing the introduced cells from being recognized by the host immune system.

Antibodies

The invention is also determined by polypeptides, polypeptide fragments or variants of SEQ ID NO: 2 and / or SEQ ID NO: 19 and / or epitopes of the invention (e.g., immunoassays well known in the art for assaying specific antibody-antigen binding). And T-cell antigen receptor (TCR) for immunospecific binding. Antibodies of the invention are, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, Fab expression libraries Resulting fragments, anti-idiotypic (anti-Id) antibodies (including anti-id antibodies to antibodies of the invention) and epitope-binding fragments of such antibodies. As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, ie, a molecule containing an antigen binding site that immunospecifically binds an antigen. Immunoglobulin molecules of the invention may be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2) or immune, IgA ( IgA1 and IgA2) or subclasses. Immunoglobulins can have both heavy and light chains. The arrangement of IgG, IgE, IgM, IgD, IgA and IgY heavy chains can be paired with light chains in kappa or lambda form.

Most preferably, the antibody is a human antigen binding antibody fragment of the invention, including but not limited to Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibody, disulfide- Fragments comprising linked Fvs (sdFv) and VL or VH domains are included. Antigen-binding antibody fragments comprising single chain antibodies may comprise only the variable region or may comprise this variable region in combination with all or a portion of the hinge region, CH1, CH2 and CH3 domains. The present invention also encompasses any combination of variable and hinge regions, CH1, CH2 and CH3 domains. Antibodies of the invention can be derived from animals, including birds and mammals. Preferably, the antibody is derived from human, mouse (mouse and rat), monkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of human immunoglobulin, and have been isolated from human immunoglobulin libraries, or are described below, for example in US Pat. No. 5,939,598 (Kucherlapatic et al.). As described, antibodies isolated from a transgenic animal for one or more human immunoglobulins that do not express endogenous immunoglobulins.

Antibodies of the invention may be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies may be specific for different epitopes of polypeptides according to the invention or specific for polypeptides of the invention and for heterologous epitopes (eg, heterologous polypeptides or solid support materials). WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147: 60-69 (1991); USP 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819 ,; Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

Antibodies of the invention may be described or specified in terms of epitopes or portions of the polypeptides of the invention to which they recognize or specifically bind. Epitope (s) or polypeptide portions can be specified as described herein, eg, by the N-terminal and C-terminal positions, by the size of the contiguous amino acid residues, or as listed in the tables and figures. Antibodies that specifically bind any epitope or polypeptide of the invention may also be excluded. Accordingly, the present invention includes antibodies that specifically bind to the polypeptides of the present invention and contemplates their exclusion.

In certain embodiments, the antibodies of the invention comprise Phe-115 to Leu-147, Ile-150 to Tyr-163, Ser-171 to Phe-194, Glu-223 to Tyr-246 and Ser-271 of the amino acid sequence in SEQ ID NO: 2. To a polypeptide comprising Phe-278. In another specific embodiment, the antibodies of the invention comprise Phe-115 to Leu-147, Ile-150 to Tyr-163, Ser-171 to Phe-194, Glu-223 to Tyr-246 and Ser- of the amino acid sequence in SEQ ID NO: 2. Binds to a polypeptide consisting of 271 to Phe-278. In a preferred embodiment, the antibody of the invention binds to a polypeptide comprising Glu-223 to Tyr-246 of SEQ ID NO: 2. In another preferred embodiment, the antibody of the invention binds to a polypeptide consisting of Glu-223 to Tyr-246 of SEQ ID NO: 2. In yet another preferred embodiment, the antibody of the invention binds to a polypeptide consisting of Phe-230 to Asn-242 of SEQ ID NO: 2. In a further preferred and customary embodiment, the antibodies of the invention inhibit one or more biological activities of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides according to the invention via specific binding. In a more preferred embodiment, the antibodies of the invention inhibit Neutrokine-alpha and / or Neutrokine-alphaSV-mediated B cell proliferation.

Antibodies of the invention may also be described or specified in terms of their cross-reactivity. Also included are antibodies that do not bind any other analog, ortholog or homologue of the polypeptide according to the invention. At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% identity to the polypeptide of the invention (in the art Antibodies that bind to polypeptides having known) and measured using the methods described herein) are also included in the present invention. In certain embodiments, the antibodies of the invention cross react with murine, rat and / or rabbit homologs of human proteins and their corresponding epitopes. Less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50% identity with the polypeptide of the present invention Antibodies that do not bind to polypeptides having a composition known in the art and measured using the methods described herein are also included in the present invention. In certain embodiments, the cross-reactivity described above may be in combination with any one, two, three, four, five or more of the specific antigenic or immunogenic polypeptides or specific antigenic and / or immunogenic polypeptides described herein. Is done in Also included are antibodies that bind to a polypeptide encoded by a polynucleotide that hybridizes with the polynucleotide of the present invention under stringent hybridization conditions as described herein. Antibodies of the invention may also be described or specified in terms of their binding affinity to the polypeptides of the invention. Preferred binding affinities include 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 , 10 ^-6 , 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M, 10 ^-10 M, 5 x 10 ^-11 M, 10 ^-11 M, 5 x 10 ^-12 M, 10 ^-12 M, And those having a dissociation constant or Kd of less than 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M and 10 ^-15 M.

The present invention also provides antibodies that competitively inhibit binding of antibodies to epitopes of the invention as determined by methods known in the art for measuring competitive binding, such as immunoassays described herein. In a preferred embodiment, the antibody competitively inhibits binding to epitopes by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50%.

Antibodies of the invention may act as agonists or antagonists of the polypeptides according to the invention. For example, the present invention includes antibodies that partially or completely block receptor / ligand interactions with the polypeptides of the present invention. Preferably the antibody of the invention binds to the antigenic epitope or portion thereof described herein. The present invention features both receptor-specific and ligand-specific antibodies. In addition, the invention features receptor-specific antibodies that do not inhibit ligand binding but inhibit receptor activation. Receptor activation (ie, signaling) can be measured by the techniques described herein or by techniques known in the art. For example, receptor activation can be measured by detecting phosphorylation of a receptor or its substrate (eg tyrosine or serine / threonine) by immunoprecipitation followed by Western blot analysis (eg as described above). In certain embodiments, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in the absence of the antibody inhibits ligand activity or receptor activity. Antibodies are provided.

In addition, the present invention features receptor-specific antibodies that inhibit both ligand binding and receptor activation, as well as antibodies that recognize receptor-ligand complexes and preferably do not specifically recognize unbound receptors or unbound ligands. It is done. Likewise, neutralizing antibodies that bind to the ligand and inhibit the binding of the ligand to the receptor, as well as agents that bind to the ligand to inhibit receptor activation but do not inhibit the ligand from binding to the receptor are included. Also included are antibodies that activate the receptor. These antibodies can act as receptor agonists, ie, enhance or activate all or part of the biological activity induced by ligand-mediated receptor activation, for example by inducing dimerization of the receptor. An antibody may be specified as an agonist, antagonist or inverse agonist for biological activity, including the specific biological activity of the peptides of the invention described herein. Such antibody agonists can be prepared using methods known in the art. See PCT Publication WO 96/40281; USP 5,811,097; Deng et al. Blood 92 (6): 1981-1988 (1998); Chen et al. Cancer Res. 58 (16): 3668-3678 (1998); Harrop et al. J. Immunol. 161 (4): 1786-1794 (1998); Zhu et al. Cancer Res. 58 (15): 3209-3214 (1998); Yoon et al. J. Immunol. 160 (7): 3170-3179 (1998); Prat et al. J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard et al. J. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al. Cytokine 9 (4): 233-241 (1997); Carlson et al. J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman et al. Neuron 14 (4): 755-762 (1995); Muller et al. Structure 6 (9): 1153-1167 (1998); Bartunek et al. Cytokine 8 (1): 14-20 (1996). The entire contents of these documents are incorporated herein by reference.

Antibodies of the invention are used in methods known in the art for purifying, detecting and tracking polypeptides of the invention, including, but not limited to, in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies are used in immunoassays to qualitatively and quantitatively measure levels of polypeptides according to the invention in biological samples. See Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor. Laboratory Press, 2nd ed. 1988). The entire contents of this document are incorporated herein by reference.

Antibodies of the invention can be used alone or in combination with other compositions. In addition, the antibodies may be recombinantly fused at the N- or C-terminus of the heterologous polypeptide or chemically bound (including covalent and non-covalent bonds) to the polypeptide or other composition. For example, the antibodies of the invention can be recombinantly fused or bound to molecules useful as labels in detection assays and to active molecules such as heterologous polypeptides, drugs or toxins. See PCT Publication WO 92/08495; WO 91/14438; WO 89/12624; USP 5,314,995 and EPO 396,387].

Antibodies of the invention include modified derivatives in which any type of molecule is covalently bound to the antibody and the antibody is not prevented from generating an anti-idiotypic response by this covalent bond. For example, but not limited to, antibody derivatives may be glycosylated, acetylated, phenylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins. Antibodies modified by the like and the like. Many chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives may contain one or more unusual amino acids.

Antibodies of the invention can be produced by any suitable method known in the art. Polyclonal antibodies directed against the antigen can be produced by a number of methods well known in the art. For example, the polypeptides of the present invention can be administered to a variety of host animals, including but not limited to rabbits, mice, rats, and the like, to induce the production of serum containing polyclonal antibodies specific for the antigen. have. Several adjuvants can be used to increase the immune response, depending on the species of the host, but are not limited to Freund's solution (complete and incomplete), inorganic gels (e.g. aluminum hydroxide), surface-active substances (e.g. lysorexi) Tin), pluronic acid polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenols and potentially useful human adjuvants (e.g. BCG (Basile Calmet-Guerin) and Corynebacterium parboom ) Is included. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including combinations of hybridomas, recombinant and phage display techniques, or a combination of these techniques. For example, known in the art and described in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., In: Monoclonal Antibodies And T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) can be used to prepare monoclonal antibodies using hybridoma technology. The entire contents of this document are incorporated herein by reference. As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to and is not a method of producing an antibody derived from a single clone, including eukaryotic, prokaryotic or phage clones.

A "monoclonal antibody" may comprise two proteins, a heavy chain and a light chain, or alternatively may consist of these.

Methods of preparing and screening specific antibodies using hybridoma technology are conventional and well known in the art and discussed in detail in Examples (eg, Example 9). As a non-limiting example, mice can be immunized with the polypeptides of the invention or cells expressing such peptides. Once an immune response is detected, for example, antigen-specific antibodies are detected in mouse serum, mouse spleens are harvested and splenocytes isolated. The splenocytes are then fused with suitable myeloma cells (eg, cells from cell line SP20 obtained from ATCC) by well known techniques. Hybridomas are selected and cloned by defined dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptide of the invention by methods known in the art. Immunization of mice with positive hybridoma clones can generally produce ascites fluid containing high levels of antibody.

Therefore, the present invention is preferably produced by fusing splenocytes isolated from mice immunized with the antigen of the present invention with myeloma cells, followed by culturing the hybridoma cells secreting the antibody of the present invention, followed by Provided are methods for preparing monoclonal antibodies, including selecting hybridomas resulting from fusion to hybridoma clones that secrete antibodies capable of binding peptides, as well as antibodies produced by such methods.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F (ab ') 2 fragments of the present invention proteolytically decompose immunoglobulin molecules using enzymes such as papain (which produces Fab fragments) or pepsin (which produces F (ab') 2 fragments). Can be generated by F (ab ') 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the invention can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles with polynucleotide sequences encoding them. In certain embodiments, such phage may be used to indicate antigen-binding domains expressed from a repertoire or combined antibody library (eg, human or mouse). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified, for example, using an antigen or labeled antigen that is bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage comprising fd and M13 expressed from phage with a disulfide stabilized Fv antibody domain, Fab or Fv, recombinantly fused to either phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare the antibodies of the invention include those described by Brinkman et al. J. Immunol. Methods 182: 41-50 (1995); Ames et al. J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al. Eur. J. Immunol. 24: 952-958 (1994); Persic et al. Gene 187 9-18 (1997); Burton et al. Advances in Immunology 57: 191-280 (1994); PCT / GB91 / 01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; And USP 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108. The entire contents of these documents are incorporated herein by reference.

As described in the literature, after phage selection, antibody coding regions from phage can be isolated and used to form human antibodies as whole antibodies or all other desired antigen binding fragments, including mammalian cells, insect cells, plant cells, It can be expressed in any preferred host, including yeasts and bacteria. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments are also described in PCT Publication WO 92/22324; Mullinax et al. BioTechniques 12 (6): 864-869 (1992); Sawai et al. AJRI 34: 26-34 (1995); And Better et al. Science 240: 1041-1043 (1998) can be used according to methods known in the art. The entire contents of these documents are incorporated herein by reference.

Examples of techniques that can be used to generate single chain Fvs and antibodies include USP 4,946,778 and 5,258,498; Huston et al. Methods in Enzymology 203: 46-88 (1991); Shu et al. PNAS 90: 7995-7999 (1993); And Skerra et al. Science 240: 1038-1040 (1998). For some uses, including the use of human in vivo antibodies and in vitro detection assays, it may be desirable to use chimeric, humanized or human antibodies. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, including, for example, antibodies having a variable region derived from a monoclonal antibody of a mouse and an immunoglobulin constant region of a human. Methods for preparing chimeric antibodies are known in the art. Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies, et al. (1989) J. Immunol. Methods 125: 191-202; And USP 5,807,715, 4,816,567 and 4,816,397]. The entire contents of each of these documents are incorporated herein by reference. Humanized antibodies are antibody molecules from non-human species antibodies that bind to the antigen of interest with one or more complementarity measuring regions (CDRs) from non-human species and backbone regions from human immunoglobulin molecules. Often, skeletal residues in the human backbone region are substituted with corresponding residues from CDR donor antibodies to modify (preferably enhance) antigen binding. These skeletal substitutions are identified by methods well known in the art, for example, by modeling the interaction of CDRs with skeletal residues to identify skeletal residues important for antigen binding and comparing sequences to specific skeletal residues at specific positions. (See, eg, Queen et al, US Pat. No. 5,585,089 and Riechmann et al., Nature 332: 323 (1988)). The entire contents of each of these documents are incorporated herein by reference. Antibodies are CDR-grafted (EP 239,400; PCT Publication WO 91/09967; USP 5,225,539, 5,530,101 and 5,585,089), surface-coated or resurfaced (EP 592,106; EP 0 519 596; Padlan Molecualr Immunology 28 (4/5): 489- 498 (1991); Studnicka et al. Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)) and chain shuffling (USP 5,565,332) It can be humanized using a variety of techniques, including.

Fully human antibodies are particularly preferred for the treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See USP 4,444,887 and 4,716,111 and PCT Publication WO 98 /. 46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. The entire contents of each of these documents are incorporated herein by reference.

In addition, human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. Mouse heavy and light chain immunoglobulin genes can be made nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region inhibit endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into undifferentiated embryonic cells to generate chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized with the selected antigen (eg, all or part of the polypeptide according to the invention) in a normal manner. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, such techniques can be used to generate therapeutically useful IgG, IgA, IgM and IgE antibodies. Such techniques for generating human antibodies are described in Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). The above techniques for preparing human antibodies and human monoclonal antibodies and protocols for preparing such antibodies are described in detail in PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent 0 598 877; U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771 and 5,939,598. The entire contents of each of these documents are incorporated herein by reference. In addition, human antibodies to specific antigens can be prepared using techniques similar to those described above from companies such as Abgenix, Inc., Fremont, Calif., And Genpharm, San Jose, Calif. You can get it.

Complete human antibodies that recognize specific epitopes can be generated using a technique called "guided selection." This method uses specific non-human monoclonal antibodies (eg mouse antibodies) to guide the selection of complete human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 (1988) ).

In addition, antibodies to the polypeptides of the invention can be used in turn according to techniques well known to those skilled in the art to produce anti-idiotypic antibodies that "mute" the polypeptides of the invention (see Greenspan & Bona). , FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, an antibody that binds to a polypeptide of the invention and competitively inhibits polypeptide multimerization and / or binding of the polypeptide to a ligand can be used to " metalogize " the polypeptide multimerization and / or binding domain and consequently. To generate anti-idiotypes that bind and neutralize the polypeptide and / or its ligands. Such neutralizing anti-idiotypes or Fab fragments of these anti-idiotypes can be used in therapeutic methods to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind the peptides of the invention and / or their ligands / receptors and thus block their biological activity.

Polynucleotides Encoding Antibodies

The present invention provides polynucleotides comprising the nucleotides encoding the antibodies of the invention and fragments thereof. In addition, the present invention is directed to binding antibodies, preferably polypeptides having the amino acid sequence of SEQ ID NO: 2, to specific binding to the polypeptide of the present invention under stringent or less stringent conditions as defined below. Polynucleotides that hybridize with the polynucleotides encoding the antibody. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 19. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 23. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 28. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 30.

Polynucleotides can be obtained by methods known in the art and the nucleotide sequence of the polynucleotides can also be determined by methods known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be derived from a chemically synthesized oligonucleotide as described, for example, in Kutmeier et al., BioTechniques 17: 242 (1994). Can be assembled. In summary this includes the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of these oligonucleotides and amplification of linked oligonucleotides by PCR.

As another alternative, the polynucleotides encoding the antibodies can be generated from nucleic acids from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin may be chemically synthesized or a suitable source (e.g., an antibody cDNA library or tissue expressing the antibody). Or hybridized to the 3 'and 5' ends of the sequence from a cDNA library generated from a cell (e.g., a hybridoma cell selected to express the antibody of the invention) or a nucleic acid isolated therefrom, preferably a poly A + RNA). It can be obtained by PCR amplification using synthetic primers that can be converted or by cloning with oligonucleotide probes specific for a particular gene sequence, for example by identifying cDNA clones from cDNA libraries encoding antibodies. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using methods well known in the art.

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, methods well known in the art for manipulation of the nucleotide sequence, such as recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, Sambrook). et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY The nucleotide sequence of the antibody can be used to generate antibodies with different amino acid sequences. For example, amino acid substitutions, deletions and / or insertions can be made.

In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains can be examined by methods well known in the art to identify the sequence of complementarity determining regions (CDRs). For example, regions of sequence overvariation can be determined by comparison with known amino acid sequences of other heavy and light chain variable regions. Using conventional recombinant DNA techniques, one or more CDRs can be humanized by inserting one or more CDRs into the backbone region, eg, into a human bone bone region, as described above. The skeletal region may be naturally occurring or may be a consensus skeletal region and is preferably a human skeletal region (see, eg, Chothia et al., J Mol. Biol. 278: 457-479 (1998) on the list of human skeletal regions). to be. Preferably, the polynucleotides produced by the combination of the backbone region and the CDRs encode an antibody that specifically binds to the polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions and preferably amino acid substitutions enhance binding of the antibody to the antigen. In addition, such methods can be used to participate in inner chain disulfide bonds to make amino acid substitutions or deletions of one or more variable region cysteine residues to produce antibody molecules lacking one or more inner chain disulfide bonds. Other variations of polynucleotides are included in the present invention and belong to the art.

In addition, techniques developed to generate “chimeric antibodies” by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity (Morrison et al., Proc., Natl Acad.Sci. 81: 851-855 (1984); Neuberger et al., Nature 32: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)). As described above, chimeric antibodies are molecules derived from different animal species that differ in different parts, such as antibodies having variable regions derived from mouse mAbs and human immunoglobulin constant regions (eg, humanized antibodies).

Alternatively, techniques for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 ( 1988) and Ward et al., Nature 334: 544-54 (1989)) can be applied to generate single chain antibodies. Single chain antibodies are formed by linking heavy and light chain fragments of the Fv region via an amino acid bridge to obtain a single chain polypeptide. Again, this. Techniques for the assembly of functional Fv fragments in E. coli can be used (Skerra et al., Science 242: 1038-1041 (1988)).

Method of producing antibody

Antibodies of the invention can be prepared by methods known in the art for the synthesis of antibodies, in particular by chemical synthesis, preferably by recombinant expression techniques.

Recombinant expression of an antibody of the invention or fragments, derivatives or analogs thereof (heavy or light chain of an antibody of the invention or a single chain antibody of the invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody. . Once a polynucleotide encoding the antibody molecule or heavy chain or light chain of the invention, or portion thereof (preferably containing heavy or light chain variable domains) is obtained, vectors for the preparation of antibody molecules are well known in the art. Known techniques can be used to produce by recombinant DNA techniques. As such, a method for preparing a protein by expressing a polynucleotide containing an antibody coding nucleotide sequence is described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention or a heavy or light chain or a heavy or light chain variable domain thereof operably linked to a promoter. Such vectors may contain nucleotide sequences encoding constant regions of antibody molecules (see, eg, PCT Publication WO 86/05807; WO 89/01036; and US Pat. No. 5,122,464) and clone the variable domains of the antibody into such vectors. Complete heavy or light chains can be expressed.

The expression vector is delivered to the host cell by conventional techniques and then the transfected cells are cultured by conventional techniques to produce the antibodies of the invention. Accordingly, the present invention includes host cells containing a polynucleotide encoding an antibody of the invention or a heavy or light chain thereof or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment for the expression of double chain antibodies, as described in detail below, vectors encoding both heavy and light chains can be expressed simultaneously in the host cell to express complete immunoglobulin molecules.

Various host-expression vector systems can be used to express the antibody molecules of the invention. Such a host-expression system exhibits a vehicle capable of generating the corresponding coding sequence and subsequently purifying but also capable of expressing the antibody molecule of the invention at that location when transformed or transfected with the appropriate nucleotide coding sequence. Indicates. These include, but are not limited to, bacteria (e.g., E. coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the antibody coding sequence, containing the antibody coding sequence. Yeast transformed with a recombinant yeast expression vector (e.g., Saccharomyces, Pichia), a recombinant viral expression vector containing an antibody coding sequence (e.g., a baculovirus), a recombinant virus expression vector containing an antibody coding sequence ( Eg, promoters derived from the genome of plant cell systems or mammalian cells infected with cauliflower mosaic virus CaMV; tobacco mosaic virus TMV or transformed with recombinant plasmid expression vectors (e.g. Ti plasmids) (e.g. metallothionein promoters) ) Or a promoter derived from a mammalian virus (eg, adenovirus late Microorganisms such as mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3 cells) with a recombinant expression construct containing a promoter; a vaccinia virus 7.5K promoter). Preferably, in particular bacterial cells (eg Escherichia coli) for the expression of a complete recombinant antibody molecule and more preferably eukaryotic cells are used for expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as major intermediate early gene promoter elements from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene 45). : 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990)).

In bacterial systems, many expression vectors can be advantageously selected depending on the use of the antibody molecule expressed. For example, it may be desirable to have a vector that induces high levels of expression of a fusion protein product that is readily purified when it is desired to produce a large amount of said protein to prepare a pharmaceutical composition of antibody molecules. Such vectors include, but are not limited to, E. coli, wherein the antibody coding sequence can be linked within the frame of the vector having the lac Z coding region individually to produce a fusion protein. E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)); pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24: 5503-5509 (1989)) and the like. The pGEX vector can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, these fusion proteins are soluble and can be readily purified by lysis in the presence of free glutathione following binding and adsorption from the lysed cells to matrix glutathione-agarose beads. The pGEX vector can contain a thrombin or Factor Xa protease cleavage site to release the target gene product cloned from the GST residue.

For insect systems, autographer california nuclear multifaceted virus virus (AcNPV) is used as a vector for expressing foreign genes. This virus grows in spordogera pruperperda cells. Antibody coding sequences can be cloned individually into non-essential regions of the virus (eg polyhedrin gene) and placed under the control of an AcNPV promoter (eg polyhedrin promoter).

For mammalian host cells, many virus-based expression systems can be used. If adenovirus is used as the expression vector, the antibody coding sequence of interest can be linked to the adenovirus transcriptional / detoxification regulatory complexes (eg, late promoters and third party leader sequences). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, region El or E3) will result in a recombinant virus that is alive in the infected host and capable of expressing the antibody molecule (see, eg, Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). It may also be necessary for efficient translation of the antibody coding sequence into which a particular initiating signal has been inserted. These signals include the ATG start codon and the contiguous sequence. In addition, the start codon must be located on the same phase as the reading frame of the desired darkening sequence to ensure translation of the complete insert. These exogenous detoxification control signals and initiation codons can be of various origins, natural or synthetic. Expression efficiency can be enhanced by insertion of appropriate transcription enhancer elements, transcription terminators, and the like (Bittner et al., Methods in Enzymol, 153: 51-544 (1987)).

In addition, host cell strains may be selected that modulate the expression of the inserted sequences or modify and process the gene product in a particular aspect desired. Such modifications (eg glycosylation) and processing (eg cleavage) of the protein product may be important for the function of the protein. Different host cells have distinctive and unique mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the foreign protein expressed. In this regard, eukaryotic host cells can be used that possess cellular mechanisms for proper processing of primary transcripts, glycosylation and phosphorylation of gene products. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hella, COS, MDCK, 293, 3T3, WI38, especially breast cancer cell lines such as BT483, Hs578T, HTB2, BT20 and T47D and CRL7030 and Hs578Bst Normal mammary cell lines such as these are included.

Stable expression is desirable for long term high yield production of recombinant proteins. For example, cell lines stably expressing antibody molecules can be produced genetically. Rather than using expression vectors containing viral replication origins, host cells can be transformed with DNA regulated by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. have. After introduction of the foreign DNA, genetically engineered cells are grown in proliferation medium for 1-2 days and then transferred to selection medium. Selection markers in recombinant plasmids provide resistance to selection and allow cells to stably integrate plasmids into their chromosomes, grow to form a focus, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer engineering cell lines expressing antibody molecules. Such engineered cell lines may be particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

Many screening systems can be used, but are not limited to herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.Acad. Sci. USA 48: 202 (1992) and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) genes can be used in tk-, hgprt- or aprt-cells, respectively. In addition, anti-metabolic resistance can be used based on selection for the following genes: dhfr (Wigler et al., Natl. Acad. Sci. USA 77: 357 (198), which provides resistance to methotrexate. O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981); gpt providing resistance to mycophenolic acid (Mulligan & Berg. Proc. Natl. Acad. Sci. USA 78: 2072 (1981)); neo which provides resistance to aminoglycoside G-418 (Clinical Pharmacy 12: 488-505; Wu and Wu, Bio therapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. 62: 191-217 (1993); May, 1993, TIB TECH 11 (5): 155-215) and hygro, which provided resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Methods commonly known in the art of recombinant DNA technology can be routinely applied to screen for recombinant clones of interest, such as, for example, Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981). The entire contents of each of these documents are incorporated herein by reference.

The expression level of the antibody molecule can be increased by vector amplification (see, for example, Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, vol. 3. (Academic Press) , New York, 1987). When a marker is amplifiable in a vector system expressing an antibody, increasing levels of inhibitors present in the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).

Host cells can be co-transfected with two expression vectors of the invention (a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide). Both vectors may contain the same selection markers that allow for the same expression of heavy and light chain polypeptides. As another alternative, a single vector can be used that encodes and expresses both heavy and light chain polypeptides. In this case, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chains (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77: 2197 (1980)). The coding sequences of the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, purification of immunoglobulin molecules by methods known in the art, for example by chromatography (eg, ion exchange, Affinity, in particular by affinity for a particular antigen according to Protein A, and by size column chromatography), centrifugation, differential solubility or other standard techniques for purification of proteins. In addition, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

The present invention provides that the antibody is recombinantly fused to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids) of the polypeptide of the invention, or Fusion proteins formed by chemical conjugation. The fusion does not necessarily need to be direct, but can be via the linker sequence. The antibody may be specific for an antigen other than the polypeptide of the invention (or a portion thereof, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more amino acids) of the polypeptide. . For example, by fusion or conjugation of a polypeptide of the invention to an antibody specific for a particular cell surface receptor, the polypeptide of the invention may target a particular cell type in vitro or in vivo. Antibodies fused or conjugated to the polypeptides of the present invention can also be used in in vitro immunoassay and purification methods according to methods known in the art (see, eg, Harbor et al .; WO 93/21232; EP439,095). Naramura et al., Immunol. Lett. 39: 91-99 (1994); US Pat. No. 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol.146 2446-2452 (1991). The entire contents of each of these documents are incorporated herein by reference.

The invention also encompasses compositions comprising polypeptides of the invention fused or conjugated to antibody domains other than the variable region. For example, a polypeptide of the invention can be fused or conjugated to an antibody Fc region or portion thereof. The antibody portion fused to a polypeptide of the invention may comprise any combination of constant regions, hinge regions, CH1 domains, CH2 domains and CH3 domains or complete domains or portions thereof. In addition, the polypeptide may be fused or bound to a portion of the antibody to form a multimer. For example, an Fc moiety fused to a polypeptide of the invention can form a dimer via disulfide bonds between the Fc moieties. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating polypeptides of the invention to antibody moieties are known in the art (see, eg, US Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946). ; EP 307,434; EP 367,166; WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol.154 : 5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89: 11337-11341 (1992). The entire contents of each of these documents are incorporated herein by reference.

As discussed above, a polypeptide corresponding to a polypeptide, polypeptide fragment or variant of SEQ ID NO: 2 is fused or conjugated to a portion of the antibody to increase the in vivo half-life of the polypeptide or using methods known in the art. Can be used for immunoassay. In addition, a polypeptide corresponding to SEQ ID NO: 2 may be fused or conjugated to the antibody portion to facilitate purification. In addition, as discussed above, a polypeptide corresponding to a polypeptide, polypeptide fragment or variant of SEQ ID NO: 19 may be fused or conjugated to a portion of the antibody to increase the in vivo half-life of the polypeptide or known in the art. Can be used for immunoassay. In addition, a polypeptide corresponding to SEQ ID NO: 19 may be fused or conjugated to the antibody portion to facilitate purification. As an example reported, a chimeric protein consisting of the first two domains of a human CD4-polypeptide and several domains of the constant region of the heavy or light chain of a mammalian immunoglobulin is described (EP 394,827; Traunecker et al., Nature 331: 84-86 (1988)). Polypeptides of the invention fused or conjugated to an antibody having a disulfide-conjugated dimer structure (due to IgG) may also be more effective at binding and neutralizing monomeric secreted proteins or molecules other than fragments thereof (Fountoulakis et. al., J. Biochem. 270: 3958-3964 (1995)). In many cases, the Fc portion in the fusion protein can be beneficial for treatment and diagnosis and as a result can provide, for example, improved pharmacokinetics (EP A 232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein is expressed, deleted and purified. For example, the Fc moiety can inhibit treatment and diagnosis when the fusion protein is used as an antigen for immunization. In drug development, antagonists of hIL-5 have been identified by fusing human proteins such as, for example, hIL-5 with the Fc moiety for purposes of high throughput screening assays (see Bennett et al., J. Molecular). Recognition 8: 52-58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

 In addition, the antibodies or fragments thereof of the invention can be fused with marker sequences such as peptides to facilitate purification. In a preferred embodiment, the marker amino acid sequence is exemplified by a hexa-histidine peptide such as a tag provided by the pQE vector (QIAGEN, Inc., 91311 Chasward Eaton Avenue 9259, California) among many commercially available. See, eg, Gentz et al., Proc. Natl. Acad. Sci. As described in USA 86: 821-824 (1984), hexa-histidine provides convenient purification of fusion proteins. Other peptide tags useful for purification include, but are not limited to, "HA" tags (Wilson et al., Cell 37: 767 (1984)) and "flag" tags corresponding to epitopes derived from influenza hemagglutinin protein. Included.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical trial procedure to determine the efficacy of, for example, the treatment method being practiced. The antibody can be conjugated to a detectable substance to facilitate detection. Examples of detectable materials include various enzymes, prosthetic molecules, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography and nonradioactive paramagnetic metal ions. A detectable substance can be conjugated or bound indirectly through an intermediate (eg, a linker known in the art) or directly to an antibody (or fragment thereof) using techniques known in the art. See, for example, US Pat. No. 4,741,900 for metal ions that can be antibody bound for use as a diagnostic agent in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase. Examples of suitable submolecular complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, monosyl chloride or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin and aequorin. Examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc.

In addition, the antibody or fragment thereof may be conjugated to a therapeutic moiety, such as a cytotoxin (eg, cytostatic or apoptosis), to a therapeutic or radioactive metal ion (eg, an alpha-radiator such as ²¹³ Bi). Cytotoxins or cytotoxic agents include all substances harmful to cells. Examples include paclitaxol, cytosalacin B, gramicidine D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydrate Oxy anthracin dione, mitoxanthrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and furomycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolic products (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, ThioEpa chlorambucil, melphalan, carmustine (BSNU) and romustine (CCNU), cyclotosamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum ( II) (DDP) cisplatin), anthracycline (e.g. daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly called actinomycin), bleomycin, Mithramycin and Anthramycin (AMC) and antimitotic agents such as vincristine and vinblastine.

It is to be understood that the conjugates of the present invention can be used to alter a given biological response and the therapeutic or drug moiety is limited to conventional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Examples of such proteins include toxins such as abrin, lysine A, Pseudomonas exotoxin or diphtheria toxin; Tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet induced growth factor, tissue plasminogen activator or apoptosis agent (e.g. TNF-alpha, TNF-beta, AIM (see WO 97/33899) ), AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6: 1567-1574 (1994)), VEGI (WO 99/23105), CD40 ligand, thrombicides Or antiangiogenic agents (eg, angiostatin or endostatin); or biological response modifiers, such as lipocaine, intorkin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL- 6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors.

The antibody may also be conjugated to a solid support which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moieties to antibodies are well known (see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623- 53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (Eds.), Pp. 475- 506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Raidolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982)).

Alternatively, the antibody can be conjugated to a second antibody to form antibody heterozygotes, as described in Segal US Pat. No. 4,676,980. The entire contents of these patents are incorporated herein by reference.

Antibodies can be used as therapeutic agents either alone or in combination with a therapeutic moiety conjugated thereto or in combination with cytotoxic factor (s) and / or cytokine (s).

Immunophenotyping

Antibodies of the invention can be used for immunophenotyping of cell lines and biological samples. The translation products of the genes according to the invention may be useful as cell specific markers and more particularly as cell markers that are differentially expressed at different stages of differentiation and / or maturation of a particular cell type. Monoclonal antibodies directed against specific epitopes or combinations of epitopes allow for the selection of cell populations expressing the markers. Various techniques using monoclonal antibodies can be used for the selection of cell populations expressing markers, including magnetic separation using antibody-coated magnetic beads, "panning" with antibodies adhered to a solid matrix (ie, plate). and fluoro cytometry (see, eg, US Pat. No. 5,985,660 and Morrison et al., Cell. 96: 737-49 (1999)).

These techniques are “non-self” in transplantation for the prevention of cell and graft-versus-host disease (GVHD) as can be seen as hematologic malignancy (ie minimal residual disease (MRD) in patients with acute leukemia). Allows selection of specific groups of cells. Alternatively, these techniques allow the selection of hematopoietic stem and progenitor cells that may undergo proliferation and / or differentiation as seen in human umbilical cord cells.

Assay of Antibody Binding

Antibodies of the invention can be assayed for immunospecific binding by methods known in the art. Immunoassays that can be used include, but are not limited to, Western blotting, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel dispersion precipitation reactions, immunodispersion Assays, hemagglutination assays, complement-fix assays, immunoradiometric assays, fluorescent immunoassays, Protein A immunoassays, and the like. Such assays are conventional and well known in the art (see, for example, Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The entire contents of this document are incorporated herein by reference. Illustrative immunoassays are briefly described below but are not intended to limit the invention.

Immunoprecipitation protocols generally describe a population of cells in RIPA buffer (1% NP-40 or Triton X-100, 1% sodium) supplemented with protein phosphatase and / or protease inhibitors (e.g., EDTA, PMSF, Aprotinin, Sodium Vanadate). In oxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate (pH 7.2), 1% trasylol), add the antibody to the cell lysate, and then at 4 ° C. for a period of time (eg, 1 to 1). 4 hours), add Protein A and / or Protein G Sepharose beads to the cell lysate, incubate at 4 ° C. for at least about 1 hour, wash the beads in lysis buffer, and then wash the beads in SDS / Resuspension in sample buffer. The ability of the antibody to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art are well aware of variables that can be altered to increase the binding of the antibody to the antigen and to reduce the background (eg, preliminary removal of cell lysate with Sepharose beads). For details on immunoprecipitation protocols, see Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blotting assays generally prepare protein samples, electrophores the protein samples on a polyacrylamide gel (eg, 8% to 20% SDS-PAGE depending on the molecular weight of the antigen), and sample is nitro from the polyacrylamide gel. Transfer to a membrane such as cellulose, PVDF or nylon, block the membrane in blocking solution (e.g. PBS + 3% BSA or fat free milk), wash the membrane in wash buffer (e.g. PBS-Tween 20) and wash the membrane in blocking buffer Block with diluted primary antibody (the antibody in question), wash the membrane in wash buffer, and wash the membrane in an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase) or radioactive molecule (eg ³² P or ¹²⁵ ) diluted in blocking buffer Blocking the primary antibody (eg, anti-human antibody) bound to I) with a secondary antibody, washing the membrane in wash buffer and detecting the presence of antigen. Those skilled in the art are familiar with the parameters that can be altered to increase the detected signal and reduce background noise. Further details on western blotting can be found in Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISA prepares the antigen, coats the wells of the 96 well microtiter plate with the antigen, adds the antibody of interest to a detectable compound such as an enzyme substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well and Culturing for a period of time and detecting the presence of antigen. In ELISA the antibody does not need to be bound to a detectable antibody. Instead, secondary antibodies (recognizing the antibody) bound to the detectable compound can be added to the wells. In addition, instead of coating the wells with the antigen, the antibodies may be coated with the wells. In this case, the secondary antibody bound to the detectable compound can be added after adding the antigen of interest to the coated well. Those skilled in the art are familiar with variables that can be altered to increase the detected signal, as well as other variables of ELISA known in the art. Further details on ELISA can be found in Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of the antibody to the antigen and the dissociation rate of the antigen-antibody interaction can be measured by competitive binding assays. One example of a competitive binding assay is radioimmunoassay comprising culturing a labeled antigen (eg ³ H or ¹²⁵ I) with the antibody in the presence of an increased amount of unlabeled antigen and detecting the antibody bound to the labeled antigen. to be. From this data the affinity and binding dissociation rate of the antibody for a particular antigen can be determined by the Scatchard float analysis. Competition with secondary antibodies can also be measured using radioimmunoassays. In this case, the antigen is cultured in the presence of the corresponding antibody bound to the labeled compound (eg, ³ H or ¹²⁵ I) and an increased amount of unlabeled secondary antibody.

Therapeutic uses

The invention also relates to an antibody-use regimen comprising administering an antibody of the invention to an animal, preferably a mammal, most preferably a human patient, for the treatment of one or more of the diseases, disorders or conditions described. Therapeutic compounds of the invention include, but are not limited to, the antibodies of the invention (including fragments, analogs, and derivatives thereof as described herein) and the nucleic acids encoding the antibodies of the invention (such as described herein) Fragments, analogs and derivatives and anti-idiotypic antibodies). Antibodies of the invention can be used to treat, inhibit or prevent a disease, disorder or condition associated with aberrant expression and / or activity of a polypeptide of the invention, including one or more of the diseases, disorders or conditions described herein. Examples of autoimmune diseases, diseases or conditions associated with the disease or disorder include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune thrombocytopenia, hemolytic anemia, antiphospholipid syndrome , Dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatoid heart disease, glomerulonephritis (eg IgA nephropathy), multiple sclerosis, neuritis, uveitis keratoconjunctivitis, polygrine dysplasia, purpura (e.g. ), Radar disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barr syndrome, insulin-dependent diabetes mellitus and autoimmune inflammation, autoimmune thyroiditis, hypothyroidism (ie Hashimoto thyroiditis, systemic lupus erythematosus, Goodpasture syndrome) , Swelling, receptor autoimmunity (eg, (a) Grave's disease, (b) myasthenia gravis and (c) insulin resistance), autologous Immune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis / dermatitis, pernicious anemia, idiopathic Edison disease, infertility, glomerulonephritis (e.g. Primary glomerulonephritis and IgA nephropathy), lactose swelling, Sjogren's syndrome, diabetes and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, endocrine disorders, Vitiligo, vasculitis, post-MI, cardiac incision syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy and other inflammatory, granulomatous, degenerative and atrophic diseases.

In certain embodiments, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another particular embodiment, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosus.

Treatment and / or prevention of a disease, disorder or condition associated with aberrant expression and / or activity of a polypeptide of the invention includes, but is not limited to alleviating the symptoms associated with their disease, disorder or condition. In addition, the antibodies of the present invention can be used to target and kill cells expressing Neutrokine-alpha at the surface and / or cells with Neutrokine-alpha bound to the surface. Antibodies of the invention may be provided in a pharmaceutically acceptable composition as known in the art or as described in the art.

As a summary of the therapeutic use of the antibodies of the present invention, the polynucleotides or polypeptides of the present invention can be bound locally or systemically in the body or for example complement (CDC) or effector cells ( Binding by direct cytotoxicity of the antibody as mediated by ADCC). Some of these methods are described in more detail below. Those skilled in the art according to the teachings provided herein will be well aware of how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without heavy experimental burden.

Antibodies of the invention can be combined with other monoclonal or chimeric antibodies or hematopoietic growth factors (e.g., IL-2, IL-3 and IL-7) that act to increase the number or activity of effector cells that interact with the antibody. Or in combination with lymphokine.

Antibodies of the invention may be administered alone or in combination with other types of treatment (eg, radiation therapy, chemotherapy, hormone therapy, immunotherapy, anti-tumor agents, antibiotics and immunoglobulins). In general, it is desirable to administer the same species lineage or product of species reactivity (in the case of antibodies) as that of the patient. Thus, in a preferred embodiment, human antibodies, fragments, derivatives, analogs or nucleic acids are administered to a human patient for treatment or prophylaxis.

High affinity for the polypeptides or polynucleotides, fragments or regions thereof of the invention for both immunoassays for polynucleotides or polypeptides of the invention (including fragments thereof) and for the treatment of diseases associated with them and / or Or in vivo inhibition of efficacy and / or neutralizing antibodies. Such antibodies, fragments or regions are preferably affinity for the polynucleotides or polypeptides of the invention, including fragments. Preferred binding affinities include 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 , 10 ^-6 , 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M, 10 ^-10 M, 5 x 10 ^-11 M, 10 ^-11 M, 5 x 10 ^-12 M, 10 ^-12 M, And those having a dissociation constant or Kd of less than 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M and 10 ^-15 M.

Gene therapy

In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is administered to treat, inhibit or prevent a disease or condition associated with aberrant expression and / or activity of a polypeptide of the invention via gene therapy. . Gene therapy refers to a therapy performed by administering an expressed or expressable nucleic acid to a patient. In this aspect of the invention, the nucleic acids produce their encoded proteins that mediate a therapeutic effect.

Any method of gene therapy used in the art may be used in accordance with the present invention. An exemplary method is described below.

Regarding gene therapy, see generally Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); And Morgan and Anderson, Ann, Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Methods that can be used as commonly known in the field of recombinant DNA technology are described in Ausubel et al., (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); And Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprises a nucleic acid sequence encoding an antibody and the nucleic acid sequence is part of an expression vector expressing the antibody or fragment thereof or chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences are operably linked to an antibody coding region and the promoters are inducible or constitutive and optionally tissue-specific. In another specific embodiment, nucleic acid molecules are used in which the antibody coding sequence and other desired sequences are flanked by regions that promote homologous recombination at desired positions in the genome, resulting in intrachromosomal expression of the antibody encoding nucleic acid ( Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989). In certain embodiments, the expressed antibody molecule is a single chain antibody and, alternatively, the nucleic acid sequence comprises a sequence that encodes both the chain and the light chain of the antibody or fragments thereof.

Delivery of the nucleic acid into the patient can be direct, in which case the patient is exposed directly to the nucleic acid or nucleic acid-containing vector. In addition, such delivery may be indirect, in which case the cells are first transformed with nucleic acid in vitro and then transplanted into the patient. These two methods are known as in vivo or ex vivo gene therapy, respectively.

In certain embodiments, the nucleic acid sequence is administered directly in vivo and the nucleic acid is expressed in vivo to produce a product that is encoded. This is accomplished by a number of methods known in the art, for example, by constructing a nucleic acid sequence as part of an appropriate nucleic acid expression vector and by expressing a deleted or attenuated retrovirus or other viral vector (see US Pat. No. 4,980,286). Or by direct injection of the extracted DNA or by use of microparticle bombardment (e.g., gene gun; Biolistic, Dupont) to allow nucleic acid sequences to be present in the cells, thereby coating them with lipid or cell-surface receptors or transfection agents Receptor-mediated endocytosis by encapsulation in liposomes, microparticles or microcapsules or by linking nucleic acid sequences to peptides known to enter the nucleus (see, eg, Wu and Wu, J. Biol. Chem. 262: 4429). -4432 (1987) (this method can be used to target cell types that specifically express receptors) In another embodiment, the ligand may comprise a soluble viral peptide to disrupt the endosome, thereby forming a nucleic acid-ligand complex that does not degrade lysosomal degradation. In nucleic acids, nucleic acids can be targeted in vivo for cell specific uptake and expression by targeting specific receptors (see, eg, WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188 and WO 93 /). Alternatively, nucleic acids can be introduced into cells by homologous recombination and incorporated for expression in host cell DNA (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989). Zijlstra et al., Nature 342: 435-438 (1989)).

In certain embodiments, viral vectors containing nucleic acid sequences encoding antibodies of the invention are used. For example, retroviral vectors can be used (Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and the integration of the host cell DNA. Nucleic acid sequences encoding antibodies for use in gene therapy are cloned into one or more vectors, which facilitate the delivery of the gene into the patient. More details on retroviral vectors can be found in Boesen et al., Biotherapy 6: 291-302 (1994), where mdr1 is used to make hematopoietic stem cells more resistant to chemotherapy. The use of retroviral vectors to deliver genes to hematopoietic stem cells is described. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93: 644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); And Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly suitable vehicles for delivering residues to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium and cause mild disease here. Other targets of the adenovirus-utilized delivery system are the liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of infecting undivided cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3: 499-503 (1993) review adenovirus-utilized gene therapy. Bout et al., Human Gene Therapy 5: 3-10 (1994) demonstrated the transfer of genes to the respiratory epithelium of rhesus monkeys using adenovirus vectors. Other examples of using adenoviruses in gene therapy are described in Rosenfeld et al., Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993); WO 94/12649; And Wang, et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment adenovirus vectors are used.

In addition, the use of adeno-associated viruses (AAV) in gene therapy has been proposed (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993); US Pat. No. 5,436,146).

Other methods of gene therapy include delivering genes to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection or viral infection. Usually, the method of delivery involves the delivery of a selection marker to the cell. Subsequently, the cells are subjected to selection to isolate cells expressing the expressed gene. The isolated cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introductions include but are not limited to transfection, electroporation, microinjection, infection with viral or bacteriophage vectors containing nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, It may be carried out by a method known in the art including spheroplast fusion and the like. Many techniques for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993); Cohen et al., Meth. Enzymol. 217: 618-). 644 (1993); Clin., Pharmac. Ther. 29: 69-92m (1995)), can be used according to the present invention as long as the necessary development and physiological functions of the recipient cells are not destroyed. This technique should provide for stable delivery of the nucleic acid to the cell so that the nucleic acid can be expressed by the cell and is preferably inherited and expressed in the cell progeny.

The resulting recombinant cells can be delivered to the patient by various methods known in the art. Recombinant blood cells (eg hematopoietic stem or progenitor cells) are preferably administered intravenously. The planned amount of cells used depends on the desired effect, the condition of the patient and the like and can be measured by one skilled in the art.

Cells into which nucleic acids can be introduced for gene therapy purposes include, but are not limited to, all available cell types, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (eg , T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes), bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc., including several stem or progenitor cells, in particular hematopoietic stem or progenitor cells do.

In a preferred embodiment, the cells used for gene therapy are autograft to the patient.

In embodiments in which the synthesized cells are used for gene therapy, the nucleic acid sequences encoding the antibodies are introduced into the cells so that they can be expressed by the cells or their progeny and then the recombinant cells are administered in vivo for the therapeutic effect. In certain embodiments, stem or progenitor cells are used. Any stem and / or progenitor cells that can be isolated and maintained can potentially be used according to aspects of the invention (see, eg, PCT Publication WO 94/08598; Stemple and Anderson, Cell 71: 973-985 (1992) Rheinwald, Meth. Cell Bio. 21A: 229 (1980); and Pittelkow and Scott. Mayo Clinic Proc. 61: 771 (1986)).

In certain embodiments, the nucleic acid to be administered for the purpose of gene therapy includes an inducible promoter operably linked to a coding region so that expression of the nucleic acid can be regulated by controlling the presence or absence of appropriate inducers of transcription.

Demonstration of therapeutic and prophylactic activity

The compounds or pharmaceutical compositions according to the invention are preferably tested in vitro and then in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic use of a compound or pharmaceutical composition include the effect of the compound on a cell line or patient tissue sample. The effect of a compound or composition on cell lines and / or tissue samples can be measured using techniques known to those of skill in the art, including but not limited to rosette formation assays and cell lysis assays. In accordance with the present invention, in vitro assays that can be used to determine whether administration of a particular compound are indicated include the growth of a patient tissue sample in culture and exposure to the compound or administration of the compound and the effect of the compound on the tissue sample. Observed in vitro cell culture assays are included.

Therapeutic and / or Prophylactic Administration and Compositions

The present invention provides methods of treating, inhibiting and preventing by administering to a patient an effective amount of a compound or pharmaceutical composition according to the invention, preferably an antibody of the invention. In a preferred embodiment, the compound is substantially purified (eg, substantially freed from substances that limit the effect of the compound or cause undesirable side effects). Patients include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs, and the like, preferably animals and are also preferably mammals and most preferably humans.

Formulations and methods of administration that can be used when the compounds comprise nucleic acids or immunoglobulins are described above. Additional suitable formulations and routes of administration may be selected from those described below.

Various known delivery systems can be used to administer the compounds of the invention, including, for example, liposomes, microparticles, microcapsule encapsulation, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)), construction of nucleic acids as part of retroviruses or other vectors. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The compound or composition may be administered by conventional routes, for example by infusion or bolus injection, by absorption through the epithelial or mucosal system (eg oral mucosa, rectal mucosa and intestinal mucosa, etc.) and other biologically active agents. It can be administered together with. Administration can be systemic or local. It may also be desirable to administer the pharmaceutical compounds or compositions of the invention into the central nervous system by suitable routes including intraventricular and intradural injection. Intraventricular injection can be facilitated by an intraventricular catheter attached to a reservoir such as, for example, an Omaya reservoir. Pulmonary administration can also be employed using, for example, formulations with inhalers or nebulizers and nebulizers.

In certain embodiments, it may be desirable to administer the pharmaceutical compound or composition of the invention locally to the site in need of treatment. This includes, but is not limited to, for example, topical infusion during surgery, topical application (eg, in combination with postoperative wound dressings), injection, catheter means, suppository means, or implant means, where the implant is a sialic A porous, nonporous or gelatinous material comprising a membrane such as a plastic membrane or a fiber). Care should be taken to use materials which do not absorb the protein, preferably when administering a protein of the invention comprising an antibody.

In another embodiment, the compound or composition can be delivered in a vehicle, in particular liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez) -Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, pp. 317-327).

In another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al) , N. Engl. J. Med. 321: 574 (1989). In another embodiment, polymeric materials may be used (Medical Applicaion of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 ( 1985); During et al., Ann. Neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105 (1989). In another embodiment, a controlled release system can be set up near the brain that requires only a therapeutic target, ie, a portion of a systemic dose (see, eg, Goodson, in Medical Applications of Controlled Release, vol. 2, pp. 115-). 138 (1984)).

Other controlled release systems are described in Langer, Science 249: 1527-1533 (1990).

In certain embodiments where a compound of the invention is a nucleic acid encoding a protein, the nucleic acid constructs the nucleic acid sequence as part of an appropriate nucleic acid expression vector and promotes the expression vector by a retroviral vector to facilitate expression of its encoded protein. US Pat. Coating or nucleic acid sequences can be administered in conjunction with homobox-like peptides known to enter the nucleus. See Joliot et al., Proc. Natl. Acad. Sci. USA 88: 1864-1868 (1991). Alternatively, nucleic acids can be introduced into cells by homologous recombination for expression and incorporated into host cell DNA.

The present invention also provides pharmaceutical compositions. Such compositions include a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means that it can be used in animals, especially humans, and is listed in a US or other generally approved pharmacopoeia that has been approved by federal or state officials. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids and examples thereof include vegetable, synthetic or animal oils (including petroleum oil) and water such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, in particular injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, whistle, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene glycol, water, Ethanol and the like. The composition may also contain small amounts of wetting agents, emulsifiers or pH adjusters if necessary. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, fatty agents, and the like. The composition may be formulated as a suppository with traditional binders and carriers (eg, triglycerides). Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are described in E.W. Martin, "Remington's Pharmaceutical Sciences". Such compositions preferably contain the purified form of the compound in a therapeutically effective amount with a suitable amount of carrier so as to provide the patient with a form for proper administration. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also contain a solubilizer and a local anesthetic such as lignocaine to relieve pain at the injection site. Generally, the ingredients are mixed together or supplied separately in unit dosage form as anhydrous concentrates or anhydrous lyophilized powders in sealed containers such as, for example, sashes or ampoules indicating the amount of active agent. When the composition is administered by infusion, the composition may be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided so that the components may be mixed prior to administration.

The compounds of the present invention may be prepared in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like and sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 Salts formed with cations such as salts derived from ethylamino ethanol, histidine, procaine and the like.

The amount of a compound according to the invention effective for the treatment, inhibition and prevention of a disease or condition associated with aberrant expression and / or activity of a polypeptide of the invention can be measured by standard clinical techniques. In vitro assays may also optionally be used to assist in determining the optimal dose range. The exact dose to be used in the formulation also depends on the route of administration and the severity of the disease or disorder and should be determined taking into account the physician's judgment and the condition of each patient. Effective doses can be determined from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dose administered to a patient is typically 0.1 mg to 100 mg per kg body weight of the patient. Preferably, the dose administered to the patient is 0.1 mg to 20 mg, more preferably 1 mg to 10 mg per kg body weight of the patient. In general, human antibodies show a longer half-life in the human body than antibodies from other species due to responses to foreign polypeptides. Thus, lower doses and less frequent administrations of human antibodies are often possible. In addition, the dosage and frequency of administration according to the invention can be reduced by enhancing the uptake and tissue penetration (eg, into the brain) of the antibody, for example by modifications such as lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition according to the invention. Such containers may optionally be accompanied by a notice stipulated by the governmental authority that controls the manufacture, use or sale of pharmaceuticals or biological products, which reflects the government's approval for the manufacture, use or sale for administration to humans. do.

Diagnostics and Imaging

Labeled antibodies and derivatives and analogs thereof that specifically bind to the polypeptide of interest may be used for diagnostic purposes for detecting, diagnosing or monitoring a disease and / or disorder associated with aberrant expression and / or activity of the polypeptide according to the invention. Can be. The present invention provides for (a) assaying the expression of a polypeptide in an individual cell or body fluid using one or more antibodies specific for that polypeptide, and (b) comparing the level of gene expression with a standard gene expression level. It provides a method for detecting aberrant expression of the polypeptide. Here, an increase or decrease in the polypeptide gene expression level assayed compared to the standard expression level is indicative of aberrant expression.

The present invention provides for (a) assaying the expression of a polypeptide in an individual cell or body fluid using one or more antibodies specific for that polypeptide, and (b) comparing the level of gene expression with a standard gene expression level. And diagnostic assay methods for diagnosing a disease. Here, an increase or decrease in assayed polypeptide gene expression levels compared to standard expression levels is indicative of a particular disease. With respect to cancer, the presence of a relatively high amount of transcript in the biopsy tissue from an individual may be indicative of a precursor to the onset of the disease or may provide a means of detecting the disease prior to the appearance of substantial clinical symptoms. This type of more accurate diagnosis can help doctors prevent the development or continued progress of cancer by implementing preventive or aggressive treatment earlier.

Antibodies of the invention can be used to assay protein levels in biological samples using conventional immunohistochemical methods known to those skilled in the art (see, eg, Jalkanen, et al., J. Cell Biol. 101: 976-985 ( 1985); Jalkanen, et al., J. Cell. Biol. 105: 3087-3096 (1987)). Other antibody-use methods useful for detecting protein gene expression include immunoassays such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; Iodo ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ In) and Technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm Radioactive isotopes such as, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Rh; Luminescent labels such as luminol; And fluorescent labels such as fluorescein and rhodamine and biotin.

Techniques known in the art can be used to label the antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional binders (see, for example, US Pat. The entire contents of each of these patents are incorporated herein by reference.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of the polypeptide of interest in an animal, preferably a mammal, most preferably a human. In one embodiment, the diagnosis comprises (a) administering to a patient an effective amount of a labeled molecule that specifically binds the polypeptide of interest (eg, parenteral, subcutaneous or intraperitoneal), and (b) Wait for a period of time for the polypeptide to be preferentially concentrated (and unbound labeled molecules are removed to background levels), (c) measuring background levels, and (d) labeled molecules from patients And detecting. Here, detection of molecules labeled above the background level indicates that the patient has a particular disease or condition associated with aberrant expression of the polypeptide. Background levels can be measured by several methods, including comparing the amount of labeled molecule detected with a standard value previously measured for a particular system. As described herein, certain embodiments of the invention relate to quantifying or qualifying the concentration of cells of a B cell lineage or cells of a mononuclear lineage using an antibody of the invention.

As described herein, antibodies of the invention can be used to treat, diagnose, or prognose patients with immunodeficiency. Antibodies of the invention are used to treat, diagnose, and / or prognostic common variable immunodeficiency disease (CVID) or sub-diseases thereof. In certain embodiments, the antibodies of the invention are used to diagnose, prognose, treat or prevent a disease characterized by defective serum immunoglobulin production, recurrent infections and / or immune system disorders. As described herein, the antibodies of the invention are used to treat, diagnose, or prognose an individual with an autoimmune disease or condition. In certain embodiments, the antibodies of the present invention are used to treat, diagnose, and / or prognose individuals with systemic lupus erythematosus or sub-diseases thereof. In another specific embodiment, the antibodies of the present invention are used to diagnose, prognose, treat or prevent a patient suffering from rheumatoid arthritis or a subsidiary disease thereof.

It will be understood in the art that the size of the patient and the imaging system used measure the amount of imaging residues needed to produce a diagnostic image. In the case of radioisotope residues, the amount of radioactivity injected into humans is normally in the range ⁹⁹ mTc in the range of about 5-20 millicuries. The labeled antibody or antibody fragment will then preferentially accumulate at the location of the cell containing the particular protein. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982). )].

Depending on several variables, including the type of label and the mode of administration, the time interval after which the labeled molecules are preferentially concentrated at the site within the patient and the unbound labeled molecules are removed to the background level may be between 6 and 48 hours or 6 to 24 hours. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, the monitoring of the disease or condition is carried out by repeating the method of diagnosing the disease or condition, e.g., one month after the first diagnosis, six months after the first diagnosis, one year after the first diagnosis, or the like.

The presence of labeled molecules can be detected in the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and equipment that can be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scanning (eg, material radiography), magnetic resonance imaging (MRI), and ultrasound measurements.

In certain embodiments, molecules are labeled with radioisotopes and detected in patients using radioactive surgical instructions (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using fluorescence reactive scanning instructions. In another embodiment, the molecule is labeled with a positron emitting metal and detected using positron emission tomography. In another embodiment, the molecules are labeled with paramagnetic labels and detected in the patient using magnetic resonance imaging (MRI).

Kit

The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In certain embodiments, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope that specifically immunoreacts with an antibody contained in the kit. Preferably the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another particular embodiment, the kits of the invention comprise two or more antibodies (monoclonal and / or polyclonal) which recognize the same and / or different sequences or regions of the polypeptides according to the invention. In another specific embodiment, the kits of the invention contain a means for detecting binding of the antibody to the polypeptide of interest (e.g., the antibody is directed to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound). A second antibody that recognizes a first antibody that can be bound or bound to a detectable substrate).

In another particular aspect of the invention, the kit is a diagnostic kit for use in selecting serum containing antibodies specific for proliferative and / or carcinogenic polynucleotides and polypeptides. Such kits may include control antibodies that do not react with the polypeptide of interest. Such kits may comprise substantially isolated polypeptide antigens containing epitopes that specifically react with one or more anti-polypeptide antigen antibodies. In addition, such kits include means for detecting binding of the antibody to the antigen (e.g., the antibody may be linked to a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). . In certain embodiments, the kit may comprise a recombinantly generated or chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be linked to a solid support.

In a more particular embodiment, the detection means of the kit described above comprises a solid support to which the polypeptide antigen is linked. Such kits may also include non-linked reporter-labeled anti-human antibodies. In this embodiment, binding of the antigen to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further aspect, the invention comprises a diagnostic kit for use in selecting serum containing antigens of the polypeptides according to the invention. The diagnostic kit includes substantially isolated antibodies that specifically immunoreact with the polypeptide or polynucleotide and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is bound to a solid support. In certain embodiments, the antibody may be a monoclonal antibody. The detection means of the kit comprises a second labeled monoclonal antibody. As an alternative or in addition, the means for detection may comprise a labeled competitive antigen.

In one diagnostic configuration, the test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the method of the present invention. After binding specific antigen antibodies to the reagent and washing to remove unbound serum components, the reagent reacts with the reporter-labeled anti-human antibody so that the reporter binds to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. . The reagent is washed again to remove unbound labeled antibody and to determine the amount of reporter associated with the reagent. Typically, reporters are enzymes that detect by incubating the solid phase in the presence of a suitable fluorometric, luminescent or chromatographic substrate (Sigma, St. Louis, MO).

Solid surface reagents in this assay are prepared by known techniques for attaching proteins to solid support materials (e.g. polymeric beads), dip sticks, 96-well plates or filter materials. These methods of attachment generally involve covalent linkage of proteins to chemically reactive groups (eg, activated carboxyl, hydroxyl or aldehyde groups) on a solid support via nonspecific adsorption of the protein to the support or typically via free amine groups. As another alternative, streptavidin coated plates can be used in combination with biotinylated antigen.

Accordingly, the present invention provides assay systems or kits for carrying out such diagnostic methods. Kits generally include a support having a surface-bound recombinant antigen and a reporter-labeled anti-human antibody for detecting the surface-bound anti-antigen antibody.

The invention also relates to antibodies which act as agonists or antagonists of the polypeptides according to the invention. For example, the present invention includes antibodies that partially or wholly disrupt receptor / ligand interactions with the polypeptides of the present invention. Both receptor-specific and ligand-specific antibodies are included. Receptor-specific antibodies that do not inhibit ligand binding but inhibit receptor activation are included. Receptor activation (ie, signaling) can be measured by the techniques described herein or by techniques known in the art. Also included are receptor-specific antibodies that inhibit both ligand binding and receptor activation. Likewise, neutralizing antibodies that bind to the ligand and inhibit the binding of the ligand to the receptor are included as well as antibodies that bind to the ligand and thereby inhibit receptor activation but inhibit the ligand from binding to the receptor. Also included are antibodies that activate the receptor. These antibodies can act as agonists for all or part of the biological activity affected by ligand-mediated receptor activation. Antibodies may be specified as agonists or antagonists for biological activity, including the specific activities described herein. In addition, antibodies that bind Neutrokine-alpha and / or Neutrokine-alphaSV are included regardless of whether Neutrokine-alpha or Neutrokine-alphaSV binds to the Neutrokine-alpha receptor. These antibodies act as Neutrokine-alpha and / or Neutrokine-alphaSV agonists as indicated by an increase in cell proliferation in response to binding of Neutrokine-alpha and / or Neutrokine-alphaSV in the presence of these antibodies. do. Such antibody agonists can be prepared using methods known in the art. See, eg, PCT Publication WO 96/40281; U.S. Patent 5,811,097; Deng, B. et al., Blood 92 (6): 1981-1988 (1998); Chen, Z, et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop, J.A. et al., J. Immunol. 161 (4): 1786-1794 (19998); Zhu. Z. et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon, D, Y. et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat. M. et al., J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard. V. et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard, j. et al., Cytokinde 9 (4): 233-241 (1997), Carlson, N.G. et al., J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman, R. E. et al., Neuron 14 (4): 755-762 (1995); Muller, Y.A. et al., Structure 6 (9): 1153-1167 (199); Bartunek. P. et al., Cytokine 8 (1): 14-20 (1996)]. The entire contents of each of these documents are incorporated herein by reference.

More than 14 monoclonal antibodies against Neutrokine-alpha were generated. These monoclonal antibodies were named as follows: 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12, 16B4, 3D4, 16C9, 13D5, 15C10 and 12C5. Preliminary analysis of these antibodies shows that each antibody binds to Neutrokine-alpha protein in Western blot analysis and when Neutrokine-alpha protein is bound to an ELISA plate. However, further analysis of antibodies 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12 and 16B4 revealed that only antibodies 12D6, 9B6, 2E5, 10G12, 9A5 and 11G12 bind to the membrane-bound forms of Neutrokine-alpha. Appeared. Thus, a subset of monoclonal antibodies raised against Neutrokine-alpha only binds to the membrane-bound form of Neutrokine-alpha, which is primarily limited to expression in monocytes and dendritic cells as discussed herein. (Ie, this subset does not bind to the soluble form of Neutrokine-alpha corresponding to amino acids 134 to 285 of SEQ ID NO: 2).

Antibody 9B6 was found to bind specifically to the membrane-bound form of Neutrokine-alpha but not to the soluble form of Neutrokine-alpha.

Epitope mapping of antibody 9B6 showed that the antibody specifically binds to an amino acid sequence contained between amino acid residues about Ser-171 to about Phe-194 of SEQ ID NO: 2. More specifically, epitope mapping revealed that antibody 9B6 specifically binds to peptides comprising amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2.

In contrast, antibodies 16C9 and 15C10 have been shown to bind soluble forms of Neutrokine-alpha (amino acids 134 to 285 of SEQ ID NO: 2) and to inhibit Neutrokine-alpha-mediated proliferation of B cells (see Example 10 ). In addition, 15C10 antibodies have been shown to inhibit Neutrokine-alpha from binding to its receptor. Epitope maps of antibody 15C10 revealed that the antibody specifically binds to an amino acid sequence contained between amino acid residues about Glu-223 to about Tyr-246 of SEQ ID NO: 2. More specifically, epitope maps showed that antibody 15C10 specifically binds to peptides containing amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In addition, antibody 15C10 specifically binds to a peptide comprising amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2.

As described above, anti-Neutrokine-alpha monoclonal antibodies were prepared. Hybridomas producing antibodies 9B6 and 15C10 were deposited with ATCC and assigned Accession Nos. PTA-1158 and PTA-1159, respectively. In one embodiment, an antibody of the invention has one or more of the same biological characteristics as one or more antibodies secreted by a hybridoma cell line deposited with accession numbers PTA-1158 or PTA-1159. "Biological feature" refers to the in vitro or in vivo activity or properties of an antibody, for example Neutrokine-alpha (eg, the polypeptide of SEQ ID NO: 2, the mature form of Neutrokine-alpha, the membrane-bound of Neutrokine-alpha) Forms, the ability to bind soluble forms of Neutrokine-alpha (amino acids 134-285 of SEQ ID NO: 2) and the antigenic and / or epitope regions of Neutrokine-alpha, substantially neutralizing Neutrokine-alpha / Neutrokine-alpha receptor binding And the ability to block Neutrokine-alpha mediated biological activity (eg, stimulation of B cell proliferation and immunoglobulin production). Optionally, antibodies of the invention will bind to the same epitope as one or more of the antibodies specifically mentioned herein. Such epitope binding can be routinely determined using assay methods known in the art.

Thus, in one embodiment, the present invention provides antibodies that specifically bind to the membrane-bound form of Neutrokine-alpha and do not bind to the soluble form of Neutrokine-alpha. These antibodies are used as diagnostic probes for identifying and / or isolating monocyte lineages expressing, but not limited to, membrane bound forms of Neutrokine-alpha. For example, the membrane-bound form of Neutrokine-alpha is increased in activated monocytes and thus antibodies of the invention can be used to detect and / or quantify the levels of activated monocytes. In addition, antibodies that bind only to the membrane-bound form of Neutrokine-alpha may be capable of forming neoplastic, pre-carcinogenic and / or other cells (eg, monocytes and dendritic cells) that express the membrane-bound form of Neutrokine-alpha. It can be used to target toxins.

In another embodiment, the antibody of the invention specifically binds only to the soluble form of Neutrokine-alpha (amino acids 134 to 285 of SEQ ID NO: 2). These antibodies include, but are not limited to, as diagnostic probes for assaying soluble Neutrokine-alpha in biological samples and for targeting cells (eg, B cells) expressing Neutrokine-alpha receptors to toxins and / or in vitro or It has use as a therapeutic agent for reducing or blocking Neutrokine-alpha mediated biological activity (eg, stimulation of B cell proliferation and / or immunoglobulin production) in vivo.

The present invention also provides antibodies that specifically bind to the membrane-bound and soluble forms of Neutrokine-alpha.

As described above, the present invention inhibits the ability of Neutrokine-alpha and / or Neutrokine-alphaSV to bind Neutrokine-alpha receptors and / or Neutrokine-alphaSV receptors in vitro and / or in vivo. Or reducing antibodies. In certain embodiments, the antibodies of the invention inhibit or reduce the ability of Neutrokine-alpha and / or Neutrokine-alphaSV to bind Neutrokine-alpha receptors and / or Neutrokine-alphaSV receptors in a test. In another particular specific embodiment, an antibody of the invention inhibits the ability of Neutrokine-alpha and / or Neutrokine-alphaSV to bind Neutrokine-alpha receptors and / or Neutrokine-alphaSV receptors in vivo; Decrease. Such inhibition can be assayed using techniques described herein or known in the art.

The present invention also specifically binds Neutrokine-alpha and / or Neutrokine-alphaSV, although Neutrokine-alpha and / or Neutrokine-alphaSV are in vitro and / or in vivo. Or antibodies that do not inhibit the ability to bind Neutrokine-alphaSV receptors. In certain embodiments, the antibodies of the invention do not inhibit or reduce the ability of Neutrokine-alpha and / or Neutrokine-alphaSV to bind Neutrokine-alpha receptors and / or Neutrokine-alphaSV receptors in the test. In another particular specific embodiment, an antibody of the invention inhibits the ability of Neutrokine-alpha and / or Neutrokine-alphaSV to bind Neutrokine-alpha receptors and / or Neutrokine-alphaSV receptors in vivo; Do not reduce.

As described above, the present invention includes antibodies that inhibit or reduce Neutrokine-alpha and / or Neutrokine-alphaSV-mediated biological activity in vitro. In certain embodiments, the antibodies of the invention inhibit or reduce in vitro Neutrokine-alpha and / or Neutrokine-alphaSV-mediated B cell proliferation. Such inhibition can be assayed by routinely modifying the B cell proliferation assay methods described herein or known in the art. In certain other conventional embodiments, the antibodies of the invention inhibit or reduce Neutrokine-alpha and / or Neutrokine-alphaSV-mediated B cell proliferation in vivo. In certain embodiments, the antibody of the invention is 15C10 or a humanized form thereof. In another preferred embodiment, the antibody is 16C9 or a humanized form thereof. Thus, in certain embodiments of the invention, 16C9 and / or 15C10 antibodies or humanized forms thereof may bind B cell proliferation by binding to soluble Neutrokine-alpha and / or Neutrokine-alphaSV and / or agonists and / or antagonists thereof. Is used to partially or completely inhibit.

As another alternative, the present invention also specifically binds Neutrokine-alpha and / or Neutrokine-alphaSV, but not Neutrokine-alpha and / or Neutrokine-alphaSV-mediated biological activity (eg, B cell proliferation). Stimuli), which do not inhibit or decrease in vitro. In certain embodiments, the antibodies of the invention do not inhibit or reduce Neutrokine-alpha and / or Neutrokine-alphaSV-mediated biological activity in vitro. In certain other conventional embodiments, the antibodies of the invention do not inhibit or reduce Neutrokine-alpha and / or Neutrokine-alphaSV-mediated biological activity in vivo. In another specific embodiment, the antibody of the invention is 16C9 or a humanized form thereof.

As described above, the present invention includes antibodies that specifically bind in vitro and / or in vivo with the same epitope as one or more of the antibodies specifically described herein.

In certain embodiments, an antibody of the invention specifically binds in vitro to the amino acid sequence contained at amino acid residues about Ser-171 to about Phe-194 of SEQ ID NO: 2. In certain other conventional embodiments, the antibody of the invention specifically binds in vivo with an amino acid sequence contained between amino acid residues about Ser-171 to about Phe-194 of SEQ ID NO: 2. In another common particular embodiment, the antibodies of the invention specifically bind in vitro to the amino acid sequences contained in amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2. In another common particular embodiment, the antibody of the invention specifically binds in vivo with the amino acid sequence contained in amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2.

In a further particular embodiment, the antibody of the invention specifically binds in vitro to the amino acid sequence contained at amino acid residues about Glu-223 to about Tyr-246 of SEQ ID NO: 2. In another further particular embodiment, an antibody of the invention specifically binds in vivo with an amino acid sequence contained between amino acid residues about Glu-223 to about Tyr-246 of SEQ ID NO: 2. In certain other conventional embodiments, the antibodies of the invention specifically bind in vitro to the amino acid sequences contained in amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In another common particular embodiment, the antibodies of the invention specifically bind in vivo with the amino acid sequences contained in amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In another common particular embodiment, the antibodies of the invention specifically bind in vitro to the amino acid sequences contained in amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2. In another particular specific embodiment, the antibody of the present invention specifically binds in vivo with the amino acid sequence contained in amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2.

In addition, the present invention provides that the 9B6 monoclonal antibody produced by hybridoma deposited with PTA-1159 is a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2, more preferably the amino acid residue Ser- Provided are antibodies that competitively inhibit binding to a polypeptide having an amino acid sequence of 171 to Phe-194. Competitive inhibition can be measured using methods known herein, such as the competitive binding assays described herein. In a preferred embodiment, the antibody comprises at least 95%, 90 that the 9B6 monoclonal antibody binds the polypeptide of SEQ ID NO: 2, more preferably the polypeptide having amino acid sequences of amino acid residues Ser-171 to Phe-194 in SEQ ID NO: 2. Competitive inhibition of at least%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%.

In addition, the present invention provides that the 15C10 monoclonal antibody produced by hybridoma deposited with PTA-1158 is a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2, more preferably the amino acid residue Glu- Provided are antibodies that competitively inhibit binding to a polypeptide having an amino acid sequence of 223 to Tyr-246. In a preferred embodiment, the antibody comprises at least 95%, 90 that the 15C10 monoclonal antibody binds the polypeptide of SEQ ID NO: 2, more preferably a polypeptide having the amino acid sequence of amino acid residues Glu-223 to Tyr-246 in SEQ ID NO: 2. Competitive inhibition of at least%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%.

A further aspect of the invention relates to 9B6 antibodies and hybridoma cell lines expressing the antibodies. Hybridoma cell lines expressing 9B6 antibody were deposited with ATCC on January 7, 2000 and were assigned ATCC accession number PTA-1159. In a preferred embodiment, antibody 9B6 is humanized.

A further aspect of the invention relates to 15C10 antibodies and hybridoma cell lines expressing the antibodies. Hybridoma cell lines expressing 15C10 antibody were deposited with ATCC on January 7, 2000 and were assigned ATCC accession number PTA-1158. In a preferred embodiment, antibody 15C10 is humanized.

In certain embodiments, the specific antibodies described above are humanized using techniques described herein or known in the art, and then used as therapeutics as described herein.

In another particular embodiment, any of the antibodies described above is used in soluble form.

In another specific embodiment, any of the antibodies described above is conjugated to toxins or labels (as described below). The conjugated antibody is used to kill or quantify a specific cell population. In a preferred embodiment, the conjugated antibody is used to kill B cells expressing Neutrokine-alpha receptors on the surface. In another preferred embodiment, the conjugated antibody is used to quantify B cells expressing Neutrokine-alpha receptors on the surface.

In another specific embodiment, any of the antibodies described above is linked to a toxin or label (as described below). The conjugated antibody is used to kill or quantify a specific cell population. In a preferred embodiment, the conjugated antibody is used to kill monocytes expressing the membrane-bound form of Neutrokine-alpha. In another preferred embodiment, the conjugated antibody is used to quantify monocytes expressing the membrane-bound form of Neutrokine-alpha.

In addition, the antibodies of the present invention activate, but are not limited to, monocytes expressing membrane-bound forms of Neutrokine-alpha on the cell surface, block monocyte activation, and / or kill the monocyte lineage. Therapeutic and / or prophylactic agents (e.g. for treating, preventing and / or diagnosing diseases or conditions associated with myeloid leukemia, mononuclear leukemia and lymphoma, monocytosis, monocytopenia, rheumatoid arthritis and other activated mononuclear cells) Used as In certain embodiments, the antibodies of the invention immobilize complement. In another specific embodiment, as described herein, an antibody (or fragment thereof) of the invention may be a toxin, radioisotope, and cell, such as a compound that binds to and activates a heterologous polypeptide or nucleic acid (eg, an endogenous cytotoxic effector system). Toxic prodrugs).

In another embodiment, one or more monoclonal antibodies that recognize or bind Neutrokine-alpha and / or muteins thereof but do not recognize or bind Neutrokine-alphaSV and / or muteins thereof Is generated. In a related aspect, one or more monoclonal antibodies are generated that recognize or bind Neutrokine-alphaSV and / or muteins thereof but do not recognize or bind Neutrokine-alpha and / or muteins thereof. .

As discussed above, the antibodies against Neutrokine-alpha and / or Neutrokine-alphaSV according to the present invention are anti-idiotypes that "mean" Neutrokine-alpha using techniques well known to those skilled in the art. Can be used to generate antibodies (see, eg, Greenspan & Bona, FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, an antibody that binds Neutrokine-alpha and / or Neutrokine-alphaSV and competitively inhibits the multimerization and / or binding of Neutrokine-alpha and / or Neutrokine-alphaSV with a ligand is Neutrokine. -Alpha TNF multimerization and / or binding domains can be used to generate anti-idiotypes that "mute" and consequently bind to and neutralize Neutrokine-alpha and / or Neutrokine-alphaSV and / or ligands thereof. . Such neutralizing anti-idiotypes or Fab fragments thereof can be used in therapeutic methods to neutralize Neutrokine-alpha ligands. For example, such anti-idiotypic antibodies bind Neutrokine-alpha and / or Neutrokine-alpha SV or bind Neutrokine-alpha and / or Neutrokine-alphaSV receptors on the cell surface of the B cell lineage. And thereby block Neutrokine-alpha and / or Neutrokine-alphaSV mediated B cell activation, proliferation and / or differentiation.

Immune System-Related Disease Diagnosis

Neutrokine-alpha is expressed in kidney, lung, peripheral leukocytes, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages and umbilical cord blood tissues, especially in cells of mononuclear lineage. Neutrokine-alphaSV is also expressed in primary dendritic cells. In addition, Neutrokine-alpha is expressed on the cell surface of the following non-hematopoietic tumor cell lines: colon carcinoma HCT 116 (ATCC Accession No. CCL-247) and HT-29 (ATCC Accession No. HTB-38); Colon adenocarcinoma Caco-2 (ATCC Accession No. HTB-37), COLO 201 (ATCC Accession No. CCL-224) and WiDr (ATCC Accession No. CCL-218); Breast adenocarcinoma MDA-MB-231 (ATCC Accession No. HTB-26); Bladder impression carcinoma SCaBER (ATCC Accession No. HTB-3); Bladder carcinoma HT-1197 (ATCC Accession No. CRL-1473); Renal carcinoma A-498 (ATCC Accession No. HTB-44), Caki-1 (ATCC Accession No. HTB-46) and Caki-2 (ATCC Accession No. HTG-47); Renal Willims tumor SK-NEP-1 (ATCC Accession No. HTB-48); And pancreatic carcinoma Hs 766T (ATCC Accession No. HTB-134), MIA PaCa-2 (ATCC Accession No. CRL-1420) and SU.86.86 (ATCC Accession No. CRL-1837). For many immune system-related diseases, "standard" Neutrokine-alpha and / or Neutrokine-alphaSV gene expression levels (ie Neutrokine-alpha in immune system tissue or body fluids from a person without an immune system disease) and / or Or substantially altered (increased or decreased) levels of Neutrokine-alpha and / or Neutrokine-alphaSV gene expression relative to Neutrokine-alphaSV), such as immune system tissue or other cells or fluids obtained from patients with Eg, serum, plasma, urine, synovial fluid or spinal fluid). Accordingly, the present invention measures the expression level of Neutrokine-alpha and / or Neutrokine-alphaSV coding genes in immune system tissue or other cells or body fluids from humans, and the measured expression levels are determined using standard Neutrokine-alpha and / or A diagnostic method useful during the diagnosis of an immune system disease, including comparing to Neutrokine-alphaSV gene expression levels, wherein compared to a standard, the immune system disease or normal activation, proliferation, differentiation and / or lethality of the gene expression level is compared. It is instructed.

In particular, certain tissues in mammals that have cancer in cells or tissues of the immune system may have a "standard" level of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and Neutrokine-alpha and / or Neutrokine-alphaSV poly It is believed to express at markedly increased or decreased levels compared to peptide encoding mRNA. In addition, when compared to plasma from a mammal that does not have the cancer, a sensitizing level of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides may cause specific body fluids (e.g., serum, Plasma, urine and spinal fluid) or cells or tissues.

For example, as described herein, Neutrokine-alpha is highly expressed in cells of the monocyte lineage. Thus, the polynucleotides of the invention (eg, polynucleotide sequences complementary to all or a portion of Neutrokine-alpha mRNAs and / or Neutrokine-alphaSV mRNAs) and antibodies (and antibody fragments) to the polypeptides of the invention are Can be used to quantify or quantify the concentration of cells of a mononuclear lineage (eg monocyte leukocyte cells) expressing Neutrokine-alpha on the cell surface. These antibodies are further diagnosed to detect abnormalities at the level of Neutrokine-alpha gene expression or abnormalities in the structure and / or temporal, tissue, cell or subcellular locations of Neutrokine-alpha and / or Neutrokine-alphaSV. Can be used for These diagnostic assays can be performed in vivo or in vitro, such as blood samples, biopsy tissue or autopsy tissue.

In addition, as described herein, Neutrokine-alpha receptors are mainly expressed in cells of the B cell lineage. Thus, Neutrokine-alpha polypeptides of the invention (including labeled Neutrokine-alpha polypeptides and Neutrokine-alpha fusion proteins) and anti-Neutrokine-alpha antibodies against the polypeptides of the invention (anti-Neutrokine -Alpha antibody fragments) can be used to quantify or quantify the concentration of cells of the B cell lineage expressing Neutrokine-alpha (eg, B cell related leukemia or lymphoma) on the cell surface. These Neutrokine-alpha polypeptides and antibodies may further be used to detect abnormalities in the level of Neutrokine-alpha receptor gene expression or abnormalities in the structure and / or temporal, tissue, cell or subcellular locations of Neutrokine-alpha receptors. It can be used to diagnose and / or diagnose activity / defects in signaling pathways associated with Neutrokine-alpha. These diagnostic assays can be performed in vivo or in vitro, eg, in blood samples or biopsy tissue, using techniques described herein or known in the art.

In one embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention or the Neutrokine-alpha and / or Neutrokine-alphaSV agonists or antagonists (eg, anti- Neutrokine-alpha and / or Neutrokine-alphaSV antibodies) are used to treat, prevent, diagnose or prognose immunodeficient humans.

Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or Neutrokine-alpha and / or Neutrokine-alphaSV agonists or antagonists of the invention (e.g., anti-Neutrokine-alpha and / or Neutro Immunodeficiency, which may be treated, prevented, diagnosed, or prognosticed with (Kin-alphaSV antibody), include: (XLA), Bruton's disease, congenital angammaglobulinemia, semi-infant agammagglobulinemia, acquired agammagglobulinemia, adult-onset agammaglobulinemia, late ongammaglobulinemia, dysgammaglobulinemia, hypomagglobulin Hypertension, transient reduced maglobulinemia in infants, unspecific reduced maglobulinemia, agammagglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiscott-Aldrich syndrome (WAS), I gM hyperimmune deficiency immunodeficiency, IgM hyperimmune deficiency immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or elevated Ig bearing antibody deficiency, immunodeficiency with thymic cancer, Ig heavy chain Deletion, kappa chain deficiency, B cell lymphocytic proliferative disease (BLPD), selective IgM immunodeficiency, febrile angammaglobulinemia (Swiss type), retinopathy, neonatal neutropenia, severe congenital leukopenia, immunodeficiency Athymic lymphoid insufficiency-aplasia or dysplasia, ataxia-capillary dilatation, complex dwarfism, semi-lymphocytic hyperplasia syndrome (XLP), Negeloff syndrome-combinative Ig immunodeficiency syndrome, purine-based nucleoside phosphorylase deficiency ( PNP), MHC type II deficiency (Bear Lymphocyte Syndrome) and one or more immunodeficiencies selected from Severe Complex Immunodeficiency. Neunda.

According to an aspect of the present invention, an individual suffering from immunodeficiency expresses an ideally low level of Neutrokine-alpha and / or Neutrokine-alphaSV compared to a person without immunodeficiency. Any means described herein or known in the art detects Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention (e.g., Neutrokine-alpha according to the invention). And / or FACS analysis or ELISA detection of Neutrokine-alphaSV polypeptides and hybridization or PCR detection of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides according to the invention) It can be used to separately measure the expression pattern of kin-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides.

Biological samples of a person suffering from immunodeficiency are characterized by low expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV compared to the levels observed in a person who does not have immunodeficiency. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists or antagonists thereof can be used for the diagnosis and / or prognosis of immunodeficiency according to the methods of the invention. have. For example, a biological sample obtained from a person suspected of having immunodeficiency (“patient”) may be used to determine relative expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides of the invention. Analyze for The expression level of at least one of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention when expressed in a person known to not suffer from immunodeficiency. Significant differences between samples obtained from patients and controls at expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists of the invention are patients Suggests that I have immunodeficiency.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti Neutrokine-alphaSV antibody) is used to treat, diagnose, and / or prognose a person suffering from common variable reverse deficiency (CVID; also known as acquired angammaglobulinosis and acquired gammaglobulinic hypotropia) or a small group of such diseases. Used. According to an aspect of the present invention, a person with CVID or a subpopulation of a person with CVID is Neutrokine-alpha and / or anti-Neutrokine-alpha in B cells and / or mononuclear cells as compared to a person without CVID. Express abnormal levels of receptors. Any means described herein or known in the art detects Neutrokine-alpha polynucleotides or polypeptides and / or Neutrokine-alpha receptor polypeptides of the invention (e.g., Neutrokine- according to the invention). FACS analysis or ELISA detection of alpha and / or Neutrokine-alphaSV polypeptides and hybridization or PCR detection of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides according to the invention) At least B cells or components thereof For example, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides of the invention in a sample containing at least monocytes or some component thereof (eg RNA) compared to a sample containing RNA). Or to measure the expression aspect of the polypeptide and / or Neutrokine-alpha receptor polypeptide separately. A sample containing at least monocytes or some component thereof (eg, RNA) is determined to reflect Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotide or polypeptide expression and at least B cells or components thereof ( For example, if a sample containing RNA) is determined to reflect less than normal levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotide or polypeptide expression, the sample may have a CVID (i.e. acquired agammaglobulinosis Or acquired gamma globulin reduction).

Subpopulations of people suffering from CVID are characterized by high levels of expression of Neutrokine-alpha and Neutrokine-alpha receptors (NAR) in peripheral or circulating B cells as compared to those observed in people not suffering from CVID. In contrast, a person without CVID is typically characterized by low levels of Neutrokine-alpha expression in peripheral or circulating B cells and high levels of NAR expression. Thus, Neutrokine-alpha, Neutrokine-alphaSV polypeptides and / or NAR polypeptides, polynucleotides and / or polypeptides of the invention and / or agonists or antagonists thereof are specific for a subpopulation of CVIDs according to the invention. Can be used for diagnosis. For example, a sample of peripheral B cells obtained from a person (patient) suspected of having CVID may be a relative of the Neutrokine-alpha, Neutrokine-alphaSV and / or NAR polynucleotides and / or polypeptides of the invention. The level of expression can be analyzed. The expression level of one or more of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention in a person known to not suffer from CVID (“control”). Significant differences between samples obtained from patients and controls at the expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists of the present invention are as follows. It suggests that you have CVID.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti- Neutrokine-alphaSV antibodies) are used to diagnose, prognose, treat or prevent diseases characterized by defective serum immunoglobulin production, recurrent infections and / or immune system disorders. Furthermore, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV). Antibodies) include infections of joints, bones, skin and / or parotid glands, blood-borne infections (eg, sepsis, meningitis, septic arthritis and / or osteomyelitis), autoimmune diseases (eg, diseases described herein), inflammatory diseases And malignant tumors and / or all diseases, disorders or conditions associated with these infections, diseases, disorders and / or malignancies, including but not limited to CVID, another primary immunodeficiency syndrome, HIV disease, CLL, recurrent bronchitis, sinusitis , Otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe herpes zoster), and / or pneumonia caries.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti Neutrokine-alphaSV antibody) is used to treat, diagnose or prognose an individual with an autoimmune disease or condition.

Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention or Neutrokine-alpha and / or Neutrokine-alphaSV agonists or antagonists of the invention (e.g., anti-Neutrokine-alpha and Autoimmune disease or condition that may be treated, prevented, diagnosed, or prognosticed with Neutrokine-alphaSV antibody) Autoimmune thrombocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatoid heart disease, glomerulonephritis (eg IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmitis, polyendocrine disease Purpura (eg, Henloch-Scoenlein purpura, Rae's disease, Stiff-Man syndrome, autoimmune lung inflammation, Guillain-Ba) rre) syndrome, insulin-dependent diabetes and autoimmune inflammation, autoimmune thyroiditis, hypothyroidism (ie Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture syndrome, pemphigus, receptor autoimmune (eg, (a) graves) Disease, (b) Myasthenia Gravis and (c) insulin resistance), autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, Multiple myositis / dermatitis, pernicious anemia, idiopathic Edison disease, infertility, glomerulonephritis (eg primary glomerulonephritis and IgA nephropathy), lactose swelling, Sjogren syndrome, diabetes and adrenaline-acting drug resistance (asthma or cystic fibrosis) Accompanying adrenergic drug resistance), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland abnormalities, vitiligo, vasculitis, post-MI, heart Dog Syndrome, urticaria, include atopic dermatitis, asthma, inflammatory muscle disease and other inflammatory, granulomatous Castle, atrophic and degenerative diseases.

According to this embodiment, an individual suffering from an autoimmune disease or disease expresses an ideally high level of Neutrokine-alpha, Neutrokine-alphaSV and / or NAR compared to an individual who does not have autoimmune disease or disease. . Any means described herein or known in the art detects Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or NAR polypeptides of the invention (eg, Neutrokine of the invention). FACS analysis or ELISA detection of alpha and / or Neutrokine-alphaSV polypeptides and hybridization or PCR detection of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides of the invention) It can be applied to measure the expression pattern of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or NAR polypeptides.

A biological sample of a person suffering from an autoimmune disease or disease is characterized by high expression levels of Neutrokine-alpha, Neutrokine-alphaSV and / or NAR when compared to that observed in a person who does not have autoimmune disease or disease. It is erased. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists or antagonists of the present invention are diagnosed and / or prognostic in accordance with the methods of the present invention. Can be used for For example, a biological sample obtained from a person suspected of having an autoimmune disease or disease (“patient”) can be prepared from the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides of the present invention. And / or can be analyzed for relative expression levels of NAR polypeptides. The expression level of at least one of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention in a person known to not suffer from an autoimmune disease or disease. Samples obtained from patients and controls at expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists and / or NAR polypeptides of the invention A significant difference between the two indicates that the patient suffers from immunodeficiency.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists and / or antagonists of this Neutrokine-alpha and / or Neurokine-alphaSV (eg, anti Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies) are used to treat, diagnose, and / or prognose a person with systemic lupus erythematosus or a subpopulation of the disease. In this embodiment, a small group of patients with systemic lupus erythematosus or systemic lupus erythematosus develops ideally high levels of Neutrokine-alpha and / or Neutrokine-alphaSV compared to a person without systemic lupus erythematosus. do. Any means described herein or known in the art detects Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention (e.g., Neutrokine-alpha and / or according to the invention). FACS analysis or ELISA detection of Neutrokine-alphaSV polypeptides and hybridization or PCR detection of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides according to the invention) Neutrokine-alpha of the invention in biological samples And / or to measure the expression profile of Neutrokine-alphaSV polynucleotides and / or polypeptides.

Biological samples of people with systemic lupus erythematosus are characterized by high expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV compared to those observed in people without systemic lupus erythematosus. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists or antagonists of the invention can be used to diagnose and diagnose systemic lupus erythematosus or a subclass thereof according to the methods of the invention. Can be used for prognosis. For example, a biological sample obtained from a person suspected of having systemic lupus erythematosus (“patient”) may be a relative of the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides of the invention. The level of expression can be analyzed. The expression level of at least one of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention in a person known to not suffer from systemic lupus erythematosus. Significant differences between samples obtained from patients and controls at the expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists of the present invention are as follows. It suggests suffering from immunodeficiency and subclass thereof.

In addition, there is a direct correlation between systemic lupus erythematosus or a subclass of this disease and the concentrations of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides (RNA) and / or polypeptides of the invention. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides (RNAs), polypeptides and / or agonists or antagonists of the invention, according to the method of the present invention, are severity of systemic lupus erythematosus or subclass thereof. It can be used to diagnose. For example, a biological sample obtained from a person suspected of having systemic lupus erythematosus (“patient”) may be a relative of the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides of the invention. The level of expression can be analyzed. The expression level of one or more of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention in a panel of humans known to exhibit a range of severity of systemic lupus erythematosus. According to this method, the agreement of the expression level with the characterized member of the panel indicates the severity of the disease.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti-Neutrokine- AlphaSV antibodies) are used to treat, diagnose or prognose a person suffering from rheumatoid arthritis or a subpopulation of the disease. In this embodiment, a small group of patients with rheumatoid arthritis or rheumatoid arthritis express abnormally high levels of Neutrokine-alpha and / or Neutrokine-alphaSV when compared to a person who does not have rheumatoid arthritis. Any means described herein or known in the art detects Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention (e.g., Neutrokine-alpha and / or according to the invention). FACS analysis or ELISA detection of Neutrokine-alphaSV polypeptides and hybridization or PCR detection of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides according to the invention) Neutrokine-alpha of the invention in biological samples And / or to measure the expression profile of Neutrokine-alphaSV polynucleotides and / or polypeptides.

Biological samples of people with rheumatoid arthritis are characterized by high expression levels of Neutrokine-alpha and / or Neutrokine-alpha SV when compared to those observed in people who do not have rheumatoid arthritis. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists or antagonists of the present invention may be used to diagnose and / or diagnose rheumatoid arthritis or subclasses thereof in accordance with the methods of the present invention. Or for prognosis. For example, a biological sample obtained from a person suspected of having rheumatoid arthritis (“patient”) may be a relative of the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides of the invention. The level of expression can be analyzed. The expression level of one or more of these molecules according to the invention is then compared with the expression level of the same molecule according to the invention in a person known to not suffer from rheumatoid arthritis. Significant differences between samples obtained from patients and controls at the expression levels of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists of the present invention are as follows. It is suggested that the patient suffers from rheumatoid arthritis and its subclass.

Accordingly, the present invention measures expression levels of genes encoding Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides in immune system tissue or other cells or body fluids from an individual, and measures the measured gene expression levels as standard Neutrokine- Provided diagnostic methods useful during the diagnosis of immune system diseases and / or autoimmune diseases, including cancer of the immune system and immunodeficiency, including comparing with alpha and / or Neutrokine-alphaSV gene expression levels, wherein standard and By comparison, the increase or decrease in gene expression levels is indicative of a disease of the immune system.

If the diagnosis of a disease of the immune system, including but not limited to the diagnosis of tumors, the diagnosis of immunodeficiency and / or the diagnosis of autoimmune diseases, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, and Patients exhibiting increased or decreased levels of Neutrokine-alpha and / or Neutrokine-alphaSV gene expression will experience worse clinical results compared to patients expressing the gene at levels closer to standard levels.

Analyzing or measuring the expression level of a gene encoding Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide means that the level of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or Neutrokine-alpha and / or Or the level of mRNA encoding Neutrokine-alphaSV polypeptide can be determined directly in the first biological sample (eg, by measuring absolute protein levels or mRNA levels) or relatively (eg, Neutrokine- in the second biological sample). Quantitative or qualitative measurement) by comparison with alpha and / or Neutrokine-alphaSV polypeptide levels or mRNA levels. Preferably, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels or mRNA levels in the first biological sample are measured and obtained from a second biological sample obtained from a non-disease or immune system disease. Levels from populations that do not have are averaged and compared to standard Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels or mRNA levels measured. As is recognized in the art, once standard Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels or mRNA levels are known, the standard levels can be used repeatedly as comparative standards.

Biological sample refers to any biological sample obtained from a human, body fluid, cell line, tissue culture or other source containing Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels or mRNA. As mentioned, biological samples include body fluids containing free extracellular domains of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (eg, serum, plasma, urine, synovial fluid and spinal fluid), immune system tissues, and Other tissue sources found to express the complete or free extracellular domain of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are included. Methods of obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, tissue biopsy is the preferred source.

The compounds of the present invention are useful for diagnosing, prognosis or treating various immune system-related diseases in mammals, preferably humans. Such diseases include, but are not limited to, tumors (eg, B-cell and mononuclear leukemia and lymphoma) and tumor metastasis, infections caused by bacteria, viruses, and other parasites, immunodeficiency, inflammatory diseases, lymphadenopathy, autoimmune diseases ( Eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease and inflammatory muscle disease) and graft-versus-host disease.

Whole cell RNA is described by Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987) can be isolated from biological samples using suitable techniques such as the one-step guanidinium-thiocyanate-phenol-chloroform method. The levels of mRNA encoding Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are then assayed using appropriate methods. The method includes Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription in combination with polymerase chain reaction (RT-PCR) and reverse transcription in combination with ligase chain reaction (RT-LCR). Included.

Assays for Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels in a biological sample can be performed using antibody-using techniques. For example, expression of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides in tissues can be studied by classical immunohistochemical methods (Jalkanen, M., et al., J. Cell. Biol. 101). : 976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105: 3087-3096 (1987)). Another antibody-use method useful for detecting Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide gene expression includes immunoassays such as enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). . Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; Iodo ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ In) and Technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm Radioactive isotopes such as, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru; Luminescent labels such as luminol; And fluorescent labels and biotin, such as fluorescein and rhodamine.

Techniques known in the art can be applied to labeling antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional binders (see, for example, US Pat. The entire contents of each of which are incorporated herein by reference.

The tissue or cell type analyzed is generally known or estimated to express the Neutrokine-alpha gene or known or estimated to express the Neutrokine-alpha receptor gene (eg, cells of a mononuclear lineage). Cells or tissues (eg, B cell lineage and spleen cells). Examples of protein isolation methods used herein include methods described in Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. The entire contents of which are incorporated herein by reference. Isolated cells can be derived from cell culture or from a patient. Analysis of cells obtained from the culture can be used to evaluate cells that can be used as part of cell-based gene therapy techniques or to test the effect of a compound on the expression of the Neutrokine-alpha gene or Neutrokine-alpha receptor gene. This may be a necessary step.

For example, an antibody or fragment thereof as described herein can be used to quantitatively or qualitatively detect the presence of a Neutrokine-alpha gene product or a conserved variant or peptide fragment thereof. This can be achieved, for example, by immunofluorescence techniques using fluorescently labeled antibodies in conjunction with optical microscopy, flow cytometry or fluorometric detection.

Antibodies (or fragments thereof) or Neutrokine-alpha polypeptides or polynucleotides of the invention may further comprise a Neutrokine-alpha gene product or a conserved variant or peptide fragment thereof as in immunofluorescence, immunoelectron microscopy or non-immunological assays. It can be used histologically for in situ detection or binding of Neutrokine-alpha to Neutrokine-alpha receptors. In situ detection can be accomplished by removing histological specimens from the patient and applying them to the labeled antibodies or Neutrokine-alpha polypeptides of the invention. The antibody (or fragment) of the Neutrokine-alpha polypeptide is preferably applied by overlapping the labeled antibody (or fragment) on the biological sample. Through the use of such methods, the presence of Neutrokine-alpha gene products or conserved variants or peptide fragments or Neutrokine-alpha polypeptide binding as well as their distribution within the test tissue can be determined. Those skilled in the art using the present invention will understand that any of a wide variety of histological methods (eg, staining procedures) may be modified to achieve in situ detection.

Immunoassays and non-immunoassays for Neutrokine-alpha gene products or conserved variants or peptide fragments thereof typically comprise samples such as lysates of biological fluids, tissue extracts, freshly harvested cells or cells cultured in cell culture. Culturing in the presence of a detectably labeled antibody capable of identifying Neutrokine-alpha gene product or conserved variants or peptide fragments thereof and detecting the bound antibody by any of a number of techniques known in the art. do.

Immunoassays and non-immunoassays for Neutrokine-alpha receptor gene products or conserved variants or peptide fragments thereof are typically samples such as lysates of biological fluids, tissue extracts, freshly harvested cells or cells cultured in cell culture. Neutrokine-alpha polypeptides are cultured in the presence of detectable or labeled Neutrokine-alpha polypeptides capable of identifying Neutrokine-alpha receptor gene products or conserved variants or peptide fragments thereof and are known in the art. Detection by any one of a number of techniques described.

Biological samples may be immobilized by contact with a solid phase support or carrier such as nitrocellulose or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support can then be washed with a suitable buffer and then treated with a detectably labeled anti-Neutrokine-alpha antibody or a detectable Neutrokine-alpha polypeptide. The solid phase support can then be washed twice with buffer to remove unconjugated antibody or polypeptide. The antibody is then optionally labeled. The amount of label bound on the solid support can then be detected by conventional means.

"Solid support or carrier" means any support capable of binding an antigen or an antibody. Well known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro and magnetite. The nature of the carrier may be slightly soluble or insoluble for the purposes of the present invention. The support material can have virtually any possible structural form as long as the bound molecule can bind the antigen or antibody. Thus, the support form may be spherical, such as beads, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface may be flat, such as a sheet, test strips, or the like. Preferred supports include polyesterine beads. One skilled in the art knows many other carriers suitable for binding antibodies or antigens and will be able to identify such carriers by routine experimentation.

The binding activity of many of the anti-Neutrokine-alpha antibodies or Neutrokine-alpha polypeptides obtained can be measured according to well known methods. Those skilled in the art will be able to determine effective and optimal assay conditions for each measurement by using routine experimentation.

In addition to assaying Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide levels or polynucleotide levels in biological samples obtained from individuals, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides may also be used. It can be detected in vivo by an image. For example, in one embodiment of the present invention B cell lymphoma is imaged using Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or anti-Neutrokine-alpha antibodies. In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or anti-Neutrokine-alpha antibodies and / or Neutrokine-alpha polynucleotides (eg, Neutrokine-alpha and / or Or polynucleotides complementary to all or a portion of Neutrokine-alphaSV mRNAs to image lymphomas (eg, monocytes and B cell lymphomas).

Antibody labels or markers for in vivo imaging of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides include those that are detectable by X-ray imaging, NMR, MRI, CAT-scan, or ESR. Included. For X-ray imaging, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not harmful to the patient at all.

Suitable markers for NMR and ESR include those having a detectable characteristic spin such as deuterium that can be incorporated into the antibody by labeling nutrients for the relevant hybridomas. When using in vivo imaging to detect increased levels of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides for human diagnosis, the use of human antibodies or "humanized" chimeric monoclonal antibodies is recommended. It may be desirable. Such antibodies can be prepared using techniques described herein or known in the art. For example, methods for preparing chimeric antibodies are known in the art (see, eg, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Cabilly et al., US Patents) 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)).

In addition, any Neutrokine-alpha polypeptide can be administered in which presence can be detected. For example, Neutrokine-alpha polypeptides labeled with a radiocontrast or other suitable compound can be administered and imaged as discussed above for labeled antibodies. Moreover, these Neutrokine-alpha polypeptides can be used for in vitro diagnostic procedures.

Radioisotopes (e.g., ¹³¹ I, ¹¹² In, ^99m Tc, ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), Indium ( ^{115 m} In, ^{113 m} In, ¹¹² In, ¹¹¹ In) and technetium ( ⁹⁹ Tc, ^{99 m} Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo) , Xenon ( ¹³³ Xe), Fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru), Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide-specific antibodies or antibody fragments labeled with a suitable detection image residue such as a radio-contrast agent or a substance detectable by nuclear magnetic resonance It is introduced into the mammal, for example parenterally, subcutaneously or intraperitoneally, so that it can be tested for immune system diseases. It will be understood in the art that the size of the patient and the imaging system used measure the amount of imaging residues needed to produce a diagnostic image. In the case of radioisotope residues, the amount of radioactivity injected into a human subject is generally in the range of ⁹⁹ mTc in the range of about 5-20 milliliters. The labeled antibody or antibody fragment will then preferentially accumulate at the location of the cell containing Neutrokine-alpha protein. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982). )].

In the context of antibodies, one of the methods by which anti-Neutrokine-alpha antibodies can be detectably labeled is by linking the antibody to an enzyme and linking the product to an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked"). Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin. Pathol. 31: 507-520 (1978); Butler, J. E., Meth. Enzymol. 73: 482-523 (1981); Maggio, E. (ed.). 1980, Enzyme Immunoassay, CRC Press. Boca Raton, FL.,; Ishikawa, E. et al., (Eds.). 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce a chemical moiety that can be detected by, for example, spectrophotometer, fluorometer or visual means. Enzymes that can be used to detectably detect an antibody include maleate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, Dehydrogenase, trios phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonucleases, urease, catalase, glucose-6- Phosphate dehydrogenase, glucoamylase and acetylcholinesterase, including but not limited to. In addition, detection can be accomplished by chromatographic methods using chromogenic substrates for enzymes. Detection can also be accomplished by visually comparing the degree of enzymatic reaction of the substrate with similarly prepared standards.

In addition, detection can be accomplished using several different immunoassays. For example, Neutrokine-alpha can be detected by radioimmunoassay (RIA) by radiolabeling an antibody or antibody fragment (see, eg, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioactive isotopes can be detected by means including, but not limited to, gamma counters, scintillation counters, or radiographs.

Antibodies can also be labeled with fluorescent compounds. Fluorescence labeled antibodies can detect the presence of the antibody due to fluorescence when exposed to light of a suitable wavelength. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanine, allophycocyanin, optalaldehyde and fluorescamine.

The antibody can also be detectably labeled using a fluorescence emitting metal such as ¹⁵² Eu or a lanthanide metal. These metals can be linked to the antibody using metal chelation groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In addition, an antibody can be detectably labeled by linking it to a chemiluminescent compound. The presence of chemiluminescent-tagged antibodies is then measured by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, terromatic acridinium esters, imidazoles, acridinium salts and oxalate esters.

Likewise, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins increase the efficacy of chemiluminescence reactions. The presence of the bioluminescent protein is measured by detecting the presence of luminescence. Bioluminescent compounds important for the purposes of the label include, but are not limited to, luciferin, luciferase, and acuorin.

Treatment of Immune System-Related Diseases

As noted above, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and polypeptides and anti-Neutrokine-alpha antibodies are abnormally high in Neutrokine-alpha and / or Neutrokine-alphaSV activity. It is useful for the diagnosis of symptoms involving low expression. In cells and tissues in which Neutrokine-alpha and / or Neutrokine-alphaSV are expressed and whose activity is regulated by Neutrokine-alpha and / or Neutrokine-alphaSV, compared to standard or "normal" levels in an individual Pathology associated with a body system in which the substantially altered (increased or decreased) expression level of Neutrokine-alpha and / or Neutrokine-alphaSV is expressed and / or active in Neutrokine-alpha and / or Neutrokine-alphaSV. It is self-evident.

In addition, those skilled in the art will appreciate that the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the present invention are members of the TNF family, so that the extracellular domains of the individual proteins are neutralized by Neutrokine-alpha and / or Neutrokine- by proteolytic cleavage. It can be released in soluble form from cells expressing alphaSV such that Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (especially soluble forms of the individual extracellular domains) can be transferred to cells, tissues of individuals from foreign sources. Or when added to the body, it will be understood that the polypeptide exerts regulatory activity against all of the individual's target cells. In addition, such type II transmembrane protein expressing cells can be added to an individual's cells, tissues or body, such that the added cells bind cells expressing receptors for Neutrokine-alpha and / or Neutrokine-alphaSV. And thereby cells expressing Neutrokine-alpha and / or Neutrokine-alphaSV can initiate action (eg, proliferation or cytotoxicity) against receptor-containing target cells.

In one embodiment, the present invention relates to a composition containing a polypeptide of the invention (eg, a Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or antimicrobial in combination with a heterologous polypeptide, heterologous nucleic acid, toxin or prodrug). A composition containing Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies) to target cells (e.g., B cells or Neutrokine-alpha expressing Neutrokine-alpha and / or Neutrokine-alphaSV receptors) And / or monocytes expressing the cell surface bound form of Neutrokine-alphaSV). Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies of the invention are via hydrophobic, hydrophilic, ionic and / or covalent interactions. And may be combined with heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs.

In one embodiment, the invention relates to a polypeptide of the invention (eg, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or anti-Neutrokine-alpha and / or anti-) coupled to a heterologous polypeptide or nucleic acid. Neutrokine-alphaSV antibody) is provided to specifically deliver a composition of the present invention to a cell. As one example, the present invention provides a method of delivering a therapeutic protein into a target cell. As another example, the present invention delivers single-stranded nucleic acids (eg, antisense or ribozymes) or double-stranded nucleic acids (eg, DNA capable of integrating into the genome of cells or replicating into episomes and transcribed) into target cells. Provide a way to.

In another aspect, the invention provides a polypeptide of the invention (eg, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibody). Provided are methods for specifically destroying cells (eg, destroying tumor cells) by administering in combination with toxins or cytotoxic prodrugs.

In certain embodiments, the present invention provides for the identification of cells of the B cell lineage (eg, B cell associated leukemia or lymphoma) by administering Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides in combination with toxins or cytotoxic prodrugs. It provides a way to destroy the enemy.

In another specific embodiment, the present invention provides for the identification of cells of a mononuclear lineage (eg, mononuclear leukemia or lymphoma) by administering Neutrokine-alpha and / or Neutrokine-alphaSV antibodies in combination with toxins or cytotoxic prodrugs. It provides a way to destroy the enemy.

Toxins "are compounds that bind and act on endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents) or Refers to any molecule or enzyme that causes death and does not normally exist within or on the surface of a cell Toxins that can be used in accordance with the methods of the invention include, but are not limited to, radioisotopes, native or known in the art. Antibodies that bind to the induced endogenous cytotoxic effector system (or complement fixation containing a portion thereof), thymidine kinase, endonucleases, RNAse, alpha toxin, lysine, avrine, Pseudomonas exotoxin A, diphtheria Toxins, saporins, momordines, gelonins, pore antiviral proteins, alpha-sarsine and cholera toxins. In addition, cell proliferation inhibitors, or cell killing agent, a therapeutic agent or a radioactive metal ion (e.g., ²¹³ Bi and alpha same-emitter or ^{103 Pd, 133 Xe, 131 I} , 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ Yttrium, ¹¹⁷ Tin, Radioisotopes such as ¹⁸⁶ Rhenium, ¹⁶⁶ Holmium and ¹⁸⁸ Rhenium), Luminol Bioluminescent labels and fluorescent labels such as fluorescein and rhodamine.

Techniques known in the art can be used to label the antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional binders (see, eg, US Pat. The entire contents of each of these patents are incorporated herein by reference. Cytotoxins or cytotoxic agents include all substances harmful to cells. Examples include paclitaxol, cytosalacin B, gramicidine D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydrate Oxy anthracin dione, mitoxanthrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and furomycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolic products (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechloretamine, thio Efa chlorambucil, melphalan, carmustine (BSNU) and romustine (CCNU), cyclotosamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II (DDP) cisplatin), anthracycline (e.g. daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly called actinomycin), bleomycin, mitra Mycin and anthracycin (AMC) and antimitotic agents (eg, vincristine and vinblastine).

"Cytotoxic prodrug" means a nontoxic compound that is converted into a cytotoxic compound by enzymes normally present in the cell. Cytotoxic prodrugs that may be used in accordance with the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide, mitomycin C, cytosine arabinosides, daunorubicin and doxorubicin Phenoxyacetamide derivatives of

In addition, symptoms caused by a decrease in Neutrokine-alpha and / or Neutrokine-alphaSV activity in the individual than standard or normal levels, in particular immune system diseases, may be caused by Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (soluble cells In the form of cells expressing an extradomain or a complete protein) or by administration of an agonist. Thus, the present invention provides an effective amount of a pattern for increasing Neutrokine-alpha and / or Neutrokine-alphaSV activity levels in an individual in need of increased levels of Neutrokine-alpha and / or Neutrokine-alphaSV activity. Neutrokine-alpha and / or Neutrokine-alphaSV comprising administering to the individual a pharmaceutical composition comprising an isolated Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or an agonist thereof Provided are methods for treating an individual in need of increased levels of activity. In addition, symptoms caused by an increase in Neutrokine-alpha and / or Neutrokine-alphaSV activity in individuals above standard or normal levels, in particular immune system diseases, may be caused by Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (soluble cells It is believed that it can be treated by administration of an antagonist (eg, an anti-Neutrokine-alpha antibody) or in the form of cells expressing an extradomain or a complete protein. Thus, the present invention provides an effective amount of a pattern for reducing Neutrokine-alpha and / or Neutrokine-alphaSV activity levels in an individual in need of reduced levels of Neutrokine-alpha and / or Neutrokine-alphaSV activity. Neutrokine-alpha and / or Neutrokine-alphaSV activity, comprising administering to the individual a pharmaceutical composition comprising an isolated Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or an antagonist thereof It provides a method of treating an individual in need of a reduced level.

The agonists of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or Neutrokine-alpha and / or Neutrokine-alphaSV of the invention can be used for the treatment of infectious bacteria. For example, infectious diseases can be treated by increasing the response response, particularly by increasing the proliferation and differentiation of B cells. The immune response can be increased by enhancing the current immune response or initiating a new immune response. As another alternative, agonists of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or Neutrokine-alpha and / or Neutrokine-alphaSV may also directly infect infectious organisms without necessarily causing an immune response. Can be suppressed.

The virus may be used to treat a disease or condition that can cause a disease or condition that can be treated with Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists of Neutrokine-alpha and / or Neutrokine-alphaSV. One example. Examples of viruses include, but are not limited to, the following DNA and RNA virus and viral families: abroviruses, adenoviruses, arenaviruses, arteriviruses, birnaviruses, bunyaviruses, calciviruses, sirs Coviruses, Coronaviruses, Dengue, EBV, HIV, Flaviviruses, Hepadnaviruses (Hepatitis), Herpesviruses (e.g. Paramyxoviruses, Morbiliviruses, Rhabdoviruses), Orthomyxoviruses (e.g. Influenza A, Influenza B and parainfluenza), papilloma virus, papovaviruses, parvoviruses, piconaviruses, poxviruses (e.g., head or vaccinia), leoviruses (e.g. rotavirus), retroviraceae (HTLV-I, HTLV) -II, lentivirus) and togaviruses (eg rubyvirus). Viruses in these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, ocular infections (eg, conjunctivitis, keratitis) , Chronic Fatigue Syndrome, Hepatitis (A, B, C, E, Chronic Active, Delta), Type B Japanese Encephalitis, Junin, Chikungunya, Rift Valley, Yellow Fever, Meningitis, Opportunistic Infection (eg AIDS) , Pneumonia, Burkitt's lymphoma, chicken pox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, polio, leukemia, rubella, sexually transmitted infections, skin diseases (eg, Kaposi's bumps) and viral infections. Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV treat, prevent, diagnose, and / or detect the symptoms or diseases Can be used to In certain embodiments, the Neutrokine alpha polynucleotide, polypeptide or agonist is used to treat, prevent and / or diagnose meningitis, dengue fever, EBV and / or hepatitis (hepatitis B). In a further particular embodiment, the Neutrokine alpha polynucleotide, polypeptide or agonist is used to treat a patient who is unresponsive to one or more other commercial hepatitis vaccines. In a further particular embodiment, the Neutrokine alpha polynucleotide, polypeptide or agonist is used to treat, prevent and / or diagnose AIDS. In a further particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV and / or Neutrokine-alpha receptor polynucleotides, polypeptides, agonists and / or antagonists treat, prevent and / or diagnose Cryptosporidium patients Used to

Similarly, it can cause a disease or condition and be treated with agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or Neutrokine-alpha and / or Neutrokine-alphaSV. Possible bacteria or fungi include, but are not limited to, the following Gram-negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (eg, Corynebacterium, Mycobacterium) , Norcadia), Cryptococcus neoformans, Aspergillus disease, Basillaseae (e.g. Anthrax, Clostridium), Bacteroidaceae, Blastomyses, Bordetella disease, Borrelia (e.g. Burelia Burg) Doperi) Brucella disease, Candida disease, Campylobacter, Coccidioidosis, Yeast Bacterium, Dematosis, E. coli (e.g. enterotoxic E. coli and intestinal bleeding E. coli), enterobacteriaceae (Crepsiella, Salmonella (e.g. Salmonella typhi and Salmonella paratypi), Serratia, Yersinia) , Helicobacter, Legionella disease, Leptospirosis, Listeria (e.g. Listeria monocytogenes), Mycoplasma thales, Bacillus, Cholera, Neisseria (e.g. Acinetobacter, Gonorrhea, Meningococcal infection), Meningococcal, Pastella SeAH infections (e.g., Actinobacillus, Haemophilus (e.g. Haemophilus influenzae), Pastella), Pseudomonas, Rickettsia, Chlamydia, Syphilis, Shigella species, Staphylococcus, Meningococcus, Pneumococcus And Streptococcus (eg Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause, but are not limited to, the following diseases: bacteremia, endocarditis, ocular infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg AIDS-associated infections), crude Peritonitis, prosthetic-associated infections, raster disease, airway infections (e.g., whooping cough or empyema), sepsis, Lyme disease, mycozoa, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (type A and B meningitis) ), Chlamydia, syphilis, diphtheria, leprosy, tuberculosis, tuberculosis, lupus, botulism, spinal cord, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted infection, skin disease (e.g., cellulitis, dematosis) , Urinary tract infection, wound infection. Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV treat, prevent, diagnose, and / or detect the symptoms or diseases Can be used to In certain embodiments, Neutrokine alpha polynucleotides, polypeptides or agonists thereof are used to treat, prevent and / or diagnose tetanus, diphtheria, botulism and / or meningitis B.

Furthermore, parasites that cause diseases or conditions and can be treated by Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV These include, but are not limited to, the following families or rivers: amoeba, barb's fever, coccidiosis, cryptosporidiosis, dientamoebaosis, trade, in vitro parasites, giardiosis, parasites, leiche Maniasis, terreriasis, toxoplasmosis, tripanosomia, trichomonas and sporeworms (eg, trichophytes, tropics, silos and ovary fever). These parasites can cause a variety of diseases or conditions, including but not limited to: amelioration, thrombiculosis, eye infections, intestinal diseases (eg, diarrhea, giardiosis), liver disease, lung diseases, opportunistic infections (eg , AIDS association), malaria, pregnancy complications and toxoplasmaemia. Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV treat, prevent, diagnose, and / or detect the symptoms or diseases Can be used to In certain embodiments, Neutrokine alpha polynucleotides, polypeptides or agonists thereof are used to treat, prevent and / or diagnose malaria.

In another embodiment, agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or Neutrokine-alpha and / or Neutrokine-alphaSVs, as well as otitis infection (eg, otitis) It is used to treat, prevent and / or diagnose infection by Streptococcus pneumoniae and other pathogenic organisms.

In certain embodiments, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti-Neutrokine- AlphaSV antibodies) are used to treat or prevent diseases characterized by defective serum immunoglobulin production, recurrent infections and / or immune system disorders. Furthermore, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (eg anti-Neutrokine-alpha and / or anti-Neutrokine-alpha) SV antibodies) include infections of joints, bones, skin and / or parotid glands, blood-mediated infections (eg, sepsis, meningitis, septic arthritis and / or osteomyelitis), autoimmune diseases (eg, diseases described herein), inflammation Diseases and malignancies and / or all diseases, disorders or conditions associated with these infections, diseases, disorders and / or malignancies, including but not limited to CVID, other primary immunodeficiency syndrome, HIV disease, CLL, recurrent bronchitis, sinusitis, It is used to treat or prevent otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster) and / or pneumocytis carnie.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof are one of the following diseases, conditions or conditions associated therewith. It can be used to diagnose, prognose, treat or prevent abnormalities: primary immunodeficiency, immune-mediated thrombocytopenia, kawasaki syndrome, bone marrow transplantation (eg, new bone marrow transplantation in adults or children), chronic B-cell lymphoid Leukemia, HIV infection (eg, HIV infection in adults or children), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

In addition, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and / or polypeptides and / or agonists and / or antagonists of the invention may be associated with one or more of the following diseases, disorders or conditions Can be used to diagnose, prognose, treat, or prevent: Guillain-Barr syndrome, anemia (eg, anemia associated with parvovirus B19, stable multiple myeloma patients at high risk of infection (eg, recurrent infections), autoimmune hemolytic anemia (E.g., excitatory autoimmune hemolytic anemia), thrombocytopenia (e.g. neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., CMV-negative recipients of cytomegalovirus (CMV) -positive organs), Gamma globulin reduced syndrome (e.g. gamma globulin reduced newborns with risk factors for infection or disease), epilepsy (e.g. refractory epilepsy), systemic vasculitis syndrome, myasthenia gravis (e.g. myasthenia gravis) Decompensation in ryeokjeung), dermis, reduction increases, and polymyositis.

Further preferred embodiments of the invention include, but are not limited to, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides and functional agonists thereof Includes uses for such purposes as:

Administration to animals (e.g. mice, rats, rabbits, hamsters, guinea pigs, pigs, smile pigs, chickens, camels, goats, horses, cattle, sheep, dogs, cats, non-human primates and humans, most preferably humans) Thereby enhancing the immune system and generating increased amounts of one or more antibodies (eg, IgG, IgA, IgM and IgE), inducing higher affinity antibody production (eg, IgG, IgA, IgM and IgE) Increase response. In certain common embodiments, the Neutrokine-alpha polypeptides and / or agonists thereof of the present invention are administered to enhance the immune system and produce increased amounts of IgG. In certain other conventional embodiments, the Neutrokine-alpha polypeptides of the invention and / or agonists thereof are administered to enhance the immune system and produce increased amounts of IgA. In another particular specific embodiment, the Neutrokine-alpha polypeptides and / or agonists thereof of the present invention are administered to enhance the immune system and produce increased amounts of IgM.

Animals that are unable to produce or impair functional endogenous antibody molecules but are capable of producing human immunoglobulin molecules by means of an immune system that has been reconstituted or partially reconstituted from other animals, including but not limited to those described above Administration to animals, including animals and also transgenic animals (see, eg, WO / 9824893, WO / 9634096, WO / 9633735, and WO / 9110741).

Vaccine adjuvant that enhances immune response to specific antigens. In certain embodiments, the vaccine adjuvant is a Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide described herein. In another particular embodiment, the vaccine adjuvant is the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides described herein (ie, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides are genetic vaccine adjuvants). . As discussed herein, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides include, but are not limited to, liposome delivery, recombinant vector delivery, injection of explanted DNA, and total gene delivery. It can be administered using known techniques.

Adjuvants to Enhance Tumor-Specific Immune Responses.

Adjuvant to enhance anti-viral immune response. Anti-viral immune responses that can be augmented using the compositions of the present invention as adjuvants include, but are not limited to, viruses and virus associated diseases or conditions described herein or known in the art. In certain embodiments, the compositions of the present invention are used as an adjuvant for enhancing an immune response against a virus, disease or condition selected from the group consisting of AIDS, meningitis, dengue disease, EBV and hepatitis (eg, hepatitis B). In another specific embodiment, the compositions of the present invention are HIV / AIDS, Respiratory Syndrome Virus, Dengue Disease, Rotavirus, Type B Japanese Encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chic Yongyunya It is used as an adjuvant for enhancing the immune response against a virus, disease or condition selected from the group consisting of Rift valley, herpes simplex and yellow fever. In another specific embodiment, the compositions of the present invention are used as an adjuvant to enhance the immune response to HIV gp 120 antigen.

An adjuvant for enhancing an anti-bacterial or anti-fungal immune response. Anti-bacterial or anti-fungal immune responses that can be augmented using the ancestors of the present invention as an adjuvant include viruses and virus associated diseases or conditions described herein or known in the art. In certain embodiments, the compositions of the present invention are used as an adjuvant for enhancing an immune response against bacteria, fungi, diseases or conditions selected from the group consisting of tetanus, diphtheria, botulism and meningitis B. In another specific embodiment, the compositions of the present invention are cholera, bacillus, Salmonella typhi, Salmonella paratypy, Myseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella species, enterotoxic E. coli. E. coli and intestinal bleeding It is used as an adjuvant for enhancing the immune response to bacteria, fungi, diseases or conditions selected from the group consisting of E. coli, Borrelia burgdorferi and malaria.

Adjuvant to enhance anti-parasitic immune response. Anti-parasitic immune responses that can be augmented using the compositions of the present invention as an adjuvant include parasitic and parasitic associated diseases or conditions described in the art or known in the art. In certain embodiments, the compositions of the present invention are used as an adjuvant to enhance an immune response against parasites. In another specific embodiment, the compositions of the present invention are used as an adjuvant to enhance the immune response to plasmodium (malaria).

As a stimulator of B cell responses to pathogens.

As an agent to strengthen an individual's immune status prior to receiving immunosuppressive therapy.

As an agent to induce higher affinity antibodies.

As an agent for increasing serum immunoglobulin concentrations.

As an agent to promote recovery of immunocompromised humans.

As an agent for increasing immunoreactivity between aging populations.

As an immune system enhancer before, during or after bone marrow transplantation and / or other transplantation (eg, allogeneic or xenotransplantation). In the context of transplantation, the compositions of the present invention may be administered prior to, concurrent with and / or after transplantation. In certain embodiments, the compositions of the invention are administered after implantation, but prior to the onset of recovery of the T-cell population. In another specific embodiment, the compositions of the present invention are administered preferentially after the recovery of the T cell population begins after transplantation but before the B cell population is fully recovered.

As an agent to enhance immunoresponsiveness in B cell immunodeficiency patients, such as patients undergoing partial or complete splenectomy. B-cell immunodeficiency that can be mitigated or treated by administering Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides of the present invention is not limited to these but severely complex immunodeficiency (SCID) -reflective , SCID- autosomal, adenosine deaminase deficiency (ADA deficiency), semi-anemia-magglobulinemia (XLA), Bruton's disease, congenital angamma-globulinemia, semi-acute infantile angamma-globulinemia, acquired agammaglobulinemia, no Gamma globulinemia, late agammaglobulinemia, aberrant gamma globulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia in infants, nonspecific hypogammaglobulinemia, agammagglobulinemia, common variable immunodeficiency syndrome (CVID) (acquired acquired) ), Wiscott-Aldrich syndrome (WAS), IgM hyperimmune deficiency syndrome, IgM hyperimmune hyperdeficiency immunodeficiency syndrome, selective IgA deficiency, IgG subtype Rheumatic deficiency (with or without IgA deficiency), normal or elevated Ig bearing antibody deficiency, immunodeficiency with thymic cancer, Ig heavy chain deletion, kappa chain deficiency, B cell lymphocytic proliferative disease (BLPD), selective IgM immunodeficiency , Febrile angammagglobulinemia (Swiss type), reticular dysplasia, neonatal neutropenia, severe congenital leukopenia, thymic lymphadenopathy with acute immunodeficiency-aplastic or dysplastic, ataxia-capillary dilatation, dwarfism, semi-lymphatic Cell proliferative syndrome (XLP), Negeloff syndrome-combinative Ig immunodeficiency, purine-based nucleoside phosphorylase deficiency (PNP), MHC type II deficiency (bare lymphocyte syndrome) and severe combined immunodeficiency syndrome.

As an agent to enhance immune responsiveness in patients with acquired B cell function. Symptoms due to acquired deficiency of B cell function that can be reduced or treated by administering Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides of the present invention include, but are not limited to, HIV infection, AIDS , Bone marrow transplantation and B cell chronic lymphocytic leukemia (CLL).

As an agent for enhancing immune responsiveness in patients with transient immunodeficiency. Temporary immunodeficiency-induced symptoms that can be alleviated or treated by administering Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof, include, but are not limited to, viral infections (eg, , Recovery from malnutrition, recovery from malnutrition, or symptoms associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.

As modulator of antigen presentation by monocytes, dendritic cells and / or B-cells. In one embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (in soluble, membrane-bound or transmembrane form) or polynucleotides enhance antigen presentation or enhance antigen presentation in vitro or in vivo. Antagonize In addition, in related embodiments, augmentation or antagonism of antigen presentation may be useful in anti-tumor treatment or in regulating the immune system.

As a mediator of mucosal immune responses. The expression of Neutrokine-alpha by monocytes and the responsiveness of B cells to these factors suggest that Neutrokine alpha is involved in the signal exchange between B cells and monocytes or their differentiated progeny. This activity is achieved in many ways similar to the CD40-CD154 signaling between B and T cells. Thus, Neutrokine-alpha may be an important modulator of T cell independent immune responses against peripheral pathogens. In particular, a special group of B cells (CD5 +), which are linked to mucosal sites and responsible for much of innate immunity in humans, can respond to Neutrokine-alpha and thus enhance a person's protective immune status.

An agent that allows an individual's immune system to develop a humoral response (ie TH2) as opposed to a TH1 cell response.

As a means of inducing tumor proliferation and thereby making the tumor more sensitive to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and therefore substantially resistant to all anti-neoplastic therapies. If these cells proliferate more rapidly, their sensitivity profiles will surely change.

As a B cell specific binding protein to which specific activators or inhibitors of cell growth can be bound. This result will focus on B cell populations in which the activity of the activator or inhibitor is normal, diseased or neoplastic.

As a means of detecting B-lined cells with their specificity. Such applications may require labeling the protein with biotin or other agents (eg, as described herein) to provide a means of detection.

As a stimulator of the B cell population in pathologies such as AIDS, chronic lymphatic diseases and / or common variable immunodeficiency.

As part of a B cell selection device with the function of separating B cells from a heterogeneous mixture of cell types. Neutrokine-alpha can bind to a solid support, to which cells specifically bind. Unbound cells are washed and bound cells are eluted sequentially. Non-limiting use of this screening makes it possible, for example, to purify tumor cells from bone marrow or peripheral blood prior to transplantation.

As a therapy for the generation and / or regeneration of lymphocytic tissue against surgery, trauma and genetic defects.

As gene-available therapy for genetic disorders causing immunodeficiency, as observed among SCID patients.

As antigen for the production of antibodies to inhibit or enhance Neutrokine-alpha mediated responses.

As a means to activate monocytes / macrophages to defend against parasitic diseases affecting monocytes such as Leshmania.

For pretreatment of bone marrow samples prior to transplantation. This treatment increases B cell expression and thus promotes recovery.

As a means of regulating secreted cytokines induced by Neutrokine-alpha.

Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides of the invention can be used to regulate IgE concentrations in vitro or in vivo.

In addition, the Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof may be used to treat, prevent and / or diagnose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis and eczema.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, diagnose, and / or alleviate selective IgA deficiency.

In certain other embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, diagnose, and / or alleviate vasozygous ataxia.

In another specific embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, diagnose and / or alleviate common variable immunodeficiency.

In another particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, diagnose, and / or alleviate semi-angammaglobulinemia. .

In another specific embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are for treating, preventing, diagnosing and / or alleviating severe complex immunodeficiency (SCID). Administered.

In another specific embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof are administered to treat, prevent, diagnose, and / or alleviate Wiscot-Aldrich. .

In another specific embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof may be used to treat, prevent, diagnose, and / or alleviate semi-Ig deficiency with excess IgM. Is administered.

In another particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) are used for chronic myelogenous leukemia, acute myeloid From leukemia, leukemia, histoblastic leukemia, mononuclear leukemia (eg acute mononuclear leukemia), leukemia paranoia, sealing mononuclear leukemia and / or other monocytes and / or mononuclear cells and / or tissues It is administered to treat, prevent, diagnose and / or alleviate induced leukemia.

In another particular embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof treat a mononuclear leukemia response as seen for example in tuberculosis. To prevent, diagnose, and / or alleviate.

In another specific embodiment, the Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides or agonists thereof of the invention have mononuclear leukocytosis, mononuclear leukopenia, mononucleosis and / or mononucleosis. Is administered to treat, prevent, diagnose and / or alleviate.

In certain embodiments, the Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides and / or anti-Neutrokine-alpha antibodies and / or agonists or antagonists of the invention are primary B lymphatic diseases and / or diseases And / or to treat, prevent, diagnose, and / or alleviate symptoms associated therewith. In one embodiment, such primary B lymphatic diseases, diseases and / or symptoms are characterized by complete or partial loss of humoral immunity.

Primary B lymphatic diseases, diseases and / or conditions associated therewith, which are characterized by complete or partial loss of humoral immunity and which can be prevented, treated, detected and / or diagnosed with the compositions of the present invention, are not limited thereto. Semi-angammaglobulinemia (XLA), severe combined immunodeficiency (SCID), and selective IgA deficiency.

In a preferred embodiment, the Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides and / or agonists and / or antagonists treat, prevent and / or treat diseases, conditions or conditions associated with one or more of the various mucosal membranes of the body. Or to diagnose. These diseases or conditions include, but are not limited to, mucositis, mucosal debridement, mucous colitis, mucosal skin leishmaniasis (e.g. American Leishmaniasis, Leishmaniasis Americana, Nasopharyngeal Leishmaniasis and New) World Leishmaniasis), mucosal skin lymph node syndrome (e.g. Kawasaki disease), small intestinal mucositis, mucous epithelial carcinoma, mucous epithelial tumor, mucosal epithelial cerebral disorder, mucosal adenocarcinoma, mucous degeneration, mucous degeneration, myxomatous degeneration , Myxoma, mucosal degeneration (e.g., cystic degeneration), mucosal invasion (e.g., type I mucosal invasion, type II mucosal invasion, type III mucosal invasion) and mucolytic disease, mucosal enteritis Small intestinal mucositis, mucopolysaccharide deposition (eg, type I mucopolysaccharide deposition, ie Hurler syndrome), type I mucopolysaccharide deposition (ie, Scheie syndrome or type V mucopolysaccharide deposition), type II Mucopolysaccharide Deposition Hunter syndrome, type III mucopolysaccharide deposition (ie, Sanfilippo syndrome), type IV mucopolysaccharide deposition (ie, Morquio syndrome), type VI mucopolysaccharide deposition (ie, Marote) Maroteaux-Lamy syndrome), type VII mucopolysaccharide deposition (ie, mucopolysaccharide deposition due to beta-glucuronidase deficiency) and mucosulfate deposition, mucopolysacchariduria, mucoconjunctivitis, Purulent mucinosis, bacillus (i.e., conjunctival bacillus), mucosal disease (i.e. bovine viral diarrhea), mucous colitis (e.g., colonic mucositis and mucosal colitis), and mucus (e.g. cystic fibrosis, pancreatic Cystic fibrosis, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoadhesion and stickiness). In very preferred embodiments, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides, polypeptides and / or antagonists and / or agonists thereof are used, in particular in combination with chemotherapy, to treat, prevent and / or diagnose mucositis. do.

In a preferred embodiment, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides, polypeptides and / or agonists and / or antagonists treat, prevent and / or treat a disease or condition or symptoms associated with sinusitis Used to diagnose.

Further symptoms, diseases or conditions that can be treated, prevented and / or diagnosed by Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides, polypeptides and / or agonists and / or antagonists thereof are osteomyelitis.

Further symptoms, diseases or conditions that can be treated, prevented and / or diagnosed by Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides, polypeptides and / or agonists and / or antagonists thereof are endocarditis. .

All of the uses described above can also be applied to veterinary medicine.

Antagonists of Neutrokine-alpha include binding and / or inhibitory antibodies, antisense nucleic acids, ribozymes and Neutrokine-alpha polypeptides of the invention. They are expected to reverse many of the activities of the ligands described above and be used clinically or substantially for the following uses:

Means for blocking foreign pathogens or various aspects of the immune response to themselves. Examples include autoimmune diseases such as lupus and arthritis, as well as immune responses to skin allergies, inflammations, intestinal diseases, wounds and pathogens. Although our current data directly demonstrate the potential role of Neutrokine-alpha in B cells and monocytes associated with pathology, the possibility that other cell types can obtain expression or response to Neutrokine-alpha. Remains. Thus, Neutrokine-alpha can be regulated by the state of the immune system and the microenvironment in which the cells are located, such as CD40 and its ligands.

Therapies for preventing B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

Graft-versus-host disease or inhibitor of graft rejection.

Therapies for B cell malignancy such as ALL, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, myeloma, multiple myeloma, Burkitt's lymphoma and EBV-transforming disease.

Therapy for chronic gamma globulin hyperemia in diseases such as unmeasured significant monoclonal gamma disease (MGUS), Waldenstrom's disease, related idiopathic monoclonal gamma disease and myeloma.

Therapies to Reduce Cell Proliferation of Giant B-Cell Lymphomas.

Means to reduce the involvement of B cells and Ig associated with chronic myeloid leukemia.

Immunosuppressant (s).

Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides or antagonists of the invention can be used to control IgE concentrations in vitro or in vivo.

In another embodiment, administration of the Neutrokine-alpha or Neutrokine-alphaSV polypeptides or polynucleotides or antagonists of the invention treats IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis and eczema. , Prophylaxis and / or diagnosis.

Inhibitors of signaling pathways involving ERK1, COX2 and cyclin D2 associated with Neutrokine-alpha induced B cell activation.

Such uses apply to a variety of hosts. Such hosts include, but are not limited to, humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, smile-pigs, chickens, goats, cattle, sheep, dogs, cats, rain Human primates and humans. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In the most preferred embodiment, the host is human.

Agonists and antagonists can be used in compositions with pharmaceutically acceptable carriers as described herein.

Antagonists are, for example, macrophages and their progenitor cells and neutrophils, basophils, type B lymphocytes and some T-cell subclasses, such as activated and CD8 cytotoxic T cells, in certain autoimmune and chronic inflammatory and infectious diseases. It can be used to inhibit Neutrokine-alpha-mediated and / or Neutrokine-alphaSV-mediated chemotaxis and activation of natural killer cells. Examples of autoimmune diseases include multiple sclerosis and insulin-dependent diabetes. Antagonists can also be used to treat, prevent and / or diagnose infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by inhibiting the growth and activation of mononuclear phagocytes. They can also be used to treat, prevent and / or diagnose idiopathic eosinophilic syndrome by inhibiting eosinophilic leukocyte production and migration. It is also possible to treat endotoxin shock by inhibiting the migration of macrophages and their production of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention by antagonists. Antagonists can also be used to treat atherosclerosis by inhibiting monocyte infiltration in the arterial walls. In addition, the antagonist inhibits chemokine-induced mast cells and basophils degranulation and histamine secretion to treat, prevent, and treat histamine-mediated allergic reactions and immunological disorders, including late allergic reactions, chronic urticaria and atopic dermatitis. It can be used to diagnose. It can also treat IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema. In addition, antagonists can be used to treat, prevent and / or diagnose chronic and acute inflammation by inhibiting the attraction of monocytes to the wound site. They can also be used to regulate normal pulmonary phagocyte populations because chronic and acute inflammatory lung disease is associated with the sequestration of mononuclear phagocytes in the lung. Antagonists can also be used to treat, prevent and / or diagnose rheumatoid arthritis by inhibiting the induction of monocytes into the synovial fluid of the patient's joints. Monocyte influx and activation play an important role in the development of degenerative and inflammatory arthrosis. Antagonists can be used to block toxic cascades, the main cause of which is IL-1 and TNF, which inhibit the biosynthesis of other inflammatory cytokines. In this way antagonists can be used to prevent inflammation. Antagonists can also be used to inhibit prostaglandin-independent fever induced by Neutrokine-alpha and / or Neutrokine-alphaSV. In addition, antagonists can be used to treat, prevent and / or diagnose patients with bone marrow abnormalities (eg, aplastic anemia and myelodysplastic syndromes). Antagonists can also be used to treat, prevent and / or diagnose asthma and allergies by preventing eosinophil accumulation in the lung. Antagonists can also be used to treat, prevent and / or diagnose subepithelial basement membrane fibrosis, which is a major feature of asthmatic lungs. In addition, antagonists can be used to treat, prevent, and / or diagnose lymphomas (eg, but not limited to, many of the various lymphomas described herein).

All uses described above can be applied to veterinary medicine. In addition, all uses described herein may also be applied to veterinary medicine.

Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention are associated with various immune system-associated diseases and / or associated with these diseases in mammals, preferably humans. It can be used to treat, prevent and / or diagnose symptoms. Many autoimmune diseases occur by inappropriate recognition of themselves as foreign substances by immune cells. Such inadequate recognition results in an immune response that induces destruction of host tissue. Thus, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists thereof of the invention capable of inhibiting the immune response, in particular the proliferation of B cells and / or the production of immunoglobulins, and / or Alternatively, administration of the antagonist may be an effective therapy for treating and / or preventing autoimmune diseases. Thus, in a preferred embodiment, the polypeptide fragments of the Neutrokine-alpha and / or Neutrokine-alphaSV antagonists of the invention (eg, Neutrokine-alpha and / or Neutrokine-alphaSV and anti-Neutrokine-alpha antibodies are Used to treat, prevent and / or diagnose autoimmune diseases.

Autoimmune diseases that can be treated, prevented and / or diagnosed with Neutrokine-alpha polynucleotides, polypeptides and / or antagonists (eg, anti-Neutrokine-alpha antibodies) and conditions associated with these diseases include Autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune thrombocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatoid heart disease , Glomerulonephritis (eg IgA nephropathy), multiple sclerosis, neuritis, uveitis keratoconjunctivitis, polyendocrine dysplasia, purpura (eg Henro-Skoenlein purpura), Later's disease, stiff-man syndrome, autoimmune pneumonia, guilin- Barr's syndrome, insulin dependent diabetes mellitus and autoimmune inflammatory eye disease.

Additional (possibly) autoimmune diseases that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (ie, Hashimoto thyroiditis (commonly characterized by For example cell-mediated and expressed in humoral thyroid cytotoxicity, Goodpasture syndrome (commonly represented as an anti-basement membrane antibody), Celtic swelling (often characteristically expressed as polar cell dissociative antibody, for example), Receptor autoimmunity (eg, (a) Grave's disease (commonly referred to as, for example, a TSH receptor antibody), (b) myasthenia gravis (commonly referred to as, for example, an acetylcholine receptor antibody), and (c) insulin resistance (Often characterized by phagocytosis of, for example, antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characteristic of, eg, antibody- As indicated by the phagocytosis of sensitized platelets).

Additional (possibly) autoimmune diseases that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to, rheumatoid arthritis (commonly characterized as immune complexes in joints for example) Scleroderma with anti-collagen antibodies (commonly characterized as, for example, nucleolus and other nuclear antibodies), mixed connective tissue diseases (commonly characterized as, for example, extractable nuclear antigens such as ribonuclear proteins) ), Polymyositis / dermatitis (commonly referred to as, for example, histone ANA), pernicious anemia (commonly referred to as, for example, anti-wall cells, microsomes and endogenous factor antibodies), idiopathic Edison disease (often characteristic) Such as for example humoral and cell-mediated adrenal cytotoxicity), infertility (commonly characterized as anti- sperm antibodies for example), glomerulonephritis (eg, Primary glomerulonephritis and IgA nephropathy (commonly characterized as eg, glomerular basement membrane antibody or immune complex), ileal swelling (commonly characterized as IgG and complement at basement membrane, for example) and Sjogren's syndrome (often characteristically For example as multiple tissue antibodies and / or as specific non-histone ANA (SS-B), diabetes (commonly represented as, for example, cell-mediated and humoral islet cell antibodies) and adrenergic drug resistance ( Adrenergic drug resistance with asthma or cystic fibrosis) (commonly characterized as beta-adrenergic receptor antibodies, for example).

Additional (possibly) autoimmune diseases that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to, chronic active hepatitis (commonly characterized as smooth muscle antibodies for example), primary bile Sex cirrhosis (often characteristically indicated as a mitochondrial antibody), endocrine gland abnormalities (often characteristically indicated as a specific tissue antibody in some cases), alum (often characteristically indicated as eg melanocyte antibodies), Vasculitis (commonly represented as eg Ig in the vessel wall and complement and / or low serum complement), post-MI (commonly represented as eg myocardial antibody), cardiac incision syndrome (commonly characterized as eg myocardium) Urticaria (commonly characterized, for example, as IgG and IgM antibodies against IgE), atopic dermatitis (often characteristic For example as IgG and IgM antibodies to IgE), asthma (commonly indicated as IgG and IgM antibodies to IgE, for example), inflammatory myopathy and other inflammatory, granulomatous, degenerative and atrophic diseases Included.

In a preferred embodiment, autoimmune diseases and disorders and / or symptoms associated with these diseases and disorders are treated, prevented and / or diagnosed using anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSVs.

In a preferred embodiment, rheumatoid arthritis is treated, prevented and / or diagnosed using anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention.

In a preferred embodiment, lupus is treated, prevented and / or diagnosed using anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention.

In certain preferred embodiments, nephritis associated with lupus is treated, prevented and / or diagnosed using anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies and / or other antagonists of the invention.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or antagonists thereof (eg, anti-neucaine-alpha and / or anti-neucaine-alphaSV antibodies) may be systemic lupus erythematosus. And / or to treat or prevent a disease, condition or condition associated therewith. Lupus-associated diseases, disorders or conditions that can be treated or prevented with Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or antagonists thereof of the present invention include, but are not limited to, hematological diseases ( Eg, hemolytic anemia, leukopenia, lymphopenia and thrombocytopenia), immunological diseases (eg anti-DNA antibodies and anti-Sm antibodies), rashes, photosensitivity, mouth ulcers, arthritis, fever, fatigue, weight loss, meningitis (Eg, meningitis), kidney disease (eg nephritis), neurological disease (eg, sudden seizures, peripheral neuropathy, CNS-associated diseases), gastrointestinal disease, Raynaud's phenomenon and pericarditis. In a preferred embodiment, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or antagonists thereof (e.g., anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies) are combined with systemic lupus erythematosus. Used to treat or prevent associated kidney disease. In the most preferred embodiment, Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides or antagonists thereof (eg, anti-Neutrokine-alpha and / or anti-Neutrokine-alphaSV antibodies) are systemic erythematous Used to treat or prevent nephritis associated with lupus.

Similarly, allergic reactions and symptoms such as asthma (particularly allergic asthma) or other dyspnea may also be attributed to the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists thereof of the present invention and And / or antagonists. In addition, these molecules can be used to treat, prevent, and / or diagnose hypersensitivity, hypersensitivity to an antigenic molecule, or blood group incompatibility.

Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof also exhibit organ rejection or graft-versus-host disease (GVHD) and / or symptoms associated therewith. Can be used to treat, prevent and / or diagnose. Organ rejection is caused by the destruction of host immune cells of the transplanted tissue via an immune response. Similarly, the immune response is also involved in GVHD but in this case the explanted immune cells destroy the host tissue. Administration of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists of the invention which inhibit the immune response, in particular the proliferation, differentiation or chemotaxis of T-cells It may be an effective therapy for preventing organ rejection or GVHD.

Similarly, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention can also be used to modulate inflammation. For example, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention can inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules include chronic prostatitis, granulomatous prostatitis and malacoplastia, inflammation associated with infection (eg, septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, Chronic and acute inflammatory conditions or cytokines (eg, TNF or IL-1), including complement-mediated supercharge rejection, nephritis, cytokine or chemokine induced lung wounds, inflammatory bowel disease, Crohn's disease It can be used to treat, prevent and / or diagnose chronic and acute inflammatory conditions due to overproduction.

In certain embodiments, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies of the invention are used to treat, prevent, control, detect, and / or diagnose inflammation.

In certain embodiments, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies of the invention are used to treat, prevent, control, detect, and / or diagnose inflammatory diseases.

In another particular embodiment, the anti-Neutrokine-alpha antibodies and / or anti-Neutrokine-alphaSV antibodies of the invention are used to treat, prevent, control, detect and / or diagnose allergic and / or hypersensitivity reactions.

Antibodies to Neutrokine-alpha and / or Neutrokine-alphaSV bind AR and / or inhibit their activity to Neutrokine-alpha and / or Neutrokine-alphaSV to prevent neutrophils from penetrating into the lungs after injury. Can be treated, prevented and / or diagnosed. The agonists and antagonists of the invention can be used, for example, in compositions with pharmaceutically acceptable carriers, as described below.

Neutrokine-alpha and / or Neutrokine-alphaSV and / or Neutrokine-alpha receptor polynucleotides or polypeptides and / or agonists and / or antagonists thereof may be used to treat diseases and conditions (eg, bronchial, For example, pulmonary and bronchial infections) and other diseases and conditions associated with other respiratory diseases and diseases. In certain embodiments, such diseases and disorders include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (eg bronchial pneumonia, bronchitis, tuberculous bronchial pneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyp, bronchiectasis ( E.g., bronchiectasis, columnar bronchiectasis and cystic bronchiectasis, bronchial adenocarcinoma, bronchial cholangiocarcinoma, bronchitis (e.g., exudative bronchitis, fibrotic bronchitis, and proliferative bronchitis), bronchial-bubble carcinoma, Bronchial asthma, bronchitis (e.g., asthmatic bronchitis, castellanic bronchitis, chronic bronchitis, croupous bronchitis, fibrosis bronchitis, hemorrhagic bronchitis, algal infectious bronchitis, obstructive bronchitis, hyperplastic bronchitis, gastric bronchitis, rotative bronchitis and allergic bronchitis), Bronchocentrifugal granulomatosis, Bronchial edema, Bronchial esophagus, Tracheal carcinoma, Tracheal cyst, Bronchial Syndrome, bronchomalacia, bronchiopharymia (eg bronchial aspergillosis), bronchopulmonary spirokitaosis, hemorrhagic bronchitis, bronchial seborrhea, bronchial spasms, bronchial wall bleeding, bronchial stenosis, biot breathing, bronchial breathing , Coussuma breathing, Coussuma-Kien breathing, respiratory acidosis, respiratory alkalosis, neonatal respiratory distress syndrome, respiratory failure, respiratory sclerosis, respiratory syncytial virus, and the like.

In certain embodiments, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists of the present invention treat, prevent and / or diagnose chronic obstructive pulmonary disease (COPD). Used to

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the present invention are used to treat, prevent and / or diagnose fibrosis and the conditions associated therewith. Examples of fibrosis include, but are not limited to, cystic fibrosis (including cystic fibrosis of the pancreas, Clark-Hardfield syndrome, fibrocystic disease of the pancreas, mucoadhesion and adhesion), endocardiomyofibrosis, idiopathic Posterior peritoneal fibrosis, meninges fibrosis, mediastinal fibrosis, measurable subepithelial fibrosis, periphery fibrosis, peripheral fibrosis, pipestem fibrosis, restorative fibrosis, subepidermal fibrosis and simmus clay pipestem fibrosis.

TNF family ligands are known to be one of the most pleiotropic cytokines, including many cellular responses (including cytotoxicity, antiviral activity, immunomodulatory activity and transcriptional regulation of several genes) (DV Goeddel et al., "Tumor Necorsis Factors: Gene Structure and Biological Activities. "Symp. Quant. Biol. 51: 587-609 (1986). Cold Spring Harbor: B. Beutler and A. Cerami.Annu. Rev. Biochem. 57: 505-518 (1988 LJ Old.Sci. Am. 258: 59-75 (1988); W. Fiers, FEBS Lett. 285: 199-224 (1991)). TNF family ligands comprising Neutrokine-alpha and / or Neutrokine-alphaSV of the present invention induce these various cellular responses by binding to TNF family receptors. Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are believed to induce an efficient cellular response including all genotypes, phenotypes and / or morphological changes for cells, cell lines, tissues, tissue cultures or patients. Lose. As mentioned, such cellular cellular responses include diseases associated with increased apoptosis or suppression of apoptosis as well as normal physiological responses to TNF family ligands. Apoptosis-programmed cell death is a physiological mechanism implicated in the lack of peripheral B and / or T lymphocytes of the immune system and its dysregulation can induce many other pathogenesis processes (JC Ameisen, AIDS 8: 1197-1213 (1994)). PH Krammer et al., Curr. Opin. Immunol. 6: 279-289 (1994)).

Diseases associated with increased cell survival or inhibition of apoptosis that can be diagnosed, treated or prevented with Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof Examples include cancer (eg, follicular lymphoma, carcinoma with p53 mutations and hormone-dependent tumors, including but not limited to colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, bowel cancer, testicular cancer, Gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, epithelioma, osteoblastoma, osteoclast, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer), autoimmune diseases (e.g., systemic erythema) Reflection lupus and immune associated glomerulonephritis, rheumatoid arthritis), viral infections (eg, herpes virus, pox virus and adenovirus), inflammation, graft-versus-host Ring, include acute transplant rejection and chronic graft're added. Thus, in a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof are used to treat, prevent and / or diagnose autoimmune diseases. And / or growth of various cancers described herein, including, for example, lymphocytic leukemia (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and cystic lymphomas, Used to inhibit development and / or metastasis. In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides of the invention are cancer cells or tissues (B cell lineage associated cancers (eg, CLL and MLL), lymphoid leukemia or lymphoma) Is used to activate, differentiate, or multiply, thereby making the cell more susceptible to cancer therapy (eg, chemotherapy or radiotherapy).

Moreover, in another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the present invention grow, develop and / or metastasize in malignant and related diseases. Examples of related diseases include leukemia (acute leukemia (e.g. acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myeloid monocytic leukemia, monocytic leukemia and red leukemia). ) And chronic leukemia (eg, including chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), erythrocytosis, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom giant globulin Hypertensive tumors including, but not limited to, hypertension, including but not limited to, fibrosarcoma, mucous sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Epithelial sarcoma, lymphangiosarcoma, lymphangiosarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma , Gland carcinoma, sebaceous gland carcinoma, papilloma sarcoma, papillary adenocarcinoma, cystic carcinoma, spinal carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, chorioepithelioma, normal somatoma, embryonic sarcoma, Wilm tumor, uterine cancer, testicular tumor, Lung carcinoma, non-small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, cranioblastoma, epithelial carcinoma, pineal carcinoma, hemangioblastoma, auditory neuroma, oligodendroma, male hemangioma, melanoma, neuroblastoma and retinoblastoma Can be mentioned.

Diseases associated with increased apoptosis that can be diagnosed, treated or prevented with Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and agonists and antagonists thereof, include AIDS, neurodegenerative diseases (e.g., , Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinal pigmentosa, cerebellar degeneration, myelodysplastic syndromes (e.g. aplastic anemia), ischemic injury (e.g. wounds caused by myocardial infarction, stroke and reperfusion injury), Toxin-induced liver disease (eg, alcohol-induced disease), septic shock, cachexia and loss of appetite. Thus, in a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the present invention treat, diagnose and / or Used to prevent

In a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or agonists and / or antagonists thereof (eg, anti-Neutrokine-alpha antibodies) are human histoblastic lymphoma U-937. Inhibit the growth of cells in a dose-dependent manner. In a further preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or agonists and / or antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) of the invention comprise PC-3 cells, Inhibits the growth of HT-29 cells, HeLa cells, MCF-7 cells and A293 cells. In a very preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides and / or agonists and / or antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) may be used for prostate cancer, colon cancer, uterine cancer. Used to inhibit the growth, development and / or metastasis of species and breast carcinomas.

Thus, in a further preferred embodiment, the invention provides an effective amount of Neutrokine-alpha and / or Neutrokine-alphaSV or Neutrokine-alpha and / or cells expressing Neutrokine-alpha and / or Neutrokine-alphaSV receptors. Or agonists or antagonists thereof that can increase or decrease Neutrokine-alphaSV mediated signaling, thereby enhancing apoptosis induced by TNF family ligands. Preferably used to treat, prevent and / or diagnose diseases in which decreased apoptosis or reduced cytokine and adsorption molecule expression is observed by increasing or decreasing Neutrokine-alpha and / or Neutrokine-alphaSV mediated signaling. . Agonists or antagonists can include monoclonal antibodies against soluble forms of Neutrokine-alpha and / or Neutrokine-alphaSV and Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides.

In a further aspect, the invention provides an effective amount of Neutrokine-alpha and / or Neutrokine-alphaSV or Neutrokine-alpha and / or Neutrokine to cells expressing Neutrokine-alpha and / or Neutrokine-alphaSV receptors. -A method for inhibiting apoptosis induced by TNF family ligands, including administering an agonist or antagonist that can increase or decrease alphaSV mediated signaling. Preferably it is used to treat, prevent and / or diagnose diseases in which increased apoptosis or NF-kappaB expression is observed by increasing or decreasing Neutrokine-alpha and / or Neutrokine-alphaSV mediated signaling. Agonists or antagonists can include monoclonal antibodies against soluble forms of Neutrokine-alpha and / or Neutrokine-alphaSV and Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides.

Since Neutrokine-alpha and / or Neutrokine-alphaSV belong to the TNF superfamily, the polypeptide must also regulate angiogenesis. In addition, because Neutrokine-alpha and / or Neutrokine-alphaSV inhibit immune cell function, the polypeptide has a wide range of anti-inflammatory activity. Neutrokine-alpha and / or Neutrokine-alphaSV can be used as angiogenesis inhibitors to stimulate the infiltration and activation of host defense cells (eg, cytotoxic T cells and macrophages) and to inhibit tumor angiogenesis. Can be treated, prevented and / or diagnosed. Those skilled in the art will recognize other noncancerous conditions that do not require blood vessel proliferation. They can also be used to enhance host defense against resistant chronic and acute infections (eg, myobacterial infections) through the attraction and activation of microbial leukocytes. In addition, Neutrokine-alpha and / or Neutrokine-alphaSV may be induced by inhibition of IL-2 biosynthesis for the treatment of T-cell mediated autoimmune bill ring and lymphoid lymphoma (eg, chronic lymphocytic lymphoma (CLL)). It can be used to inhibit T-cell proliferation. Neutrokine-alpha and / or Neutrokine-alphaSV can also be used to promote wound healing through both restoration of connective tissue and debris clearance that promote inflammatory cells. In the same way, Neutrokine-alpha and / or Neutrokine-alphaSV can also be used to treat, prevent and / or diagnose other fibrotic diseases including cirrhosis, osteoarthritis and pulmonary fibrosis. Neutrokine-alpha and / or Neutrokine-alphaSV also increases the presence of eosinophils with the unique function of killing larvae of parasites that penetrate tissues, such as in schistosomiasis, trichinosis and roundworm. In addition, Neutrokine-alpha and / or Neutrokine-alphaSV regulate the activation and differentiation of several hematopoietic progenitor cells, for example by secreting mature white blood cells from bone marrow after chemotherapy, i.e. regulating hematopoiesis with the recruitment of stem cells. Can be used to Neutrokine-alpha and / or Neutrokine-alphaSV can also be used to treat, prevent and / or diagnose sepsis.

The polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are useful for diagnosing and treating or preventing a wide range of diseases and / or conditions. Such diseases and conditions include, but are not limited to, cancers (eg, immune cell-related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancers associated with mutations or modifications of p53, brain cancer, bladder cancer, cervical cancer, Colon cancer, colon cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, gastric cancer, etc., lymphocytic proliferative diseases (e.g. lymphadenitis), microorganisms (e.g., viruses, bacteria, etc.) infections (e.g., HIV-1 infection, HIV- 2 infection, herpesvirus infection (including but not limited to HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human papilloma virus infection , Hepatitis (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori infection, permeable Staphylococcus, etc., parasite infection, nephritis, bone disease (e.g. osteoporosis), arteriosclerosis, pain, cardiovascular diseases (e.g. neovascularization, Angiogenesis or reduced circulation (eg ischemic disease (eg myocardial) Infarction, stroke, etc.), AIDS, allergies, inflammation, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinal pigmentosa, cerebellar degeneration), graft rejection (acute and chronic), graft-versus-host Diseases, diseases caused by myelodyscopy disorders (eg, aplastic anemia, etc.), destruction of joint tissues in rheumatism, liver diseases (eg, acute and chronic hepatitis, liver damage and cirrhosis), autoimmune diseases (eg, multiple sclerosis, rheumatism) Arthritis, systemic lupus erythematosus, immunocomplex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Haimoto's thyroiditis, etc., cardiomyopathy (e.g., dilated cardiomyopathy), diabetes mellitus, diabetes complications (e.g. diabetes) Kidney disease, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, sepsis shock and ulcerative colitis.

The polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are useful for promoting angiogenesis, wound healing (eg, wounds, burns and fractures). The polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are also useful as adjuvants for enhancing an immune response against a particular antigen, an antiviral immune response.

More generally, the polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are useful for modulating (ie, increasing or decreasing) an immune response. For example, the polynucleotides and / or polypeptides of the invention are useful for preparing or recovering from surgery, trauma, radiotherapy, chemotherapy and transplantation or for promoting immune responses and / or recovery in the elderly and immunocompromised persons. Can be used to Alternatively, the polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune diseases. In certain embodiments, the polynucleotides and / or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions as described herein or known in the art.

Preferably, agonists or antagonists of Neutrokine-alpha and / or Neutrokine-alphaSV polynucleotides or polypeptides and / or Neutrokine-alpha and / or Neutrokine-alphaSV (eg anti-Neutrokine-alpha) Treatment with the antibody comprises administering an effective amount of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide or an agonist or antagonist thereof to the patient or removing the cells from the patient and the Neutrokine-alpha and / or Or by supplying Neutrokine-alphaSV polynucleotides and resupplying the engineered cells to the patient (ex vivo therapy). In addition, as described herein, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides or polynucleotides can be used as an adjuvant in a vaccine to induce an immune response to an infectious disease.

Formulation and Administration

The Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide composition (preferably containing a polypeptide that is a soluble form of Neutrokine-alpha and / or Neutrokine-alphaSV extracellular domains) is clinically determined for each patient. Conditions (especially side effects of treatment with Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides alone), delivery sites, methods of administration of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide compositions It is formulated and administered in a pattern consistent with good medical practice, taking into account the administration schedule, and other factors known to the physician. Accordingly, in consideration of these, the "effective amount" of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides for the purposes herein is determined.

As a general suggestion, the total pharmaceutically effective amount of parenteral administered Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides per dose may vary from about 1 microgram / kg body weight per patient, although this may vary depending on therapeutic judgment. 10 mg / kg / day. More preferred doses are at least 0.01 mg / kg / day and most preferably about 0.01 to 1 mg / kg / day for humans.

In another embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention are administered to a human at a dose of 0.0001 to 0.045 mg / kg / day, preferably 0.0045 to 0.045 mg / kg / day Is administered at a dose of, more preferably, about 45 micrograms / kg / day, and in mice a dose of about 3 mg / kg / day.

When administered continuously, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are typically injected 1 to 4 times per day at a dose of about 1 microgram / kg / hour to about 50 micrograms / kg / hour For example, it is administered by continuous subcutaneous infusion using a minipump. Intravenous sac solutions may also be used.

The duration of treatment and the interval at which post-treatment reactions are needed to observe changes vary depending on the desired effect.

In certain embodiments, the total pharmaceutically effective amount of parenterally administered Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides per dose may vary from about 0.1 micrograms / kg body weight per day, depending on therapeutic judgment. 45 μg / kg / day. More preferred doses are at least 0.1 μg / kg / day and most preferably about 0.01 to 50 micrograms / kg / day for humans. Neutrokine-alpha and / or Neutrokine-alphaSV can be administered by continuous infusion, several divided injections per day (e.g., three or more times daily or two times daily), once daily or divided intervals (e.g., twice daily). , Once a day, every other day, twice a week, once a week, once a week, once a month, once every two months and once every quarter). For continuous infusion, Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides typically range from about 0.001 micrograms / kg / hour to about 10 micrograms / kg / hour to about 50 micrograms / kg / hour. Doses are administered 1 to 4 times per day, or by continuous subcutaneous infusion, eg using a minipump.

Effective doses of the compositions according to the invention to be administered can be determined by methods well known to those skilled in the art taking into account such variables as biological half-life, bioavailability and toxicity. Such measurements are within the skill of one of ordinary skill in the art, particularly in view of the detailed description provided herein.

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides during treatment may also play an important role in determining therapeutically and / or pharmacologically effective methods of administration. Dose changes, such as repeated administration of relatively low doses of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides over a relatively long period of time, may result in relatively high doses of Neutrokine-alpha and / or Neutrokine-alpha over a relatively short period of time. Repeated administration of SV may provide a therapeutically and / or pharmacologically distinct effect from the effect achieved (see, eg, serum immunoglobulin level experiments described in Example 6).

Freireich, E.J. Using the equivalent surface area dose conversion factors described in et al., Cancer Chemotheraphy Reports 50 (4): 219-44 (1966), those skilled in the art will appreciate that Neutrokine-alpha and / or Neutrokine-alpha in a given experimental system. Data obtained using SV can be easily converted into accurate estimates of pharmaceutically effective amounts of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides to be administered per dose in other experimental systems. Experimental data obtained via administration of Neutrokine-alpha in mice (see Example 6) are shown in Neutrokine-alpha in rats, monkeys, dogs and humans via the conversion factors provided by Freireich et al. To an accurate estimate of the pharmaceutically effective dose of The conversion table below (Table 3) summarizes the data provided by Freyrach. Table 3 provides factors suitable for converting doses expressed in mg / kg from one species to equivalent surface area doses expressed in mg / kg from another species.

Equivalent surface area dose conversion factor. Mouse (20g) Rat (159g) Monkey (3.kg) Piece (8kg) Person (60kg) mouse One 1/2 1/4 1/6 1/12 Rat 2 One 1/2 1/4 1/7 monkey 4 2 One 3/5 1/3 dog 6 4 5/3 One 1/2 Person 12 7 3 2 One

Thus, for example, using the conversion factors provided in Table 3, 1.5 mg / kg in monkeys because the dose of 50 mg / kg in mice is (50 mg / kg) x (1/4) = 12.5 mg / kg Is switched to the appropriate dose. As a further example, doses of 0.02, 0.08, 0.8, 2 and 8 mg / kg in mice are 1.667 micrograms / kg, 6.67 micrograms / kg, 66.7 micrograms / kg, 166.7 micrograms / kg and 0.667 in humans, respectively. The same effect is seen with a dose of micrograms / kg.

Pharmaceutical compositions containing the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention may be oral, rectal, parenteral, subcutaneous, intranasal, vaginal, intraperitoneal, topical (powders, ointments, Drops or transdermal patches), oral or oral or nasal sprays (eg, via aspiration of vapor or powder). In one embodiment, “pharmaceutically acceptable carrier” means any type of nontoxic solid, semisolid or liquid filler, diluent, encapsulating agent or formulation aid. In certain embodiments, “pharmaceutically acceptable” means that it can be used in an animal, especially a human, and is listed by a US or other generally approved pharmacopoeia that has been approved by federal or state officials. Non-limiting examples of suitable pharmaceutical carriers according to this embodiment are described in E.W. Martin, "Remington's Pharmaceutical Sciences" and include sterile liquids such as vegetable, synthetic or animal oils (including petroleum oil), such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially in injectable solutions. The composition may contain small amounts of wetting or emulsifying agents or pH buffers if necessary. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

As used herein, the term “parenteral” refers to a mode of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV compositions of the invention (including polypeptides, polynucleotides and antibodies and agonists and / or antagonists thereof) are administered subcutaneously.

In another preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV compositions of the invention (including polypeptides, polynucleotides and antibodies and agonists and / or antagonists thereof) are administered intravenously.

Neutrokine-alpha and / or Neutrokine-alphaSV compositions of the invention are also suitably administered by a sustained release system. Suitable examples of sustained release compositions include suitable polymeric materials (e.g., semipermeable polymer matrices shaped like films or microcapsules), suitable hydrophobic materials (e.g. as emulsions in acceptable oils) or ion exchange resins and slightly soluble derivatives. (Eg, soluble salts).

Sustained release materials include polylactide (US Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22: 547-556 ( 1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) Or poly-D-(-)-3-hydroxybutyl acid (EP 133,988).

Sustained release compositions also include compositions of the invention entrapped in liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein) and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patents 4,485,045 and 4,544,545 and EP 102,324. Usually, liposomes are bovine (about 200-800 angstroms) monolayers with lipid content of at least about 30 mole percent cholesterol and the selected ratio can be adjusted for optimal Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide therapy. .

In another embodiment, the fatty composition of the present invention comprises a crystalline form known in the art.

In yet further embodiments, the compositions of the present invention are delivered via a pump (Langer, Supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)).

Other controlled release systems are described in Langer, Science 249: 1527-1533 (1990).

For parenteral administration, in one embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides generally contain a polypeptide of the desired purity to the patient at a pharmaceutically acceptable carrier, i.e. at the dose and concentration employed. Formulated by mixing in a unit dose injectable form (solution, suspension or emulsion) with a carrier which is nontoxic and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidants and other compounds that are known to be harmful to the polypeptide.

Generally, formulations are prepared by uniformly and densely mixing Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides with liquid carriers or finely divided solid carriers or both. The product is then shaped into the desired formulation, if necessary. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the patient. Examples of such carrier vehicles include water, saline, ring gel solutions and dextrose solutions. Non-aqueous vehicles such as nonvolatile oils and ethyl oleate are also useful for liposomes as well as herein.

The carrier suitably contains small amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are nontoxic to patients at the dosages and concentrations employed and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof; Antioxidants such as ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); Proteins (eg, serum albumin, gelatin or immunoglobulins); Hydrophilic polymers (eg, polyvinylpyrrolidone); Amino acids (glycine, glutamic acid, aspartic acid or arginine); Monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose, sucrose or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; County ions such as sodium; Preservatives such as cresol, phenol, chlorobutanol, benzyl alcohol and parabens; And / or nonionic surfactants such as polysorbates, poloxamers or PEGs.

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides typically have a pH of about 3 to 10 or a pH of 3 to 8, more preferably a pH of 5 to 8, most preferably 6 to 7 in the vehicle. At a pH of about 0.001 mg / ml to 100 mg / ml or 0.1 mg / ml to 100 mg / ml, preferably 1 to 10 mg / ml or 1 to 10 mg / ml. It will be understood that Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide salts are formed when certain of the above excipients, carriers or stabilizers are used.

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides to be used for therapeutic administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes (eg 0.2 micron membranes). Therapeutic Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide compositions are generally placed in a container with a sterile entry opening, such as a vial with a stopper penetrated by an intravenous solution bag or a hypodermic syringe.

Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are usually stored in single or multiple dose containers, such as sealed ampoules or vials, as aqueous solutions or as lyophilizers for regeneration. As an example of lyophilizer, a 10-ml vial is charged with 5 ml of sterile-filtered 1% (w / v) aqueous Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide solution and the resulting mixture is lyophilized. Let's do it. Infusion solutions are prepared by recovering lyophilized Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides into bacteriostatic water for injection.

As another alternative, the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides are stored in a single dose container in lyophilized form. The infusion solution is restored to a sterile carrier for injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical composition according to the invention. Optionally, the container may optionally be accompanied by a notice stipulated by the governmental authority that controls the manufacture, use or sale of a medicament or biological product, which will give government approval of the manufacture, use or sale for administration to humans. Reflect. In addition, the polypeptides of the present invention can be used in combination with other therapeutic compounds.

The compositions of the present invention can be administered alone or in combination with other adjuvants. Adjuvants that may be administered with the compositions of the present invention include, but are not limited to, alum, alum and a mixture of deoxycholate (ImmunoAg), MTP-PE (Biocine Corp), QS21 (Genentech, Inc.), BCG And MPL. In certain embodiments, the compositions of the present invention are administered in combination with QS-21. Other adjuvants that may be administered with the compositions of the invention include, but are not limited to, monophosphoryl lipid immunomodulators, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts, MF-59 and virosome adjuvants. Technology is included. Vaccines that can be administered with the compositions of the present invention include, but are not limited to, MMR (measles, mumps, rubella), polio, chicken pox, tetanus / diphtheria, hepatitis A, hepatitis B, haemophilus influenza , Vaccines and / or PNEUMOVAX-23 ^™ to protect against whooping cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, spermitis, rabies, typhoid fever and whooping cough. The combination may be administered simultaneously (eg as a mixture), separately but simultaneously or sequentially or sequentially. This includes administering the combined formulations together as a therapeutic mixture and also administering the combined formulations separately but simultaneously (eg, via separate intravenous lines to the same person). Administration in the "blended" state further includes administering one of the compounds or agents first followed by the second administration separately.

In another particular embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^™ to treat, prevent and / or diagnose infection and / or all diseases, conditions and / or symptoms associated therewith. In one embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^™ to treat, prevent and / or diagnose Gram-positive bacterial infections and / or all diseases, conditions and / or symptoms associated therewith. In another embodiment, the compositions of the present invention can be used in combination with PNEUMOVAX-23 ^™ to treat, prevent, and / or treat all diseases, disorders and / or symptoms associated with one or more bacterial members of the genus Enterococcus and / or Streptococcus. And / or diagnose. In another embodiment, the compositions of the present invention can be used in combination with PNEUMOVAX-23 ^™ to treat, prevent and / or diagnose infection and / or all diseases, disorders and / or symptoms associated with one or more bacterial members of Group B Streptococcus. do. In another embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^™ to treat, prevent and / or diagnose infections and / or all diseases, disorders and / or symptoms associated with Streptococcus pneumoniae.

The compositions of the present invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, chemotherapeutic agents, antibiotics, antiviral agents, steroidal and nonsteroidal anti-inflammatory agents, conventional immunotherapeutic agents and cytokines. The combination may be administered simultaneously (eg as a mixture), separately but simultaneously or sequentially or sequentially. This includes administering the combined agents together as a therapeutic mixture and also administering the combined agents separately but simultaneously (eg, via separate intravenous lines to a human). Administration of the “blended” state further includes administration of one of the compounds or agents first and a second of administration separately.

In one embodiment, the compositions of the present invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that can be administered with the compositions of the present invention include, but are not limited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta) , LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (WO 96/14328 ), AIM-I (WO 97/33899), AIM-II (WO 97/34911), APRIL (J. Exp. Med. 188 (6): 1185- 1190), endocine-alpha (WO 98/07880), OPG and Neutrokine-alpha (WO 98/18921), OX40 and nerve growth factor (NGF) and Fas, Soluble forms of CD30, CD27, CD40 and 4-IBB, TR2 (WO 96/34095), DR3 (WO 97/33904), DR4 (WO 98) / 32856), TR5 (International Patent Publication No. WO 98/30693), TR6 ( Patent Publication No. WO 98/30694), TR7 (International Patent Publication No. WO 98/41629), TRANK, TR9 (International Patent Publication No. WO 98/56892), TR10 (International Patent Publication No. WO) 98/54202), 312C2 (WO 98/06842) and TR12.

In a preferred embodiment, the composition of the present invention comprises a CD40 ligand (CD40L), a soluble form of CD40L (eg AVREND ^™ ), a biologically active fragment, variant or derivative of CD40L, an anti-CD40L antibody (eg an agonistic antibody or antagonistic antibody) And / or anti-CD40 antibodies (eg, agonistic antibodies or antagonistic antibodies).

In certain embodiments, the compositions of the present invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and / or protease inhibitors. Nucleoside reverse transcriptase inhibitors that can be administered in combination with the compositions of the present invention include, but are not limited to, RETROVIR ^™ (dozibudine / AZT), VIDEX ^™ (didanocin / ddI), HIVID ^™ (Zalcitabine / ddC) , ZERIT ^™ (Stavudine / d4T), EPIVIR ^™ (Lamivudine / 3TC) and COMBIVIR ^™ (Zidovudine / Lamivudine). Nonnucleoside reverse transcriptase inhibitors that can be administered in combination with the compositions of the present invention include, but are not limited to, VIRAMUNE ^™ (nevirapine), RESCRIPTOR ^™ (delaviradine), and SUSTIVA ^™ (epavirenz). . Protease inhibitors that can be administered in combination with the compositions of the invention include, but are not limited to, CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir), INVIRASE ^™ (saquinavir), and VIRACEPT ^™ (Nlpina) Bir). In certain embodiments, the antiretroviral agent, nucleoside reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor and / or protease inhibitor are combined with the compositions of the present invention to treat, prevent and / or diagnose AIDS and / or to treat HIV infection. It can be used to treat, prevent and / or diagnose.

In another embodiment, the compositions of the present invention can be administered in combination with an anti-opportunistic infectious agent. Anti-opportunistic agents that can be administered in combination with the compositions of the present invention include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE ^TM , DAPSONE ^TM , PENTAMIDINE ^TM , ATOVAQUONE ^TM , ISONIAZID ^TM , RIFAMPIN ^TM , PYRAZINAMIDE ^TM , ETHAMBUTOL ^TM , ^{rIFABUTIN TM, CLARITHROMYCIN TM, AZITHROMYCIN TM} , GANCICLOVIR TM, fOSCARNET TM, CIDOFOVIR TM, fLUCONAZOLE TM, iTRACONAZOLE TM, KETOCONAZOLE TM, aCYCLOVIR TM, FAMCICOLVIR TM, PYRIMETHAMINE TM, lEUCOVORIN TM, NEUPOGEN TM ( Phil Grass team / G-CSF) And LEUKINE ^™ (sargramostim / GM-CSF). In certain embodiments, the compositions of the present invention are used in combination with TRIMETHOPRIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ and / or ATOVAQUONE ^™ to prophylactically treat, prevent and / or diagnose opportunistic pneumocytis carini pneumonia. In another specific embodiment, the compositions of the present invention are used in combination with ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ and / or ETHAMBUTOL ^™ for prophylactically treating, preventing and / or diagnosing opportunistic mycobacterium avium complications. do. In another specific embodiment, the compositions of the present invention are used in combination with RIFABUTIN ^™ , CLARITHROMYCIN ^™ and / or AZITHROMYCIN ^™ to prophylactically treat, prevent and / or diagnose opportunistic mycobacterium tuberculosis infections. In another specific embodiment, the compositions of the present invention are used in combination with GANCICLOVIR ^™ , FOSCARNET ^™ and / or CIDOFOVIR ^™ to prophylactically treat, prevent and / or diagnose opportunistic cytomegalovirus infections. In another specific embodiment, compositions of the present invention is used to treat, prevent and / or ^TM FLUCONAZOLE, ITRACONAZOLE ^TM and / or KETOCONAZOLE ^TM and formulated to diagnose opportunistic fungal infection prophylactically. In another specific embodiment, the compositions of the present invention are used in combination with ACYCLOVIR ^™ and / or FAMCICOLVIR ^™ for prophylactically treating, preventing and / or diagnosing opportunistic herpes simplex virus type I and II infections. In another specific embodiment, the compositions of the present invention are used in combination with PYRIMETHAMINE ^™ and / or LEUCOVORIN ^™ for prophylactically treating, preventing and / or diagnosing opportunistic toxoplasmagondi infection. In another specific embodiment, the compositions of the present invention are used in combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to prophylactically treat, prevent and / or diagnose opportunistic bacterial infections.

In a further aspect, the compositions of the present invention are administered in combination with an antiviral agent. Antiviral agents that can be administered with the compositions of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, and lemantidine.

In a further embodiment, the compositions of the present invention are administered in combination with antibiotics. Antibiotics that can be administered with the compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactams (glycopeptides), beta-lactamases, clindamycin, chloramphenylcol, cephalosporins, ciprofloxacin, Erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinazolone, rifampin, streptomycin, sulfonamide, tetracycline, trimetapririm, trimethoprim-sulfamtoxazole and vancomycin.

Conventional non-specific immunosuppressive agents that can be administered in combination with the compositions of the present invention include, but are not limited to, steroids, cyclosporin, cyclosporine analogs, cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathio Prin, FK-506, 15-deoxyspergualin and other immunosuppressive agents that act by inhibiting the function of the responding T cells.

In certain embodiments, the compositions of the present invention are administered in combination with an immunosuppressant. Immunosuppressants that can be administered with the compositions of the invention include, but are not limited to, ORTHOCLONE ^™ (OKT3), SANDIMMUNE ^™ / NEORAL ^™ / SANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CELLCEPT ^™ (mycophenol) Rate), azathioprine, glucocorticosteroids and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents can be used to inhibit rejection of organ or bone marrow transplants.

In a preferred embodiment, the compositions of the present invention are administered in combination with steroid therapy. Steroids that can be administered in combination with the compositions of the present invention include, but are not limited to, oral corticosteroids, prednisone and methylprednisolone (eg, IV methylprednisolone). In certain embodiments, the compositions of the invention are administered in combination with prednisone. In a further particular embodiment, the compositions of this invention are administered in combination with prednisone and an immunosuppressant. Immunosuppressants that can be administered with the compositions of the present invention and prednisone include, but are not limited to, azathioprine, cyclophosphamide and cyclophosphamide IV. In another particular embodiment, the compositions of this invention are administered in combination with methylprednisolone. In a further particular embodiment, the composition of the present invention is administered in combination with methyl prednisone and an immunosuppressive agent. Immunosuppressants that can be administered in combination with the compositions of the present invention and methylprednisone include, but are not limited to, azathioprine, cyclophosphamide and cyclophosphamide IV.

In a preferred embodiment, the composition of the present invention is administered in combination with an antimalarial agent. Antimalarial agents that can be administered with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine and / or quinacrine.

In a preferred embodiment, the compositions of the present invention are administered in combination with NSAIDs.

In a typical embodiment, the compositions of the present invention are administered in combination with one, two, three, four, five, ten or more of the following drugs: NRD-101 (Hoechst Marion Roussel), Dimethaid, Oxa Prozin Potassium (Monsanto), Mecassermine (Chiron), T-614 (Toyama), Pemetrexed Disodium (Eli Lilly), Ateleutone (Abbott), Valdecosib, Eltenac (Byk Gulden) ), Campus, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS-Biomedix) , IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco), Paclitaxol (Angiotech), DW-166HC (Dong Wha), Darbupelon mesylate (Warner-Lambert), soluble TNF-receptor 1 ( Synogen; Amgen, Institute for Pharmaceutical Research (IPR-6001), Hoffman-La Roche, Scotia Pharmaceuticals (EF-5), Boehringer Ingelheim (BIIL-284), Boehringer Ingelheim (BIIF-1149), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau) and Butiksoko Bit propionate (WarnerLambert).

In a preferred embodiment, the compositions of the present invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomaleate, auranopine, cyclosporine, penicila Min, azathioprine, antimalarial drugs (eg, those described herein), cyclophosphamide, chlorambucil, gold, ENBREL ^™ (Etanercept), anti-TNF antibodies and prednisolone.

In a more preferred embodiment, the composition of the present invention is administered in combination with an antimalarial agent, methotrexate, anti-TNF antibody, ENBREL ^™ and / or suprapsalazine. In one embodiment, the compositions of this invention are administered in combination with methotrexate. In another embodiment, the compositions of the present invention are administered in combination with an anti-TNF antibody. In another embodiment, the compositions of this invention are administered in combination with methotrexate and anti-TNF antibodies. In another embodiment, the compositions of the present invention are administered in combination with suprapsalazine. In another specific embodiment, the compositions of this invention are administered in combination with methotrexate, anti-TNF antibodies and suprasalazine. In another embodiment, the compositions of this invention are administered in combination with ENBREL ^™ . In another embodiment, the compositions of this invention are administered in combination with ENBREL ^™ and methotrexate. In another embodiment, the compositions of this invention are administered in combination with ENBREL ^™ , methotrexate and suplasalazine. In another embodiment, the compositions of this invention are administered in combination with ENBREL ^™ , methotrexate and suplasalazine. In another embodiment, one or more antimalarial agents are combined with one of the combinations described above. In certain embodiments, the compositions of the present invention are administered in combination with antimalarial agents (eg hydroxychloroquine), ENBREL ^™ , methotrexate and supposalazine. In another particular embodiment, the compositions of the present invention are administered in combination with antimalarial agents (eg hydroxychloroquine), sulfasalazine, anti-TNF antibodies and methotrexate.

In a further aspect, the compositions of the present invention are administered in combination or alone with one or more intravenous immunoglobulin agents. Intravenous immune globulin preparations that may be administered with the compositions of the present invention is not limited to these but include ^{GAMMAR TM, IVEEGAM TM, SANDOGLOBULIN TM} , GAMMAGARD S / D TM and ^TM GAMIMUNE. In certain embodiments, the compositions of the present invention are administered in combination with an intravenous immunoglobulin agent in a transplantation therapy (eg bone marrow transplant).

CD40 ligand (CD40L), soluble form of CD40L (eg AVREND ^™ ), biologically active fragments, variants or derivatives of CD40L, anti-CD40L antibodies (eg agonistic or antagonistic antibodies) and / or anti-CD40 antibodies (eg , Antagonistic or agonistic antibodies).

In a further embodiment, the compositions of the present invention are administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that can be administered with the compositions of the present invention include, but are not limited to, glucocorticoids and nonsteroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, acrylic acetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, Pyrazole, pyrazolone, salicylic acid derivatives, thiazinecarboxamide, e-acetamidocapronic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amicsetrin, bendazac, benzidamine, butane Colum, Ifenpyramid, Ditazole, Emorphazon, Guazulene, Nabumethone, Nimesulide, Orgotane, Oxaceprole, Paraniline, Peri Soxal, Pipeoxime, Proquazone, Proxazole and Tenidab Included.

In another embodiment, the compositions of this invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); Antiestrogens (eg tamoxifen); Antimetabolites (eg, fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamic acid, placamycin, mercaptopurine and 6-thioguani); Cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, esturamustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin and Vincristine sulfate); Hormones (eg, methoxyprogesterone, esturamustine phosphate sodium, ethynyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol disulfate, chlorotrianicene and testosterone); Nitrogen mustard derivatives (eg, mephalen, korambucil, mechlorethamine (nitrogen mustard) and thiotepa); Steroids and combinations (eg, betamethasone sodium phosphate); And others (eg, dicarbazine, asparaginase, mitotaine, vincristine sulfate, vinblastine sulfate and etoposide).

In certain embodiments, the compositions of this invention are administered in combination with each of the combinations of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or components of CHOP. In another embodiment, the compositions of this invention are administered in combination with rituximab. In a further embodiment, the compositions of the present invention are combined with rituximab and CHOP or administered with every respective combination of components of rituximab and CHOP.

In a further embodiment, the compositions of the present invention are administered in combination with cytokines. Cytokines that may be administered with the compositions of the invention include, but are not limited to, GM-CSF, G-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40 , CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TNF-beta. In another embodiment, the compositions of the present invention include, but are not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL- Administration with all interleukins, including 20, IL-21 and IL-22. In a preferred embodiment, the compositions of the present invention are administered in combination with IL4 and IL10. Both IL4 and IL10 have been observed by the inventors to enhance Neutrokine-alpha mediated B cell proliferation.

In a further aspect, the compositions of the present invention are administered with chemokines. In another embodiment, the compositions of the present invention are administered with chemokine beta-8, chemokine beta-1 and / or macrophage inflammatory protein-4. In a preferred embodiment, the composition of the present invention is administered in combination with chemokine beta-8.

In a further embodiment, the compositions of the present invention are administered in combination with an IL-4 antagonist. IL-4 antagonists that can be administered with the compositions of the invention include, but are not limited to, soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides, biologically induced by IL-4 Anti-IL-4 receptor antibodies that bind IL-4 receptors without conducting signals, anti-IL4 antibodies that block binding of IL-4 to one or more IL-4 receptors, and IL-4 receptors that bind IL-4 receptors Included are muteins of IL-4 that do not conduct biological signals induced by. Preferably, the antibody used according to this method is a monoclonal antibody (including, for example, antibody fragments as described herein).

In a further aspect, the compositions of the present invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that can be administered with the compositions of the present invention include, but are not limited to, LEUKINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FILGRASTIM ^™ ).

In a further embodiment, the compositions of the present invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered with the compositions of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF -8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.

In addition, the compositions of the present invention may be administered alone or in combination with other methods of treatment, including but not limited to radiotherapy. Such combination therapy may be administered sequentially and / or simultaneously.

Agonists and Antagonists-Assays and Molecules

The invention also provides compounds that increase or block the action of Neutrokine-alpha or Neutrokine-alphaSV polypeptides on cells such as interaction with Neutrokine-alpha or Neutrokine-alphaSV binding molecules such as receptor molecules. Provided are methods of screening for identifying compounds. Agonists are compounds that increase the natural biological action of Neutrokine-alpha or Neutrokine-alphaSV or act in a similar way to Neutrokine-alpha or Neutrokine-alphaSV, while antagonists reduce or eliminate this action. It is a compound.

In another aspect, the invention provides a method of identifying receptor proteins or other ligand-binding proteins that specifically bind to Neutrokine-alpha or Neutrokine-alphaSV polypeptides. For example, cell membranes, such as membranes or preparations thereof, can be prepared from cells expressing molecules that bind Neutrokine-alpha or Neutrokine-alphaSV. This preparation is incubated with labeled Neutrokine-alpha or Neutrokine-alphaSV, and the Neutrokine-alpha or Neutrokine-alphaSV bound to the receptor or other binding protein is isolated and characterized according to conventional methods known in the art. Check it. As another alternative, Neutrokine-alpha or Neutrokine-alphaSV polypeptides are bound to a solid support so that the binding molecules dissolved from the cells bind to the column and are then eluted and characterized according to conventional methods.

In the assays of the present invention for agonists or antagonists, cell membranes such as membranes or agents thereof are Neutrokine-alpha or Neutrope, such as molecules of signaling or regulatory pathways regulated by Neutrokine-alpha or Neutrokine-alphaSV. It can be prepared from cells expressing a molecule that binds to keen-alphaSV. The agent is incubated with labeled Neutrokine-alpha or Neutrokine-alphaSV in the absence or presence of a candidate molecule which may be a Neutrokine-alpha or Neutrokine-alphaSV agonist or antagonist. The ability of a candidate molecule to bind a binding molecule is manifested by reduced binding of the labeled ligand. Molecules that bind without inducing the effects of Neutrokine-alpha on binding to Neutrokine-alpha or Neutrokine-alphaSV binding molecules are likely to be good antagonists. Molecules that bind well and exhibit the same or closely related effects as Neutrokine-alpha or Neutrokine-alphaSV are agonists.

Neutrokine-alpha or Neutrokine-alphaSV-like effects of potential agonists and antagonists, for example, measure the activity of a second messenger system after interaction of a candidate molecule with a cell or an appropriate cellular agent and determine the effect of Neutrokine- It can be measured by comparing the effect of a molecule to induce the same effect as alpha or Neutrokine-alphaSV or Neutrokine-alpha or Neutrokine-alphaSV. In this regard, second messenger systems that may be useful include, but are not limited to, AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.

Another example of an assay for Neutrokine-alpha or Neutrokine-alphaSV antagonists is a receptor molecule or recombinant Neutrokine-alpha or Neutrokine-alphaSV membrane-bound to Neutrokine-alpha or Neutrokine-alphaSV and a potential antagonist. It is a competition assay that binds to receptor molecules under conditions suitable for competition inhibition assays. Neutrokine-alpha or Neutrokine-alphaSV can be labeled, for example, by radioactivity and thus the efficacy of a potential antagonist by accurately measuring the number of Neutrokine-alpha or Neutrokine-alphaSV molecules bound to the receptor molecule. Can be evaluated.

Potential antagonists include small organic molecules, peptides, polypeptides (eg, IL-13), and antibodies that bind or inhibit the activity of the polypeptide of the invention. In addition, potential antagonists bind to the same site on the binding molecule, such as the receptor molecule, without inducing Neutrokine-alpha or Neutrokine-alphaSV induced activity and thereby remove Neutrokine-alpha or Neutrokine-alphaSV from the binding. It may be a small organic molecule, peptide, polypeptide or antibody such as a closely related protein that excludes and inhibits the action of Neutrokine-alpha or Neutrokine-alphaSV.

Other potential antagonists include antisense molecules. Antisense technology can be used to regulate gene expression via antisense DNA or RNA or through triple-helix formation. Antisense techniques are described, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression." CRC Press, Boca Raton, FL (1988). Antisense technology can be used to regulate gene expression via antisense DNA or RNA or through triple-helix formation. Antisense techniques are described, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression." CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, in Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); And Dervan et al., Science 251: 1360 (1991). This method is based on the binding of polynucleotides to complementary DNA or RNA. For example, the 5 'coding portion of a polynucleotide encoding the extracellular domain of a polypeptide of the invention can be used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to complement regions of genes involved in transcription thereby inhibiting the transcription and production of Neutrokine-alpha and / or Neutrokine-alphaSV. Antisense RNA oligonucleotides hybridize with mRNA in vivo and block the translation of mRNA molecules into Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit the production of Neutrokine-alpha and / or Neutrokine-alphaSV.

In one embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, the vector or portion thereof is transcribed to produce an antisense nucleic acid (RNA) of the invention. Such vectors contain sequences encoding Neutrokine-alpha and / or Neutrokine-alphaSV antisense nucleic acids. Such vectors can be maintained as episomes or integrated into chromosomes as long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by standard recombinant DNA techniques in the art. Vectors can be plasmids, viruses, and the like, known in the art for use in replication and expression in vertebrate cells. Expression of the sequences encoding Neutrokine-alpha and / or Neutrokine-alphaSV or fragments thereof may be possible with any promoter known in the art to act in vertebrates, preferably human cells. Such promoters may be inducible or constitutive. These promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310 (1981), promoters contained in the 3 ′ long terminal repeats of Loose sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980), herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296: 39-42 (1982) and the like.

Antisense nucleic acids of the invention comprise sequences complementary to at least a portion of the RNA transcripts of the Neutrokine-alpha and / or Neutrokine-alphaSV genes. However, absolute complementarity is desirable but not necessarily required. As referred to herein, a “complementary to at least a portion of an RNA” refers to a sequence that has sufficient complementarity to hybridize with the RNA to form a stable double strand, wherein the double strand Neutrokine-alpha and / or Neutrokine For alphaSV antisense nucleic acids, single strands of double stranded DNA can be tested or assayed for triple strand formation. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the larger the hybridizing nucleic acid, the more base mismatch with Neutrokine-alpha and / or Neutrokine-alphaSV RNA that the nucleic acid contains and can form a stable double stranded (or triple stranded). One skilled in the art can determine the acceptable level of mismatch by using standard procedures for measuring the melting point of hybridized complexes.

Oligonucleotides complementary to the 5 'end of the message (e.g., the 5' untranslated sequence up to and including the AUG initiation codon) work most efficiently when inhibiting translation. However, sequences complementary to the 3 'non-translated sequence of mRNA have also been shown to be effective in inhibiting translation of mRNA (Wagner, R., 1994, Nature 372: 333-335). Thus, oligonucleotides complementary to the 5'- or 3'-non-coding non-coding regions of Neutrokine-alpha and / or Neutrokine-alphaSV described in FIGS. 1A-B and 5A-B, respectively, are endogenous Neutrokine-alpha. And / or antisense methods that inhibit the translation of Neutrokine-alphaSV mRNA. Oligonucleotides complementary to the 5 ′ non-toxic region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are less efficient translation inhibitors but can be used in accordance with the present invention. Antisense nucleic acids, whether or not designed to hybridize to the 5'-, 3'- or coding regions of Neutrokine-alpha and / or Neutrokine-alphaSV mRNAs, should be at least 6 nucleotides in length and preferably Oligonucleotides consisting of 6 to about 50 nucleotides in length. In certain aspects oligonucleotides consist of at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the present invention may be single stranded or double stranded DNA or RNA or chimeric mixtures or derivatives or variants thereof. Oligonucleotides can be modified, for example, at base residues, sugar residues or phosphate backbones to enhance the stability, hybridization, and the like of the molecule. Oligonucleotides include peptides (eg, to target host cell receptors in vivo), cell membranes (Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre; et al., Proc. Natl. Acad. Sci. 84: 648-652 (1987); PCT Publication No. WO88 / 09810 published Dec. 5, 1988) or the blood-brain barrier (April 25, 1988) Transport promoters, hybridization-triggered cleavage agents (Krol et al., BioTechniques 6: 958-976 (1988)) or intervening agents via published PCT Publication No. WO89 / 10134) (Zon, Pharm. Res) 5: 539-549 (1988)). To this end, oligonucleotides may be linked to other molecules such as peptides, hybridization triggered crosslinkers, transport agents, hybridization triggered cleavage agents, and the like.

Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydrate Oxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylquinosine, inosine, N6-isopentenyladenin, 1- Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquiosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenyne, uracil- 5-oxyacetic acid (v), Waibutoxin, pseudouracil, quiocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thioura Sil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N One or more modified base residues selected from the group consisting of -2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine.

Antisense oligonucleotides may also include one or more modified sugar residues selected from the group consisting of, but not limited to, arabinose, 2-fluoroarabinose, xylose, and hexose.

In another embodiment, the antisense oligonucleotides include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphoramidate, methylphosphonate, alkyl One or more modified phosphate backbones selected from the group consisting of phosphoester and formacetal or analogs thereof.

In another embodiment, the antisense oligonucleotide is an alpha-anomeric oligonucleotide. Alpha-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNAs, and in contrast to normal beta-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15: 6625-6641 ( 1987)). Oligonucleotides are 2-0-methylribonucleotides (Inoue et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett. 215: 327-330 (1997).

The polynucleotides of the present invention can be synthesized by standard methods known in the art, for example, using automated DNA synthesizers (such as those available from BioSearch, Applied Biosystems, etc.). By way of example, phosphorothioate oligonucleotides are described in Stein et al., Nucl. Acids Res. 16: 3209 (1988)], and methylphosphonate oligonucleotides can be prepared using controlled pore free polymeric supports and the like [Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85: 7448-7451 (1988).

Antisense nucleotides complementary to the Neutrokine-alpha and / or Neutrokine-alphaSV coding region sequences can be used, while antisense nucleotides complementary to the transcribed non-toxic region are most preferred.

Potential antagonists according to the invention are also catalytic RNAs or ribozymes (see PCT International Publication No. WO90 / 11364 published October 4, 1990; Sarver et al., Science 247: 1222-1225 (1990) ). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to degrade Neutrokine-alpha and / or Neutrokine-alphaSV mRNAs, it is preferred to use hammerhead ribozymes. Hammerhead ribozymes cleave mRNA at the commanded position by contiguous regions forming base pairs complementary to the target mRNA. The only requirement is that the target mRNA has a two base 5'-UG-3 'sequence. The construction and production of hammerhead ribozymes is well known in the art and described in more detail in Haseloff and Gerlach, Nature 334: 585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within the nucleotide sequence of Neutrokine-alpha and / or Neutrokine-alphaSV (FIGS. 1A-B and 5A-B, respectively). Preferably, this ribozyme is such that the cleavage recognition site is located near the 5 'end of Neutrokine-alpha and / or Neutrokine-alphaSV mRNA, ie to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts. Manipulated to allow.

As with the antisense method, the ribozymes of the invention can be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) and can be used to produce Neutrokine-alpha and / or Neutrokine-alphaSV in vivo. Must be delivered to the expressing cell. DNA constructs encoding ribozymes can be inserted into cells in the same manner as described above for the insertion of antisense encoding DNA. Preferred delivery methods are DNA constructs that "encode" ribozymes such that the transfected cells can provide sufficient amounts of ribozymes to destroy endogenous Neutrokine-alpha and / or Neutrokine-alphaSV messages and inhibit translation. Is used under the control of strong constitutive promoters (eg, pol III or pol II promoters). Unlike antisense molecules, ribozymes are catalysts, so lower intracellular concentrations are needed for efficiency.

Endogenous gene expression can also be reduced by inactivating or "knocking out" Neutrokine-alpha and / or Neutrokine-alphaSV genes and / or promoters thereof using target homologous recombination [see: Smithies et al., Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51: 503-512 (1987); Thompson et al., Cell 5: 313-321 (1989). The entire contents of each of these documents are incorporated herein by reference. For example, the non-functional mutant polynucleotides of the present invention contiguous by DNA homologous to the endogenous polynucleotide sequence can be used to express the polypeptide of the present invention in the presence or absence of a selection marker and / or a negative selection marker. Cells can be transfected. In another embodiment, knockout occurs in cells containing, but not expressing, genes of interest using techniques known in the art. Insertion of the DNA construct through target homologous recombination results in inactivation of the target gene. This method is particularly suitable in the field of research and agriculture, where modifications of embryonic stem cells can be used to generate new animals with inactivated target genes (see Thomas & Capecchi 1987 and Thompson 1989, supra). However, this method can be commonly applied and used in humans, provided that the recombinant DNA construct is administered or targeted directly to the site of interest in vivo using appropriate viral vectors known to those skilled in the art. The entire contents of these documents are incorporated herein by reference.

In another embodiment, the antagonist according to the invention is a soluble form of Neutrokine-alpha and / or Neutrokine-alphaSV (eg, ligand binding domains of Neutrokine-alpha and / or Neutrokine-alphaSV, TNF conserved domains). Fragments of Neutrokine-alpha and / or ligand binding domains of Neutrokine-alpha and / or Neutrokine-alphaSV, TNF conserved domains and / or extracellular domains described in FIGS. Fragments of Neutrokine-alphaSV described in FIGS. 5A-B). Soluble forms of Neutrokine-alpha and / or Neutrokine-alphaSV that may be naturally present or synthesized are Neutrokine-alpha and / or Neutrokine-alphaSV receptors (e.g. DR5 (see WO 98/41629). ), TR10 (cf. 98/54202); 312C2 (cf. WO 98/06842) and TRN, TR11SV1 and TR11SV2 (US Patent Application No. 09 / 176,200) for natural Neutrokine-alpha and / or Antagonizes Neutrokine-alpha and / or Neutrokine-alphaSV mediated signaling by competing with Neutrokine-alphaSV or by forming multimers that can or cannot bind to receptors but cannot induce signal conduction These antagonists inhibit Neutrokine-alpha and / or Neutrokine-alphaSV mediated stimulation of lymphocyte (eg B-cell) proliferation, differentiation and / or activation. , CD30) and Neutrokine-alpha-Fc and / or Neutrokine-alphaSV-Fc fusion Antibodies specific for the sum protein.

TNF family ligand "means a natural, recombinant and synthetic ligand capable of binding to members of the TNF receptor line and inducing and / or blocking ligand / receptor signaling pathways. The TNF ligand family includes, but is not limited to, TNF- Alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in the complex heterotrimer LT-alpha2-beta), FasL, CD40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (WO 97/33899), AIM-II (WO 97/34911), APRIL (J. Exp. Med. 188 (6) : 1185-1190), endocine-alpha (International Publication No. WO 98/07880), Neutrokine-alpha (International Publication No. WO 98/18921), CD27L, CD30L, 4-1BBL, OX40L, CD27, CD30, 4- 1BB, OX40 and Nerve Growth Factor (NGF) In a preferred embodiment, the Neutrokine-alpha and / or Neutrokine-alphaSV TNF family ligands of the invention are DR5 (International Publication No. WO 98 /). 41629), TR10 (WO 98/54202), 312C2 (WO 98/06842) and TR11, TR11SV1 and TR11SV2 (US Patent Application 09 / 176,200).

Antagonists of the invention also include antibodies specific for the TNF family receptor or Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention. Antibodies according to the invention can be prepared according to any of a variety of standard methods using the Neutrokine-alpha and / or Neutrokine-alphaSV immunogens of the invention. As mentioned, such Neutrokine-alpha and / or Neutrokine-alphaSV immunogens include the complete Neutrokine-alpha and / or described in FIGS. 1A-B (SEQ ID NO: 2) and 5A-B (SEQ ID NO: 19), respectively. Neutrokine-alphaSV polypeptides (which may or may not include a leader sequence) and, for example, ligand binding domains, TNF-conserved domains, extracellular domains, transmembrane domains and / or intravesicular domains or combinations thereof Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide fragments comprising a.

Polyclonal and monoclonal antibody agonists or antagonists according to the invention are described in Tartaglia and Goeddel, J. Biol. Chem. 267 (7): 4304-4307 (1992); Tartaglia et al., Cell 73: 213-216 (1993) and PCT Application WO 94/09137, which are preferably specific for polypeptides of the invention having the amino acid sequence of SEQ ID NO: 2. (Ie, specifically bind). As used herein, the term "antibody" (Ab) or "monoclonal antibody" (mAb) refers to a complete molecule capable of binding an antigen as well as fragments thereof (eg, Fab and F (ab ') fragments). Fab, Fab 'and F (ab') fragments are removed more quickly from the circulation and lack Fc fragments and may have weaker nonspecific tissue binding of complete antibodies (Wahl et al., J. Nucl. Med. 24: 316-325 (1983).

In a preferred method, the antibody according to the invention is mAb. Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256: 495-497 (1975) and US Patent No. 4,376,110 ,; harlow et al., Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988: Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses.Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody Technology," In "Laboratory Techiques in Biochemistry and Molecular Biology , Volume 13 (Burdon et al., Eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds that bind Neutrokine-alpha and / or Neutrokine-alphaSV domains are also candidate agonists and antagonists according to the present invention. Such binding compounds can be captured using a yeast two-hybrid system (Fields and Song, Nature 340: 245-246 (1989)). Modifications of the yeast two-hybrid system have been published by Roger Brent and colleagues (Gyuris, Cell 75: 791-803 (1993); Zervos et al., Cell 72: 223-232 (1993)). Preferably, the yeast two-hybrid system is used according to the invention to capture compounds that bind to the ligand binding domain, extracellular, intracellular, transmembrane and death domains of Neutrokine-alpha and / or Neutrokine-alphaSV. do. Such compounds are good candidate agonists and antagonists according to the present invention.

For example, using the two-hybrid assay described above and using the extracellular or intracellular domains or portions of Neutrokine-alpha and / or Neutrokine-alphaSV receptors or portions thereof, Neutrokine-alpha and / or Neutrokine -Can identify cellular proteins that interact with alpha SV receptors in vivo. Such assays can also be used to identify ligands with potential efficacy or antagonistic activity of Neutrokine-alpha and / or Neutrokine-alphaSV receptor action. This screening assay was previously used to identify proteins that interact with the cytoplasmic domain of mouse TNF-RII and as a result two receptor bound proteins were identified (Rothe et al., Cell 78: 681 (1994)). . Such protein and amino acid sequences that bind to the cytoplasmic domain of Neutrokine-alpha and / or Neutrokine-alphaSV receptors are good candidate agonists and antagonists according to the present invention.

Other screening techniques include cells expressing the polypeptide of the invention in a system for measuring extracellular pH changes caused by receptor activation, as described, for example, in Science, 246: 181-296 (1989). Eg, transfected CHO cells). As another example, a potential agonist or antagonist may be contacted with a cell expressing a polypeptide of the invention and a second messenger response (eg signal conduction) may be measured to determine whether the potential antagonist or agonist is effective.

Agonists according to the invention include TNF family ligand peptide fragments, transforming growth factors, neurotransmitters (eg glutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells and anti- Natural and synthetic compounds such as metabolites are included. Preferred agonists include chemotherapeutic drugs such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Other agonists include ethanol and -amyloid peptides (Science, 267: 1457-1458 (1995)).

Preferred agonists are fragments of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention that stimulate lymphocyte (eg B cell) proliferation, differentiation and / or activation. Further preferred agonists include polyclonal and monoclonal antibodies or fragments thereof produced against Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention. Agonist antibodies generated against TNF family receptors are described by Tartaglia et al., Proc. Natl. Acad. Sci. USA 88: 9292-9296 (1991); And Tartaglia et al., J. Biol. Chem. 267: 4304-4307 (1992) (another reference: WO 94/09137).

In further embodiments, immunomodulatory molecules such as IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha may be lymphocytes (eg, B cells). ) Can be used as agonists of the Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of the invention that stimulate proliferation, differentiation and / or activation. In certain embodiments, IL4 and / or IL10 are used to enhance Neutrokine-alpha- and / or Neutrokine-alphaSV-mediated proliferation of B cells.

In a further aspect of the invention, cells genetically engineered to express a polypeptide of the invention or cells engineered to express the polypeptide of the invention (eg knockout) are administered to a patient in vivo. . Such cells may be obtained from patients (ie, animals including humans) or MHC-compliant donors, but are not limited to fibroblasts, bone marrow cells, blood cells (ie, lymphocytes), adipocytes, myocytes, endothelial cells, etc. This may be included. Methods of transfection or transduction (using viral vectors, preferably vectors incorporating transgenes into the cell genome), including but not limited to plasmids, cosmids, YACs, extruded DNA, electroporation, liposomes, etc. These cells are genetically engineered according to recombinant DNA techniques to introduce the coding sequences of the polypeptides according to the invention into cells or disrupt the coding sequences and / or endogenous regulatory sequences associated with the polypeptides of the invention. The coding sequence of the polypeptide according to the invention can be set under the control of a potent constitutive or inducible promoter or promoter / enhancer such that the expression and preferably secretion of the polypeptide according to the invention is achieved. Engineered cells expressing and preferably secreting the polypeptides of the invention can be introduced systemically into the patient (eg, circulating or intraperitoneally).

Alternatively, cells can be incorporated into the matrix or transplanted into the body, eg, genetically engineered fibroblasts can be implanted as part of a skin graft and genetically engineered endothelial cells can be lymphatic or vascular As part of the implant (see Anderson et al., US Pat. No. 5,399,349 and Mulligan & Wilson, US Pat. No. 5,460,959). The entire contents of these documents are incorporated herein by reference.

If the cells to be administered are non-autologous or non-MHC compatible cells, these cells can be administered using well known techniques that inhibit the development of a host immune response against the introduced cells. For example, cells can be introduced in a capsule form, which allows components to be exchanged into an adjacent extracellular environment while the introduced cells are not recognized by the host immune system.

In another aspect of the invention, the activity of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides can be reduced using "dominant negative". To this end, constructs encoding defective Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides, such as mutants in which all or part of the TNF-conserved domain has been deleted, can be used in gene therapy to produce appropriate target cells. May reduce the activity of Neutrokine-alpha and / or Neutrokine-alphaSV. For example, a nucleotide sequence that directs host cell expression of Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides, in which all or part of the TNF-conserved domain has been modified or deleted, can be incorporated into monocytes or other cells or tissues. It can be introduced (by in vivo or ex vivo gene therapy as described herein or known in the art). Alternatively, targeted homologous recombination can be used to introduce the deletion or mutation into endogenous Neutrokine-alpha and / or Neutrokine-alphaSV genes in the mononuclear cells of the patient. Engineered cells will express non-functional Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides (ie, ligands (eg multimers) that can bind but cannot induce signal conduction).

Chromosome black

Nucleic acid molecules of the invention are also valuable for chromosomal identification. The sequence can specifically target and hybridize specific sites on an individual's chromosome. In addition, it is currently necessary to identify specific sites on the chromosome. Only a few are currently used to mark chromosome positions as chromosome labeling reagents based on substantial sequence data (repeat polymorphism). Mapping of DNA to chromosomes according to the present invention is an important first step in elucidating the correlation between these sequences and disease related genes.

In certain preferred embodiments in this regard, the genomic DNA of the Neutrokine-alpha and / or Neutrokine-alphaSV genes is cloned using the cDNAs and / or polynucleotides described herein. This can be accomplished using generally available libraries and various known techniques. For this purpose, genomic DNA is then used for in situ chromosome mapping according to known techniques.

In addition, in some cases, sequences for chromosomes can be mapped by making PCR primers (preferably 15-25 bp) from cDNA. By analyzing the 3 'non-toxic region of the gene by computer, one does not link one or more exons in genomic DNA, thereby rapidly selecting primers that complicate the amplification process. These primers are then used for PCR selection of somatic hybrids containing individual chromosomes. Fluorescent in situ hybridization (FISH) of cDNA clones to mid-term chromosomal dispersions can be used to provide accurate chromosomal location in one step. This technique can be used with probes from cDNAs as short as 50 or 60 bp (Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988)).

Once the exact chromosomal location of the sequence is mapped, the physical location of the sequence on the chromosome may be correlated with the genetic map data. Such data are described, for example, in V. McKusick, Mendelian Inheritance in Man] (available on-line from Welch Medical Library of John Hopkins University). Subsequently, correlations between genes designated for the same chromosomal region and the disease are identified through family analysis (cogeneity of physically adjacent genes).

Subsequently, it is necessary to determine the difference in cDNA or genomic sequence between the patient and normal persons. If a mutation is observed in all or part of the patient but not in any normal person, the mutation must be a pathogen.

Based on current analysis of physical mapping and gene mapping techniques, cDNA located precisely in the chromosomal region associated with a disease may be one of 50 to 500 potential pathogens. (This assumes a mapping analysis of 1 megabase and one gene per 20 kb).

According to the technique described above, the combination of somatic hybrids and radiation hybrids determined that the chromosomal positions of Neutrokine-alpha and Neutrokine-alphaSV were 13q34, with very high confidence.

The present invention has been described above in general, and the present invention will be more readily understood with reference to the following examples. This embodiment is provided for the purpose of illustration and not limitation. In the following examples, most of the Neutrokine-alpha polynucleotides and polypeptides of the present invention are specifically cited and described. Each example can also be used to prepare and / or investigate the Neutrokine-alphaSV polynucleotides and / or polypeptides of the invention. One of ordinary skill in the art may readily prepare Neutrokine-alphaSV using the following examples.

Example 1a: E. of "His-tagged" Neutrokine-alpha. Expression and Purification in E. coli

In this example, bacterial expression vector pQE9 (pD10) is used to see expression in bacteria (QIAGEN, Inc., see above). pQE9 encodes ampicillin antibiotic resistance ("Ampr") and is the origin of bacterial replication ("ori"), IPTG inducible promoter, ribosomal binding site ("RBS"), QIAGEN, Inc. 6 codons encoding histidine residues and suitable single restriction enzyme cleavage sites that allow purification by affinity purification methods using the product Ni-Nitrolo-Triacetic Acid ("Ni-NTA") affinity resin. It includes. These factors are arranged such that an inserted DNA fragment encoding a polypeptide can express the polypeptide with six His residues (ie, "6 X His tags") covalently attached to the amino terminus of the polypeptide.

The DNA sequence encoding the target site of the Neutrokine-alpha protein comprising an extracellular domain is a PCR oligonucleotide that anneals to the amino terminal sequence of the protein of interest and the sequence in the deposited construct located 3 'side of the cDNA coding sequence. Amplify from deposited cDNA clones using primers. In addition, additional nucleotides comprising restriction sites to facilitate cloning in the pQE9 vector are added to the 5 'and 3' primer sequences, respectively.

The 5 'primer for cloning the extracellular domain of the protein is SEQ ID NO: 5' comprising the underlined BamHI restriction site and then 18 nucleotides of the amino terminal coding sequence of the extracellular domain of the sequences shown in Figures 1A and 1B. -GTG GGA TCC A GC CTC CGG GCA GAG CTG-3 '(SEQ ID NO: 10). Of course, the point in the protein coding sequence where the 5 'primer starts can change the DNA segment encoding any desired site of the whole Neutrokine-alpha protein to be amplified shorter or longer than the extracellular domain of that form. The 3 'primer is a sequence 5'-GTG AAG CTT TTA containing an underlined HindIII restriction site followed by two stop codons and 18 nucleotides complementary to the 3' end of the coding sequence of the DNA sequence shown in Figures 1A and 1B. TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 11).

The amplified DNA fragment and vector pQE9 are digested with BamHI and HindIII, and then the digested DNA is linked together. The DNA inserted into the restriction digested pQE9 vector is positioned with the protein coding region downstream of the IPTG-inducible promoter and inserted in the frame of the starting AUG and six histidine codons.

Then the competent mixture was prepared using a standard procedure as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). this. E. coli cells are transformed. this. E. coli uses strain M15 / rep4 comprising multiple copies of plasmid pREP4 that expresses lac inhibitors and confers kanamycin resistance (“Kan ^r ”) in carrying out the exemplary embodiments herein. This strain, the only of the many strains useful for expressing proteins, is available from Qiagen Inc. (see above). Transformants are identified through their activity of proliferating in LB plate media in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the presence of cloned DNA is confirmed by restriction enzyme analysis, PCR and DNA sequencing. Clones containing the desired constructs are grown overnight (“O / N”) via liquid culture in LB medium supplemented with ampicillin (100 μg / ml) and kanamycin (25 μg / ml). O / N cultures are inoculated in bulk cultures at dilution rates of about 1:25 to 1: 250. The cells proliferate such that the optical density at 600 nm (“OD600”) is between 0.4 and 0.6. Isopropyl-beta-D-thiogalactopyranoside (“IPTG”) is then added at a final concentration of 1 mM to inactivate the lacI inhibitors to induce transcription from the lac inhibitor susceptible promoter. The cells are then incubated for 3-4 hours. The cells are harvested by centrifugation.

The cells are then stirred in 6M guanidine-HCl (pH 8) at 4 ° C. for 3-4 hours. Cell debris is removed by centrifugation and the supernatant containing protein is injected onto a nickel-nitrilo-triacetic acid (“Ni-NTA”) affinity resin column (Qiagen Inc., see above). Proteins with 6x His tags can be purified with a simple one step procedure by binding to Ni-NTA resins with high affinity (see QIAexpressionist, 1995 (Qiagen Inc., see above)). Briefly, the supernatant is injected onto a column in 6M guanidine-HCl, pH 8, the column is washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally 6M guanidine-HCl, pH 5 To elute Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides.

The purified protein is then regenerated by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate (pH 6 buffer) +200 mM NaCl. Alternatively, the protein may be regenerated while immobilized on a Ni-NTA column. As recommended conditions, regeneration is carried out using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl, pH 7.4, containing a protease inhibitor. Regeneration should be carried out for at least 1.5 hours. After regeneration, the protein can be eluted by the addition of 250 mM imidazole. The imidazole is removed via a dialysis step against final PBS or 50 mM sodium acetate (pH 6 buffer) +200 mM NaCl. Purified protein is stored at 4 ° C. or frozen at −80 ° C.

Example 1b E. of Neutrokine-alpha. Expression and Purification in E. coli

For bacterial expression, this example uses the bacterial expression vector pQE60 (Qiagen Inc., Chasworth 91311 Eaton Avenue 9259, CA). pQE60 encodes an ampicillin antibiotic resistance ("Ampr") gene and is a nickel origin from bacterial origin of replication ("ori"), IPTG inducible promoter, ribosomal binding site ("RBS"), and Qiagen Inc. (see above). Six codons encoding histidine residues and suitable single restriction enzyme cleavage sites that allow purification by affinity purification methods using a nitrilo-triacetic acid ("Ni-NTA") affinity resin. These factors are arranged such that an inserted DNA fragment encoding a polypeptide can express the polypeptide with six His residues (ie, "6 X His tags") covalently attached to the carboxy terminus of the polypeptide. However, in this embodiment, the polypeptide coding sequence is inserted such that the translation of six His codons is blocked so that the polypeptide does not have a 6X His tag.

A DNA sequence encoding a target site of a protein comprising an extracellular domain sequence uses a PCR oligonucleotide primer that anneals to the amino terminal sequence of the protein of interest and the sequence in the deposited construct located 3 'side of the cDNA coding sequence. Amplified from deposited cDNA clones. In addition, additional nucleotides comprising restriction sites to facilitate cloning in the pQE60 vector are added to the 5 'and 3' sequences, respectively.

The 5 'primer for cloning the extracellular domain of the protein is SEQ ID NO: 5 comprising the underlined Bsp HI restriction site and then 17 nucleotides of the amino terminal coding sequence of the extracellular domain in the sequences shown in FIGS. 1A and 1B. -GTG TCA TGA GCC TCC GGG CAG AGC TG-3 (SEQ ID NO: 12). Of course, the point in the protein coding sequence where the 5 'primer starts can change the desired site of the entire protein so that it can be amplified shorter or longer than the extracellular domain of that form. The 3 'primer is a sequence 5'-GTG AAG CTT TTA containing an underlined HindIII restriction site followed by two stop codons and 18 nucleotides complementary to the 3' end of the coding sequence of the DNA sequence shown in Figures 1A and 1B. TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 13).

The amplified DNA fragment and vector pQE60 are digested with BspHI and HindIII, and then the digested DNA is linked together. The DNA inserted into the restriction digested pQE60 vector is placed so that the protein coding region comprising the relevant stop codon is downstream of the IPTG-inducible promoter and inserted in the frame of the starting AUG. Associated stop codons interfere with the translation of six histidine codons downstream of the insertion point.

Then the competent mixture was prepared using a standard procedure as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). this. E. coli cells are transformed. this. E. coli uses strain M15 / rep4 comprising multiple copies of plasmid pREP4 that expresses lac inhibitors and confers kanamycin resistance (“Kan ^r ”) in carrying out the exemplary embodiments herein. This strain, the only of a number of strains suitable for expressing a protein, is available from Qiagen Inc. (see above). Transformants are identified through their activity of proliferating in LB plate media in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the presence of the cloned DNA is confirmed by restriction enzyme analysis, PCR and DNA sequencing.

Those skilled in the art will appreciate that any of a number of bacterial expression vectors can be used in place of pQE9 and pQE60 in the expression protocols presented in this example. For example, novel pHE4-based bacterial expression vectors, particularly pHE4-5 vectors, can be used for bacterial expression of this example (ATCC Accession No. 209311: and variants thereof). Plasmid DNA pHE4-5 / MPIFD23, deposited under ATCC Accession No. 209311, is a vector plasmid DNA containing an insert encoding another ORF. The construct was deposited on September 30, 1997 at the US P. G. Cultivation Collection Center (10801, Manassas University Boulevard 10801, Virginia, USA). Substitution of the unrelated ORFs of pHE4-5 with the Neutrokine-alpha ORFs of the present invention would be readily performed by those skilled in the art according to current molecular biology techniques using NdeI and Asp718 restriction sites adjacent to the unrelated MPIF ORF inserts. It can be done.

The pHE4-5 bacterial expression vector is a neomycin phosphotransferase gene, E. coli for selection. Replication origin of E. coli, T5 phage promoter sequence, two lac operator sequences, shine-dalgano sequence and lactose operon inhibitor gene (lacIq). These factors are arranged such that the insert DNA fragment encoding the polypeptide can express a polypeptide covalently linked to 6 His residues (ie, "6 X His tag") at the amino terminus of the polypeptide. The promoter and operator sequences of the pHE4-5 vector are synthesized. The synthesis of nucleic acid sequences is well known in the art (CLONETECH 95/96 catalog, p. 215-216, CLONETECH, 94303 Palo Alto East Midau Circle 1020, California, USA).

Clones containing the desired Neutrokine-alpha constructs were grown overnight (“O / N”) via liquid culture in LB medium supplemented with ampicillin (100 μg / ml) and kanamycin (25 μg / ml). Let's do it. O / N cultures are inoculated in bulk cultures at dilution rates of about 1:25 to 1: 250. The cells proliferate such that the optical density at 600 nm (“OD600”) is between 0.4 and 0.6. Isopropyl-beta-D-thiogalactopyranoside (“IPTG”) is then added at a final concentration of 1 mM to inactivate the lacI inhibitors to induce transcription from the lac inhibitor susceptible promoter. The cells are then incubated for 3-4 hours. The cells are harvested by centrifugation.

The cells are then stirred in 6M guanidine-HCl (pH 8) at 4 ° C. for 3-4 hours. Cell debris was removed by centrifugation and the supernatant containing Neutrokine-alpha was dialyzed against 50 mM sodium acetate buffer (pH 6) supplemented with 200 mM NaCl. Alternatively, the protein can be successfully regenerated by dialysis against 500 mM NaCl, 20% glycerol, 25 mM Tris HCl, pH 7.4, containing a protease inhibitor. After regeneration, the protein can be purified via ion exchange, hydrophobic interactions and size exclusion chromatography. Alternatively, purified proteins can be obtained using affinity chromatography such as antibody columns. Purified protein is stored at 4 ° C. or frozen at −80 ° C.

In some embodiments, it is also preferred to prepare expression constructs as described in this example to vary one or more of the three cysteine residues in the Neutrokine-alpha polypeptide sequence. The cysteine residues in the Neutrokine-alphapolypeptide sequence are located at positions 147, 232 and 245 in the sequence as shown in SEQ ID NO: 2 and at positions 213 and 226 of the Neutrokine-alphapolypeptide sequence as shown in SEQ ID NO: 19 (Neutrokine Since there is no residue corresponding to amino acid residues 143 to 160 of the Neutrokine-alpha polypeptide sequence in the alphaSV polypeptide sequence, the cysteine corresponding to Cys-147 in the Neutrokine-alpha polypeptide sequence is Neutrokine-alphaSV. Not present in the polypeptide sequence).

Example 2:

Cloning, Expression, and Purification of the Bacululovirus Expression System of Neutrokine-alpha Proteins

In this example, the cloned DNA encoding the extracellular domain of the protein from which the naturally-bound intracellular and transmembrane sequences are removed is inserted into the baculovirus and the baculovirus leader and summers et al., A. Manual of Methods for Baculovirus Vectors and Insect Cell Culture Precedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987)] was used to express the extracellular domain of Neutrokine-alpha protein using the plasmid shuttle vector pA2GP. do. This expression vector is a potent polyhedrin promoter of Autographa californica nuclear polyhedron virus (AcMNPV) followed by convenient signal peptides (leaders) of the baculovirus gp67 protein and BamHI, XbaI and Asp718. Restriction sites. The polyadenylation site of Simian virus 40 (“SV40”) is useful for effective polyadenylation. For easy selection of recombinant viruses, plasmids contain E. coli under weak Drosophila promoter control. The β-galactosidase gene from E. coli is included in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes come into contact with viral sequences on both sides for cell-mediated homologous recombination with wild-type viral DNA to produce a viable virus expressing the cloned polynucleotide.

In addition to the above vectors, various baculovirus vectors such as pAc373, pVL941 and pAcIM1, which are familiar to those skilled in the art, can be used as long as they provide signals such as signal peptides required for the construct and AUG of the correct frame, and appropriately located transcription, translation, secretion, etc. Can be. Such vectors are described, for example, in Luckow et al., Virology 170: 31-39 (1989).

The intracellular domain and transmembrane domain sequences that are naturally associated with the AUG initiation codon and the amino acids Gln-73 to Leu-79 (SEQ ID NO: 2) shown in FIGS. 1A and 1B are deleted and Neutrogen in the deposited clone The cDNA sequence encoding the N-terminal deletion form of the extracellular domain of the keen-alpha protein is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of this gene. The 5 'primer comprises the underlined BamHI restriction enzyme site followed by 18 nucleotides of the extracellular domain sequence of the Neutrokine-alpha protein shown in FIGS. 1A and 1B and begins with the indicated N-terminus of the extracellular domain of the protein. It has the sequence 5'-GTG GGA TCC CCG GGC AGA GCT GCA GGG C-3 '(SEQ ID NO: 14). The 3 'primer is a sequence 5'-GTG GGA TCC TTA TTA CAG CAG containing an underlined BamHI restriction site followed by two stop codons and 18 nucleotides complementary to the 3' coding sequence shown in Figures 1A and 1B. TTT CAA TGC ACC-3 '(SEQ ID NO: 15).

In other embodiments, constructs designed to express the entire estimated extracellular domain of Neutrokine-alpha (ie, amino acid residues Gln-73 to Leu-285) are preferred. Those skilled in the art will be able to use the polynucleotide and polypeptide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, to design polynucleotide primers that produce such clones.

In another preferred embodiment, the pA2GP expression construct encodes amino acid residues Leu-112 to Leu-285 of the Neutrokine-alpha polypeptide sequence set forth in SEQ ID NO: 2. In another preferred embodiment, the pA2GP expression construct encodes amino acid residues Ser-78 to Leu-285 of the Neutrokine-alphapolypeptide sequence set forth in SEQ ID NO: 2.

Amplified fragments are isolated from 1% agarose gels using a commercial kit ("Geneclean", BIO 101 Inc., Lazolla, Calif.). The fragment is then digested with BamHI and again purified on 1% agarose gel. The fragment is labeled F1.

Plasmids can be digested with the restriction enzyme BamHI and optionally dephosphorylated using bovine visceral phosphatase according to conventional procedures known in the art. DNA is then isolated from the 1% agarose gel using a commercial kit ("Geneclean", BIO 101 Inc., Lazolla, Calif.). The vector DNA is represented by "V1".

Fragment F1 and dephosphorylated plasmid V1 are linked together with T4 DNA ligase. this. E. coli HB101 or other suitable XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.). E. coli host cells are transformed with the ligation mixture and plated on culture plates. BamHI is used to digest DNA from each colony and analyze the degradation products by gel electrophoresis to identify bacteria containing plasmids with human genes. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is represented by pA2GP-Neutrokine-alpha.

Using 5 μg of plasmid pA2GP-Neutrokine-alpha and 1 μg of a commercially available linearized baculovirus DNA (“BaculoGold ™ baculovirus DNA”, Pharmingen, San Diego, Calif.), See Felgner et al., Proc. Natl.Acad.Sci. USA 84: 7413-7417 (1987) according to the lipofection method described herein. 1 μg of BaculoGold ™ virus DNA and 5 μg of plasmid pA2GP Neutrokine-alpha are mixed in sterile wells of microtiter plates containing 50 μl of serum-free Grace medium (Life Technologies Inc., Gatsburg, Mass.). . Then 10 μl of lipofectin and 90 μl of Grace's medium are added and mixed and incubated for 15 minutes at room temperature. Thereafter, the transfection mixture is added dropwise to Sf9 insect cells (ATCC CRL1711) inoculated in a 35 mm tissue culture plate containing 1 ml of serum-deleted Grace medium. The culture plate is incubated at 27 ° C. for 5 hours. The transfection solution is then separated from the culture plate and 1 ml of Grace insect medium supplemented with 10% fetal calf serum is added. The incubation is then continued for 4 days at 27 ° C.

After 4 days the supernatant is collected (as described in Summers and Smith, supra) and subjected to plaque analysis. Agarose gels with “Blue Gal” (Life Technologies Inc., Rockville, MD) can be used to easily identify and isolate gal-expressing clones that produce blue colored salt plaques (this type of A detailed description of the "plaque assay" is also given in User Guides on Insect Cell Culture and Baculovirology distributed by Life Technologies Inc. p. 9 to 10). After appropriate incubation, blue stained plaques are harvested with the tip of a micropipette (eg Eppendorf). Agar containing the recombinant virus is resuspended in a microcentrifuge tube containing 200 μl of Grace's medium, and the suspension containing the recombinant baculovirus is used to infect Sf9 cells inoculated in a 35 mm culture dish. After 4 days, the supernatant of the culture dish is collected and stored at 4 ° C. This recombinant virus is called V-Neutrokine-alpha.

Sf9 cells are grown in grace medium supplemented with 10% heat inactivated FBS to demonstrate expression of Neutrokine-alpha gene. The cells are infected with recombinant baculovirus V-Neutrokine-alpha with a multiplicity of infection (“MOI”) of about 2. If radiolabeled protein is needed, after 6 hours the medium is removed and replaced with SF900 II medium (Life Technologies Inc.), which is deleted with methionine and cysteine. 42 hours after, ³⁵ S- methionine and 5μCi ³⁵ S- cysteine (Amersham product) is added 5μCi. The cells are further incubated for 16 hours and then harvested by centrifugation. Proteins in supernatants as well as intracellular proteins are analyzed by SDS-PAGE and subsequent autoradiographs (if radiolabeled).

Microsequencing of amino acid sequences present at the amino terminus of the purified protein can be used to determine the cleavage point and length of the amino terminal sequence and secretory signal peptide of the extracellular domain of the purified protein.

As a specific experimental example, recombinant Neutrokine-alpha is purified from baculovirus infected Sf9 cell supernatant as follows. Insect cells are grown in EXCEL401 medium (JRH Scientific) supplemented with 1% (v / v) fetal calf serum. 92 hours after infection, the harvested supernatant is clarified by centrifugation at 18,000 × g and subsequent 0.45 m height filtration. In addition, a degreasing filtration step can be used to remove lipid contaminants and consequently enhance the initial yield of Neutrokine-alpha protein.

Supernatants are injected onto a set of tandem Poros HS-50 / HQ-50. Alternatively, Toyopearl QAE, Toyopearl Super Q (Tosohass), Q-Sepharose (Pharmacia), and equivalent resins may be used. This step is used as a negative purification step to remove strong anion binding contaminants. The HS / HQ effluent is adjusted to pH 7.5 with 1M Tris-HCl, pH 8, diluted with an equivalent amount of 50 mM of Tris-HCl, pH 8 and then injected onto a Poros PI-20 or PI-50 column. The PI column was first washed with 4 column volumes of 75 mM sodium chloride in 50 mM Tris-HCl (pH 7.5), followed by a step gradient of 300 mM, 750 mM, 1500 mM sodium chloride 3 to 5 column volumes in 50 mM Tris-HCl (pH 7.5). Use to elute. Neutrokine-alpha protein appears as a 17KD band in reducing SDS-PAGE and is present in the 0.75M to 1.5M sodium chloride fraction.

PI fractions are further purified through Sephacryl S100HR (Pharmacia) size exclusion column equilibrated with 0.15 M sodium chloride, 50 mM sodium acetate (pH 6). The S200 fraction is mixed with sodium chloride to a final concentration of 3M and injected onto a Toyopearl Hexyl 650C (Tosohass) column. The hexyl column is eluted with 5 to 15 column volumes using a linear gradient of 3M to 0.05M sodium chloride in 50 mM sodium acetate (pH 6). In addition, the sodium chloride gradient in the hexyl chromatography step may be replaced by a gradient of 1M to 0M ammonium sulfate in 50 mM sodium acetate (pH 6). Fractions containing purified Neutrokine-alpha as analyzed via SDS-PAGE are combined and dialyzed against a buffer containing 150 mM sodium chloride, 50 mM sodium acetate (pH 6).

The final purified Neutrokine-alpha protein expressed in the baculovirus system as described herein has an N-terminal sequence beginning with amino acid residue Ala-134 as set forth in SEQ ID NO: 2. RP-HPLC analysis shows a single peak with a purity of at least 95%. Endotoxin levels are below the detection limit in the LAL assay.

In another embodiment, recombinant Neutrokine-alpha is purified from baculovirus infected Sf9 cell supernatant containing 0.25% bovine serum as follows.

Sf9 supernatants are harvested by centrifugation at 18,000 × g. The supernatant is then treated with 10 mM calcium chloride for 10 to 15 minutes in weak alkali conditions, then centrifuged and subjected to filtration at a depth of 0.22 μm. The resulting Sf-9 cell supernatant is diluted 2-fold and injected onto a Poros PI-50 column (PE Biosystems). The column is equilibrated with 50 mM Tris pH 7.4. The PI-50 column was washed with 50 mM Tris pH 7.4 1 CV and then eluted with 1.5 M NaCl 3 CV in 50 mM NaOAc pH 6. PI fraction is injected onto a Sephacryl S200 column equilibrated with 50 mM NaOAc, pH 6, 125 mM NaCl. The S200 fraction was mixed with salts to a final concentration of 0.7 M ammonium sulfate and 0.6 M NaCl, and the Toyopearl Hexyl 650C column (Toso Haas) equilibrated with a buffer containing 0.6 M NaCl, 0.7 M ammonium sulfate in 50 mM NaOAc, pH 6 Product). This column is then washed with 2 CV of the same buffer. The recombinant Neutrokine-alpha was then eluted stepwise with 50 mM NaOAc (pH 6) 3 CV, then 20% ethanol 2 CV. Recombinant Neutrokine-alpha protein was eluted at the end of the ammonium sulfate (0.3 M to 0 M salt) gradient. Appropriate fractions were collected and dialyzed against a buffer containing 50 mM NaOAc (pH 6) and then passed through a Poros 50 HQ column. The HQ effluent was diluted to 4 ms and injected onto a Toyopearl DEAD 650M column and eluted with 25 mM NaCitrate, 125 mM NaCl.

In another embodiment, recombinant Neutrokine-alpha is expressed and purified using a baculovirus vector system in Sf + insect cells.

First, amino acid residues Ser-78 to Leu-285 of the Neutrokine-alpha polypeptides shown in FIGS. 1A and 1B (amino acid residues Ser-78 to Leu-285 of the Neutrokine-alpha polypeptide sequence shown in SEQ ID NO: 2) Polynucleotides encoding exactly the same) are subcloned into baculovirus transfer construct PSCs to generate baculovirus expression plasmids. pA2GP transfer vectors from pVL941 include the lacZ gene cloned downstream of the Drosophila heat shock promoter for selection with gp67 signal peptide, modified multiple cloning sites and blue plaques. For cloning, the PSC signal peptide coding sequence was joined to Ala-134 (SEQ ID NO: 2 and Figures 1A and 1B) of the Neutrokine-alpha password sequence using Neutrokine-alpha (SEQ ID NO: 2) sequence and pA2GP vector sequence. The method is designed to fuse and insert this fusion into the PSC baculovirus transfer plasmid. This method uses a two-step polymerase chain reaction (PCR) procedure. First, primers are designed to amplify Neutrokine-alpha sequences. The 5 'primer contains a sequence encoding Ala-134 and the following residues (5'-GGT CGC CGT TTC TAA CGC GGC CGT TCA GGG TCC AGA AG-3'; SEQ ID NO: 31) and then the PSC signal peptide C-terminus. It consists of a coding sequence. 3 'primer (5'-CTG GTT CGG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTA GAT C-3'; SEQ ID NO: 32) is the reverse complement of the pA2GP vector sequence downstream of the Neutrokine-alpha coding sequence, It then consists of a KpnI restriction enzyme site and a spacer sequence (a sequence for increasing cleavage efficiency by KpnI). PCR is carried out with pA2GP containing Neutrokine-alphaplasmid template and primers O-1887 and O-1888 and the resulting PCR product is purified using standard techniques.

Secondary PCR reactions are carried out using the PSC baculovirus transfer plasmid pMGS 12 as a template. The pMGS12 plasmid consists of an AcNPV EcoRI "I" fragment inserted into pUC8, a polyhedrin coding sequence and a polylinker site followed by an ATG initiation codon replaced with a PSC signal peptide. PCR reactions included a 5 'primer (5'-CTG GTA GTT CTT CGG AGT GTG-3'; SEQ ID NO: 33) and PSC signal peptide annealed to pMGS12, AcNPV ORF603 upstream of the native NgoM IV and EcoR V sites as templates. A 3 'primer (5'-CGC GTT AGA AAC GGC GAC C-3'; SEQ ID NO: 34) annealed to the 3 'end of the coding sequence is used.

The PCR product is further combined with the PSC signal peptide-polyhedrin upstream region PCR product to produce a PCR product in which the PSC signal peptide is seamlessly fused to Ala-134 of the Neutrokine-alpha password sequence. Since the 3 'end of the PSC signal peptide PCR product (pMGS12 / O-959 / O-1044) overlaps the 5' end of the Neutrokine-alphaPCR product made with primers O-1887 / O-1888, two PCRs The products were combined and elongated by PCR using primers O-959 and O-1888.

The overlapped stretched PCR product containing the PSC signal peptide fused to the resulting Neutrokine-alpha sequence is then inserted into baculovirus transfer plasmid pMGS12. PCR products were digested with NgoM IV and Kpn I, and fragments were purified and linked to NgoM IV-Kpn I-cleaved pMGS12. Competitive e. After transforming E. coli DH5α cells, colonies were harvested to prepare plasmid DNA microproducts. Plasmid DNA is analyzed by restriction digestion to identify several positive clones obtained after each ligation reaction, and three clones (pAcC9669, pAcC9671 and pAcC9672) are selected for mass plasmid purification. The resulting plasmid DNA is subjected to DNA sequencing to identify and sequence Neutrokine-alpha inserts.

The following steps relate to a method for recovering and purifying recombinant Neutrokine-alpha from Sf + insect cells. Unless otherwise indicated, these steps are performed at 2 to 8 ° C.

collection

Step 1. CaCl ₂ Treatment

Sf + cell supernatants were obtained by centrifugation at 8,000 × g. Recovery Buffer-1 (1M CaCl ₂ ) is added to the supernatant so that the final concentration of CaCl ₂ is 10 mM. (In another preferred embodiment, 1M ZnCl ₂ may be used instead of 1M CaCl _2. ) The pH of this solution is adjusted to 7.7 ± using Recovery Buffer-2 (1M Tris pH 8 (± 0.2)). The solution is incubated for 15 minutes and then centrifuged at 8,000xg.

refine

Step 1. Chromatography on Poros PI-50 Column

Sf + cell supernatants are injected onto a Poros PI-50 column (PE Biosystem). The column is equilibrated with PI-1 buffer (50 mM Tris, 50 mM NaCl, pH 7.4 (± 0.2)). PI-50 columns are washed with PI-1 buffer 1 to 2 CV and then eluted with a PI-2 buffer (50 mM sodium citrate, pH 6 (± 0.2)) using a 3CV linear gradient. Elution is monitored by ultraviolet (UV) absorbance at 280 nm. Appropriate fractions are collected according to the eluate peaks and analyzed by SDS PAGE. Collect the appropriate fractions.

Step 2. Chromatography on Toyopearl Hexyl 650C Column

PI harvest is mixed with a salt with a final concentration of 0.7M (NH ₄ ) ₂ SO ₄ , and HIC-1 buffer (50 mM NaOAc, 0.6M NaCl, 0.7M (NH ₄ ) ₂ SO ₄ pH 6 (± 0.2)) Inject on a Toyopearl Hexyl 650C (Toso Haas) column equilibrated with. This column is then washed with HIC-1 buffer 2CV. Recombinant Neutrokine-alpha was then eluted with HIC-2 buffer (50 mM NaOAc pH 6.0 (± 0.2)) 3 to 5 CV and 2 CV 20% ethanol washes. Elution is monitored by UV absorbance and conductivity at 280 nm. Fractions are collected along the eluate peak and analyzed by SDS-PAGE. Then the appropriate fractions are collected.

Step 3. Chromatography at SP Sepharose FF

Hexyl fractions were dialyzed and adjusted to pH 4.5 with SP-1 buffer (50 mM sodium acetate, pH 4.5 (± 0.2)), diluted to 4 ms, and then SP-1 buffer (50 mM sodium acetate, pH 4.5 (± 0.2)). Inject through the SP Sepharose (Cation Exchanger, Pharmacia) column equilibrated. Recombinant Neutrokine-alpha protein is eluted from the SP column with SP-2 buffer (50 mM sodium acetate, pH 5.5 (± 0.2)) at pH 5.5. Elution is monitored by ultraviolet absorbance at 280 nm. Fractions are collected along the eluate peak and analyzed by SDS PAGE. Collect the appropriate fractions.

Step 4. Dialysis of Recombinant Neutrokine-alpha

SP fractions are injected into a 6-8kd cutoff membrane device, followed by overnight dialysis or diafiltration using dialysis buffer (10 mM sodium citrate, 140 mM sodium chloride, pH 6 (± 0.2)).

Step 5. Filtration and Filling

The protein concentration of the recombinant Neutrokine-alpha solution from step 4 is determined by non-sinuconic acid (BCA) protein assay. Recombinant Neutrokine-alpha preparations are adjusted with appropriate buffer to final protein concentration and filtered under controlled conditions. The filtrate (bulk) is stored in a suitable sterile container of -20 ° C or lower.

In a specific embodiment, the Neutrokine-alpha protein of the invention, produced as described below, is adjusted to a final protein concentration of 1-5 mg / ml, with 10 mM sodium citrate, 140 mM sodium chloride, pH 6.0 ± (0.4 ) Is stored in a type 1 glass vial below -20 ° C.

The process is monitored for UV absorbance at 280 nm during the chromatographic treatment. Where available, in-process chromatography intermediates are used to test conductivity, pH and monitor by SDS and / or RP-HPLC.

The column and purification apparatus are washed and sterilized using 0.2M or 0.5M NaOH followed by deionized water followed by 0.1M or 0.5M acetic acid. The column apparatus and the purification apparatus are washed with deionized water and stored in a suitable stock solution if necessary. Prior to use the device was equilibrated with suitable buffer (equilibrated as described herein or as known in the art).

As another preferred embodiment, 1M ZnCl ₂ may be used instead of 1M CaCl ₂ in step 1 of the above-mentioned recovery process. In this embodiment, a combination of ZnCl ₂ and CaCl ₂ can also be used. Various combinations of 0.1M ZnCl ₂ and 0.9M CaCl ₂ can be used to recover the recombinant Neutrokine-alpha protein, for example, a combination of 0.1M ZnCl ₂ and 0.9M CaCl ₂ , 0.2M ZnCl ₂ and Combination of 0.8M CaCl ₂ , Combination of 0.3M ZnCl ₂ and 0.7M CaCl ₂ , Combination of 0.4M ZnCl ₂ and 0.6M CaCl ₂ , Combination of 0.5M ZnCl ₂ and 0.5M CaCl ₂ , 0.6M ZnCl A combination of ₂ and 0.4M CaCl ₂ , a combination of 0.7M ZnCl ₂ and 0.3M CaCl ₂ , a combination of 0.8M ZnCl ₂ and 0.2M CaCl ₂ , a combination of 0.9M ZnCl ₂ and 0.1M CaCl ₂ , and the like. It is not limited to this. However, the presence of EDTA interferes with the recovery process. Moreover, ZnCl ₂ and / or CaCl ₂ present in Recovery Buffer-1 may induce a large amount of formation of high molecular weight (or molecular mass) Neutrokine-alpha multimers.

Example 3 Cloning and Expression of Neutrokine-alpha in Mammalian Cells

Typical mammalian expression vectors include promoter factors that mediate the initiation of transcription of mRNA, promoter coding sequences, and signals necessary for polyadenylation of the transcription termination and transcript. Still other factors include enhancers, Kozak sequences and mediation sequences adjacent to RNA splicing donor sites and acceptor sites. High efficiency transcription can be achieved by early and late promoters derived from SV40, long term repeats (LTR) and cytomegalovirus (CMV) derived from retroviruses such as RSV, HTLVI, HIVI. However, cell factors can also be used (eg human actin promoter). Expression vectors suitable for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Upmasala, Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC 12MI (ATCC 67109). do. Mammalian host cells that can be used include human HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells, Chinese hamster ovary (CHO) Cells and HEK 293 cells.

Alternatively, the gene can be expressed in a stable cell line that contains a gene incorporated into the chromosome. In addition, co-transfection with selection markers such as dhfr, gpt, neomycin, hygromycin can identify and isolate transfected cells.

In addition, the transfected genes can be amplified to express large amounts of the encoded protein. DHFR (dihydrofolate reductase) markers are useful for developing cell lines that contain hundreds or even thousands of copies of the gene. Another useful selection marker is the enzyme glutamine synthetase (GS) (Murphy et al., Biochem., J 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992)). . This marker is used to propagate mammalian cells in selection medium and select the cells with the highest resistance. This cell line contains an amplification gene integrated into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are also useful for protein production.

Expression vectors pC1 and pC4 are strong promoters of Lewis sarcoma virus (LTR) (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) + fragments of CMV-enhancer (Boshart et al., Cell 41 : 521-530 (1985)). Multiple cloning sites with multiple cloning sites, eg, restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vector further comprises a 3 'intron, a polyadenylation signal and a termination signal of the rat preproinsulin gene.

Example 3 (a): Cloning and Expression in COS Cells                 

The expression plasmid pNeutrokine-α-HA is prepared by cloning a portion of the deposited cDNA encoding the extracellular domain of the protein into the expression vector pcDNAI / Amp or pcDNAIII. The extracellular domain is fused to the secreted leader sequence of the human IL-6 gene to produce the polypeptide in soluble, secreted form.

The expression vector pcDNAI / amp is (1) E. E. coli effective for propagation in E. coli and other prokaryotic cells. Origin of replication of E. coli; (2) ampicillin resistance genes for selecting plasmid containing prokaryotic cells; (3) SV40 replication origin for propagation in eukaryotic cells; (4) CMV promoter, polylinker, SV40 intron; (5) Several codons encoding hemagglutinin fragments (i.e., "HA" tags to facilitate purification) and then cDNAs are advantageously placed under the control of expression of the CMV promoter and are restricted by SV40 by restriction sites in the polylinker. Stop codons and polyadenylation signals to be operatively coupled to introns and polyadenylation signals. The HA tag corresponds to epitopes from the flu hemagglutinin protein described in Wilson et al., Cell 37: 767 (1984). When the HA tag is fused to the target protein, an antibody that recognizes an HA epitope can be used to easily detect and recover the recombinant protein. pcDNAII also includes a neomycin marker for selection.

The DNA fragment encoding the extracellular domain of the Neutrokine-alphapolypeptide is cloned into the polylinker region of the vector such that expression of the recombinant protein is induced by the CMV promoter. Plasmid construction method is as follows. Neutrokine-alpha cDNA of the deposited clones were Amplification is carried out using primers comprising an advantageous restriction site as described above for the construction of a vector for expressing Neutrokine-alpha in E. coli. Suitable primers include the following used in this example. 5, including 18 nucleotides of the 5 'coding region in the extracellular domain of the Neutrokine-alpha protein and the sequence encoding the underlined BamHI site, Kozak sequence, AUG initiation codon, secretory leader peptide from the human IL-6 gene The primer has the following sequence: 5'-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3 '(SEQ ID NO: 16). A 3 'primer comprising 18 nucleotides complementary to the underlined BamHI restriction site and the 3' coding sequence immediately preceding the stop codon has the following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC- 3 '(SEQ ID NO: 17).

PCR amplified DNA fragments and vector pcDNAI / Amp are digested with BamHI and linked. This using a coupling mixture. E. coli strain SURE (Stratagene Cloning Systems, 92037 Lazola North Torrey Pines Road 11099, California, USA) was transformed, and the transformed cultures were plated on ampicillin medium plates and incubated to propagate ampicillin resistant colonies. I was. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other techniques for the presence of fragments encoding Neutrokine-alpha extracellular domains.

To express recombinant Neutrokine-alpha, DEAE-DEXTRAN was used as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). COS cells are transfected with the expression vector as described above. Cells are cultured under conditions capable of expressing Neutrokine-alpha through this vector.

Expression of the Neutrokine-alpha-HA fusion protein is described in Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)] is used to detect by radiolabeling and immunoprecipitation. For this purpose, two days after transfection, the cells are labeled by incubating for 8 hours in a medium containing ³⁵ S-cysteine. The cells and medium were harvested and the cells were washed with RIPA buffer containing a detergent (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS as described in Wilson et al., Supra). , pH 7.5) and lysate. HA-specific monoclonal antibodies are used to precipitate proteins from cell lysates and culture media. Precipitated protein is analyzed by SDS-PAGE and autoradiography. Expression products of expected size were observed in cell lysates and not in negative controls.

Example 3 (b) Cloning and Expression in CHO Cells

Vector pC4 is used to express Neutrokine-alpha protein. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). A portion of the deposited cDNA encoding the extracellular domain is fused to the secretory leader sequence of the human IL-6 gene to produce a soluble secreted Neutrokine-alpha polypeptide. The vector plasmid contains the mouse DHFR gene located under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells transfected with this plasmid lacking dihydrofolate activity can be selected by proliferating the cells in a selection medium (α-MEM, Life Technologies) supplemented with chemotherapeutic methotrexate. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) is well known in the literature (eg, Alt, FW, Kellems, RM, Bertino, JR, and Schimke, RT, 1978, J. Biol. Chem. 253: 1357-1370, Hamlin, JL and Ma, C. 1990, Biochem. Et Biophys.Acta, 1097: 107-143, Page, MJ and Sydenham, MA 1991, Biotechnology 9: 64-68). Cells that proliferate at increasing concentrations of MTX overproduce the target enzyme DHFR as a result of amplification of the DHFR gene, thereby expressing drug resistance. If a second gene is linked to this DHFR gene, it is usually co-amplified and overexpressed. It is well known in the art that such methods can be used for the development of cell lines carrying more than 1000 copies of amplification genes. As a result, when methotrexate is removed, a cell line in which an amplification gene is introduced into one or more chromosomes of the host cell is obtained.

Plasmid pC4 is a potent promoter of the long term repeat (LTR) of Lewis sarcoma virus (Cullen, et al., Molecular and Cellular Biology, March 1985: 438-447) + human cytomegalovirus (CMV) to express the gene of interest. Fragments isolated from enhancers of adjacent early genes of Boshart et al., Cell 41: 521-530 (1985). Downstream of the promoter is a single restriction enzyme cleavage site capable of incorporating genes: BamHI, XbaI and Asp718. The plasmid also includes the 3 'intron and the polyadenylation site of the rat preproinsulin gene after this cloning site. Other high efficiency promoters can also be used for expression, such as human β-actin promoters, early or late SV40 promoters, or long terminal repeats from other retroviruses such as HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express Neutrokine-alpha in a regulated manner in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc). Natl. Acad. Sci. USA 89: 5547-5551). For polyadenylation of mRNA, other signals, such as from human growth hormone or globin genes, can also be used. Stable cell lines carrying the gene of interest integrated in the chromosome can also be selected upon simultaneous transfection with a selection marker such as gpt, G418 or hygromycin. It is advantageous to use at least one marker for the selection at first, such as G418 and methotrexate.

Plasmid pC4 is digested with the restriction enzyme BamHI and then dephosphorylated using bovine visceral phosphatase according to procedures known in the art. The vector is then separated from the 1% agarose gel.

The DNA sequence encoding the extracellular domain of Neutrokine-alpha protein is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. 5 containing 18 nucleotides of the underlined BamHI site, Kozak sequence, AUG initiation codon, sequence encoding secretory leader peptides derived from the human IL-6 gene, and the 5 'coding region of the extracellular domain of Neutrokine-alpha protein The primer has the following sequence: 5 'GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3 '(SEQ ID NO: 16). A 3 'primer containing 18 nucleotides complementary to the underlined BamHI sequence and the 3' coding sequence immediately preceding the stop codon has the following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3 ' (SEQ ID NO: 17).

The amplified fragment is digested with restriction enzyme BamHI and purified again on 1% agarose gel. The isolated fragment and the dephosphorylated vector are linked with T4 DNA ligase. Then, this. E. coli HB101 or XL-1 blue cells are transformed and restriction enzyme assay or the like is used to identify bacteria comprising fragments inserted into plasmid pC4.

Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 is cotransfected with 0.5 μg of plasmid pSVneo using lipofectin (Felgner et al., Supra). Plasmid pSV2-neo contains a neo gene derived from Tn5, which encodes an enzyme that confers resistance to a group of antibiotics, including G418, a major selectivity marker. Cells are seeded in α-MEM supplemented with 1 mg / ml G418. After 2 days, cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) infused with α-MEM supplemented with methotrexate 10 ng / ml, 25 ng / ml or 50 ng / ml + G410 1 mg / ml . After about 10-14 days, the monoclones are trypsinized and then inoculated into 6-well Petri dishes or 10 ml flasks using various concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate were again transferred to new wells of 6 wells containing higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or reversed phase HPLC analysis.

Six or more Neutrokine-alpha expression constructs have been prepared by the inventors of the present invention to readily produce Neutrokine-alpha and / or Neutrokine-alphaSV polypeptides of various sizes in various expression systems. Expression constructs are as follows: (1) pNa.A71-L285 (expressing amino acid residues Ala-71 to Leu-285), (2) pNa.A81-L285 (expressing amino acid residues Ala-81 to Leu-285), (3) pNa.L112-L285 (expressing amino acid residues Leu-112 to Leu-285), (4) pNa.A134-L285 (expressing amino acid residues Ala-134 to Leu-285), (5) pNa.L147-L285 (Expressing amino acid residues Leu-147 to Leu-285), and (6) pNa.G161-L285 (expressing amino acid residues Gly-161 to Leu-285).

In a preferred embodiment, the expression construct is used to express various Neutrokine-alphamuteins in bacterial systems, baculovirus systems and mammalian systems.

In another preferred embodiment, the construct is a heterologous polypeptide, eg, a signal peptide from human IL-6, a signal peptide from CK-β8 (CK-β8 disclosed in published PCT application PCT / US95 / 09058). Expressing Neutrokine-alpha polypeptide fragments fused at the N-terminus and / or C-terminus to amino acids -21 to -1) or human IgG Fc regions of the sequence. Other sequences known in the art can also be used.

Example 4: Tissue Distribution of Neutrokine-alpha mRNA Expression

In order to examine the expression of the Neutrokine-alpha gene in human tissue, Northern blot analysis is performed using methods described in Sambrook et al., Described above, and the like. The cDNA probe containing the full nucleotide sequence of Neutrokine-alpha protein (SEQ ID NO: 1) is labeled with ³² P using the redi prime ™ DNA labeling system (Amersham Life Science) according to the manufacturer's instructions. After labeling, the probes are purified using CHROMA SPIN-100 ™ column (Clontech Laboratories, Inc.) according to manufacturer's protocol PT1200-1. Neutrokine-alpha and / or Neutrokine-alpha mRNAs are examined in various human tissues using purified labeled probes.

Multiple tissue northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech and ExpressHyb ™ hybridization solution (Clontech) was used according to manufacturer's protocol PT1190-1. Irradiate with the labeled probe. After hybridization and washing the blots are mounted and exposed to film overnight at -70 ° C. The film is then developed according to standard procedures.

A panel of multiple tissue northern blots is probed to determine the pattern of Neutrokine-alpha and / or Neutrokine-alpha expression. As a result, prominent expression of 2.6 kb mRNA was observed in peripheral blood leukocytes, spleen, lymph nodes and bone marrow, and detectable expression in placenta, heart, lung, fetal liver, thymus and pancreas. Analysis of the cell line panel demonstrated high expression of Neutrokine-alpha and / or Neutrokine-alpha in HL60 cells and detectable levels in K562, not at all in Raji, HeLa or MOLT-4 cells. In conclusion, Neutrokine-alpha and / or Neutrokine-alpha mRNA expression appears to be concentrated in the immune system.

Example 5 Gene Therapy Using Endogenous Neutrokine-alpha Genes

Another method of gene therapy described in the present invention is described in, eg, US Pat. No. 5,641,670 (1997.6.24); International Publication No. WO96 / 29411 (published 16.9.26); International Publication No. WO 94/12650 (published 4 April 199); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); And Zijlstra et al., Nature 342: 435-438 (1989), to operably bind the promoter to the endogenous Neutrokine-alpha sequence via homologous recombination. This method involves activating a gene present in the target cell but not expressed in this cell or expressed at a lower concentration than necessary. A polynucleotide construct is prepared comprising a promoter and a target sequence homologous to the 5'-noncoding sequence of endogenous Neutrokine-alpha adjacent to the promoter. The target sequence is sufficiently close to the 5 'end of Neutrokine-alpha such that upon homologous recombination the promoter is operably linked to the endogenous sequence. The promoter and target sequence can be amplified using PCR. Preferably, the amplified promoter comprises restriction enzyme sites unique at the 5 'end and the 3' end. In addition, the 3 'end of the first target sequence comprises the same restriction enzyme site as the 5' end of the amplified promoter, and the 5 'end of the second target sequence contains the same restriction site as the 3' end of the amplified promoter. It is preferable.

The amplified promoter and the amplified target sequence are digested with appropriate restriction enzymes and then treated with bovine intestine phosphatase. The degraded promoter and the degraded target sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. The construct is size fractionated on agarose gels and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as polynucleotides extruded via electroporation. However, polynucleotide constructs may also be administered with transfection active agents such as liposomes, viral sequences, viral particles, precipitating agents and the like. Such methods of delivery are known in the art.

Once the cells have been transfected, homologous recombination will occur which operably binds the promoter to the endogenous Neutrokine-alpha sequence. As a result, Neutrokine-alpha is expressed in the cell. Expression can be detected by immunological staining or by any other method known in the art.

Fibroblasts are obtained from a subject via skin biopsy. The resulting tissue is placed in DMEM + 10% fetal calf serum. Fibroblasts at exponential or early stop phases are trypsinized and washed from plastic surfaces using nutrient media. A certain amount of cell suspension is taken for counting and the remaining cells are centrifuged. The supernatant is aspirated off and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged and the supernatant aspirated off. The cells are resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. The final cell suspension contains about 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, plasmid pUC18 (MBI Fermentas, Amherst, NY) is digested with HindIII to construct plasmids targeting Neutrokine-alpha gene loci. The CMV promoter is amplified by PCR using the XbaI site on the 5 'end and the BamHI site on the 3' end. The two Neutrokine-alpha non-coding sequences are amplified by PCR as follows: The first Neutrokine-alpha non-coding sequence (Neutrokine-alpha fragment 1) is located at the HindIII site at the 5 'end and at the 3' end. Amplification using Xba site; The second Neutrokine-alpha noncoding sequence (Neutrokine-alpha fragment 2) is amplified using the BamHI site at the 5 'end and the HindIII site at the 3' end. The CMV promoter and Neutrokine-alpha fragment are digested with the appropriate enzymes (CMV promoters-XbaI and BamHI, Neutrokine-alpha fragment 1-XbaI; Neutrokine-alpha fragment 2-BamHI) and linked together. The resulting linkage product is digested with HindIII and linked with HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with 0.4 cm electrode gap (BioRad). Final DNA concentrations are generally at least 120 μg / ml. Next, 0.5 ml of the cell suspension (containing about 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and the DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser device (BioRad). The capacitance and voltage are set at 960 μF and 250 to 300 V, respectively. As the voltage increases, cell viability decreases, but the percentage of viable cells that stably feed the introduced DNA into the genome rapidly increases. For these variables to be provided, a pulse time of about 14-20 mSec must be observed.

The electroporated cells are held at room temperature for about 5 minutes, then the contents in the cuvette are carefully taken with a sterile transition pipette. The cells are added directly to 10 ml of pre-warmed nutrient medium (DMEM with 15% bovine serum) in a 10 cm culture dish and incubated at 37 ° C. The next day, the medium is aspirated off and 10 ml of fresh medium is replaced and incubated further for 16 to 24 hours.

Subsequently, the engineered fibroblasts alone or propagated in a fused state on cytodex 3 microcarrier beads and then injected into the host. These fibroblasts produce protein products. Fibroblasts can be injected into a patient as described above.

Example 6 Neutrokine-alpha, a New Member of the Tumor Necrosis Factor Ligand Acting as B Lymphocyte Stimulator

APRIL (28.7%) (Hahne, M., et al., J. Exp. Med. 188, 1185-90 (1998)), TNF-α (16.2%) (Pennica, D., et al., Nature 312,724 -729 (1984)) and human neutrophils that share significant homology within the expected extracellular receptor-ligand binding domains for LT-α (14.1%) (Gray, Nature 312, 721-724 (1984)). A 285 amino acid protein was identified in the monocyte derived cDNA library (FIG. 7A). We refer to this as the cytokine Neutrokine-alpha (also referred to as B lymphocyte stimulator (BLyS) based on biological activity). Hydrophobicity analysis of the Neutrokine-alpha protein sequence revealed a potential transmembrane domain between amino acid residues 47-73 located in front of the non-hydrophobic amino acid, indicating that Neutrokine-alpha, like other members of the TNF ligand family, is type II. Implying a membrane binding protein (Cosman, D., Stem. Cells. 12: 440-55 (1994)). Expression of this cDNA in mammalian cells (HEK 293 and Chinese hamster ovary) and Sf9 insect cells identifies a soluble form of 152 amino acids with an N-terminal sequence beginning with an alanine residue at amino acid 134 (arrow of FIG. 7A). . Restoration of the mass-to-charge ratio identified the mass of Neutrokine-alpha to 17,038 Daltons, which is consistent with the expected mass (17037.5 Daltons) for the 152 amino acid proteins with single disulfide bonds.

Using human / hamster somatic hybrids and radiation-hybrid mapping panels, genes encoding Neutrokine-alpha have never been combined with any component of the TNF superfamily gene (Cosman, D. Stem. Cells. 12: 440-55 (1994)), which is found to be bound to the marker SHGC-36171, which maps to the human chromosome 13q34.

The expression profile of Neutrokine-alpha is determined by Northern blot (FIG. 7B) and flow cytometry (Table 5 and FIG. 8). Neutrokine-alpha is encoded by a single 2.6 kb mRNA found at high concentrations in peripheral blood leukocytes, spleen, lymph nodes and bone marrow. Lower expression concentrations are detected in the placenta, heart, lung, fetal liver, thymus and pancreas. Neutrokine-alpha in a panel of cell lines is detected in HL-60 and K562, but not in Raji, HeLa or MOLT-4 cells. This result is confirmed by flow cytometry using Neutrokine-alpha-specific mAb 2E5. As shown in Table 5, Neutrokine-alpha expression was not detected in T or B lineage cells, but only in cells of bone marrow origin. In addition, analysis of normal blood cell types showed significant expression in mononuclear cells of resting phase that were upregulated by about four fold after 3 days of cell exposure to IFN-γ (100 U / mL) (FIG. 8A). Concomitant increases in Neutrokine-alpha-specific mRNA are also observed (FIG. 8B). In contrast, Neutrokine-alpha was not expressed in freshly isolated peripheral blood granulocytes, T cells, B cells or NK cells.

Purified recombinant Neutrokine-alpha ("r Neutrokine-alpha") and activated and proliferated in a variety of cell line assays including B cells, T cells, monocytes, NK cells, hematopoietic progenitor cells and various cells of endothelial and epithelial origin Investigate whether there is activity that induces differentiation or death. Among these assays Neutrokine-alpha was cultured in the presence of Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM in which specifically purified amygdala B cells were fixed with formalin as a priming agent. One standard co-stimulation assay was found to increase B cell proliferation (Sieckmann, DG, et al., J. Exp. Med. 147: 814-29 (1978); Ringden, O., et al., Scand. J. Immunol. 6: 1159-69 (1977). As shown in FIG. 9A, recombinant Neutrokine-alpha induces dose dependent proliferation of tonsil B cells. The reaction was similar to that of rIL2 in a dose range of 0.1 to 10,000 ng / ml. Neutrokine-alpha also induces B cell proliferation when incubated with cells co-stimulated with immobilized anti-IgM (FIG. 9B). Dose dependent responses were readily observed when the amount of crosslinker was increased in the presence of fixed concentrations of IL2 or r Neutrokine-alpha.

Purified Neutrokine-alpha was biotinylated to determine if specific biological activity on B cells correlated with receptor expression. The resulting biotin-Neutrokine-alpha protein retained its biological function in standard B cell proliferation assays. Biotinylated Neutrokine-alpha binding was determined in lineage-specific assays of human whole peripheral blood cells in T cells, monocytes, NK cells and granulocytes as measured by CD3, CD14, Cd56 and CD66b, respectively. It was observed that it could not be detected (FIG. 10A). In contrast, biotinylated Neutrokine-alpha bound to peripheral CD20 ⁺ B cells. In addition, receptor expression is also detected in the B cell tumor cell lines REH, ARH-77, Raji, Namalwa, RPMI8226 and IM-9, but derived from any bone marrow tested, including THP-1, HL-60, K-562 and U-937. It is not detected even in the cell line. Representative flow cytometry profiles for myeloma cell line IM-9 and histiocyte cell line U-937 are shown in FIG. 10B. Similar results are obtained when using biologically active FLAG-tagged Neutrokine-alpha proteins in place of chemically modified Biotin-Neutrokine-alpha. Taken together, these results confirm that Neutrokine-alpha exhibits clear B cell orientation in all aspects of receptor distribution and biological activity. However, it is necessary to reveal whether cell activation can induce the expression of Neutrokine-alpha receptors in peripheral blood cells, other normal cells or existing cell lines.

To investigate the species specificity of Neutrokine-alpha, B cells of mouse spleens are cultured in the presence of human Neutrokine-alpha and SAC. As a result, it was confirmed that r Neutrokine-alpha induces in vitro proliferation of spleen B cells of the mouse and binds to cell surface receptors on the cells. Interestingly, the immature surface Ig negative B cell precursors isolated from the bone marrow of the mouse did not proliferate or bind ligand in response to Neutrokine-alpha.

To assess the in vivo activity of r Neutrokine-alpha, BALB / c mice (3 / group) were treated with buffer, or r Neutrokine-alpha 0.08 mg / kg, 0.8 mg / kg, 2 mg / kg or 8 mg / kg. Inject twice a day (intraperitoneal). Mice that received this treatment for four consecutive days were killed and various tissues and serum collected for analysis. In another embodiment, BALB / c mice may be injected (intraperitoneally) twice daily with any amount of r Neutrokine-alpha in the range of 0.01-10 mg / kg. In a preferred embodiment, BALB / c mice are injected (intraperitoneally) twice daily with any amount of r Neutrokine-alpha in the range of 0.01-3 mg / kg. , 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg , 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg , 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg , 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg and 3.0 mg / kg Including, but not limited to). In another preferred embodiment, BALB / c mice are injected (intraperitoneally) twice daily with any amount of rneutrokine-alpha in the range of 0.02-2 mg / kg (specific preferred embodiment administration in this embodiment). The amount is 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, or 0.2 mg. / Kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1.0mg / kg, 1.1mg / kg, 1.2mg / Kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg and 2.0 mg / kg , But not limited to this).

Microscopically, the effect of Neutrokine-alpha administration was observed in spleen fragments stained with conventional hematoxylin and eosin (H & E) and immunohistochemically with mAbs specific for CD45R (B220) (FIG. 11A). . The structure of the normal spleen changes with the rapid expansion of the peripheral zone of white water and the unique increase in cell fidelity of red water (FIG. 11A). Expansion of the peripheral zone appears to be the result of an increased number of lymphocytes expressing the B cell marker CD45R (B220). In addition, an appropriate number of CD45R (B220) positive cells infiltrate the lymphatic (PALS) region around the T cell enriched artery. This suggests that the white water change is due to the increase in B cell number. Concentrated packed cell populations, often filling red water pores, are not stained by CD45R (B220). Further experimentation is needed to characterize and characterize all cell types involved in the mechanism by which Neutrokine-alpha alters the structure of the spleen.

Flow cytometry of the spleens of mice treated with 2 mg / kg Neutrokine-alpha showed that Neutrokine-alpha showed a proportion of mature (CD45R (B220) ^dull , ThB ^bright ) B cells than was observed in control mice. It appears to increase about 10 fold (FIG. 11B). In addition, in further assays in which mice were treated with buffer, Neutrokine-alpha 0.08 mg / kg, 0.8 mg / kg, 2 mg / kg or 8 mg / kg, 0.08 mg / kg, 0.8 mg / kg and 2 mg / kg The ratio of mature (CD45R (B220) ^dull , ThB ^bright ) B cells, respectively, is increased about 10-fold than that observed in control mice, whereas buffer and 8 mg / kg produce mature B cells at approximately the same rate. Is observed. See Table 4.

FACS Analysis of B Cell Subjects in Mouse Spleen Neutrokine-α (mg / kg) % Mature B cells (R2) % CD45R-positive cells (R1) Control (buffer) 1.26 52.17 0.08 mg / kg 16.15 56.53 0.8 mg / kg 18.54 57.56 2mg / kg 16.54 57.55 8mg / kg 1.24 61.42

A possible consequence of mature B cells in vivo is the relative increase in serum Ig titers. Therefore, the concentrations of serum IgA, IgG and IgM are compared between buffered mice and Neutrokine-alpha treated mice (FIG. 11C). Neutrokine-alpha administration increases serum concentrations of IgA and IgM by two and five fold, respectively. Interestingly, the blood circulation concentration of IgG does not increase.

Moreover, dose-dependent responses were observed in serum IgA titers seen in mice treated with varying amounts of Neutrokine-alpha over 4 days, while a clear dose dependency was observed upon administration of the same amount of Neutrokine-alpha over 2 days. It doesn't work. When administered for 4 days, administration of Neutrokine-alpha 8 mg / kg, 2 mg / kg, 0.8 mg / kg, 0.08 mg / kg and 0 mg / kg is about 800 μg / ml, 700 μg / ml, 400 Serum IgA titers of μg / ml, 200 μg / ml and 200 μg / ml are shown. Ie, Neutrokine-alpha 8 mg / kg, 2 mg / kg, 0.8 mg / kg, 0.08 mg / kg and 0 mg / kg for 4 days yielding about 4, 3.75, 2 and minimum folds. Administration increases the IgA serum concentration above the baseline concentration observed when only buffer is administered. In another embodiment, this experiment can be performed with any r Neutrokine-alpha amount in the range of 0.01-10 mg / kg. In a preferred embodiment, Neutrokine-alpha is administered in the range of 0.01-3 mg / kg. (A specific preferred dosage in this embodiment is 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / Kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / Kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / Kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / Kg, 2.9 mg / kg and 3.0 mg / kg, but are not limited to these). In another preferred embodiment, Neutrokine-alpha is administered in the range of 0.02-2 mg / kg (specifically preferred dosages in this embodiment include 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg and 2.0 mg / kg, but are not limited to these).

The data presented herein define Neutrokine-alpha as a novel component of the TNF-ligand superfamily that induces B cell proliferation and differentiation both in vivo and in vitro. Neutrokine-alpha may be expressed by other B cell growth and differentiation factors such as IL2 (Metzger, DW, et al., Res. Immunol. 146: 499-505 (1995)), IL4 (Armitage, RJ et al, Adv. Exp. Med. Biol. 292: 121-30 (1991); Yokota, T., et al., Proc. Natl. Acad. Sci. USA 84: 5894-98 (1986)), IL5 (Takatsu, K., et al. , Proc. Natl. Acad. Sci. USA 84: 4234-38 (1987); Bertolini, JN, et al., Eur. J. Immunol. 23: 398-402 (1993), IL6 (Poupart, P. , et al., EMBO J. 6: 1219-24 (1987); Hirano, T., et al., Nature 324: 73-76 (1986)), IL7 (Goodwin, RG, et al., Proc. Natl Acad. Sci. USA 86: 302-06 (1989); Namen, AE, et al., Nature 333: 571-73 (1988)), IL13 (Punnonen, J., et al., Allergy. 49: 576. -86 (1994)), IL15 (Armitage, RJ et al., J. Immunol. 154: 483-90 (1995)), CD40L (Armitage, RJ, et al., Nature 357: 80-82 (1992); Van Kooten, C. and Banchereau, J. Int. Arch. Allergy. Immunol. 113: 393-99 (1997)) or CD27L (CD70) (Oshima, H., et al., Int. Immunol. 10: 517-26 (1998); Lens, SM, et al., Semin. Immunol. 10: 491-99 (1998)) and the monocyte-specific gene / protein expression patterns and B lymphocytes. There are differences in specific receptor distribution and biological activity. Taken together, these data suggest that Neutrokine-alpha is involved in the exchange of signals between B cells and monocytes or their differentiated progeny. All B cells can utilize signaling in this manner, but limited expression patterns and Ig secretion suggest the role of Neutrokine-alpha involved in the activation of CD5 ⁺ or "uncommon" B cell responses. These B cells provide important components for the innate immune system and provide a protective effect against pathogens of the environment through the secretion of multireactive IgM and IgA antibodies (Pennell, CA, et al., Eur. J. Immunol. 19: 1289-95 (1989); Hayakawa, K., et al., Proc. Natl. Acad. Sci. USA 81: 2494-98 (1984)). Alternatively, Neutrokine-alpha can act as a regulator of T cell independent responses in a manner similar to the CD40 and CD40L responses in T cell dependent antigen activation (van den Eertwegh, AJ et al., J. Exp. Med. 178). : 155-65 (1993); Grabstein, KH, et al., J. Immunol. 150: 3141-47 (1993). Thus, Neutrokine-alpha, its receptor or related antagonist is useful for the treatment of B cell diseases associated with autoimmune, nephrogenic and / or immunodeficiency syndromes.

Way

mouse.

BALB / cAnNCR (6-8 weeks) is a small isolated cage with recycled paper according to the standard method purchased from Charles River Laboratories, Inc. (National Research Council, Guide for the care and use of laboratory animals (1999)). And granulated rodent diet (Harlan Sprague Dawley, Inc) and drinking water bottles are provided in an arbitrary amount manner. The animal protocol used in this study was reviewed and approved by the HGS Institutional Animal Care and Use Committee.

Isolation of Full Length Neutrokine-alpha cDNA.

To find sequences homologous to the receptor binding domain of the TNF family, the BLAST algorithm was used to examine the expressed sequence tag (EST) database of human genomic science inks. Full length Neutrokine-alphaclones were identified and sequenced and deposited in GenBank (Accession No. AF132600). The Neutrokine-alpha open reading frame was designed to anneal to a 5 'primer (5'-CAG ACT GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3') and an expected downstream stop codon to anneal to the expected start codon. PCR amplification using 'primer (5'-CAG ACT GGT ACC GTC CTG CGT GCA CTA CAT GGC-3'). BamHI and Asp 718 restriction sites are attached to the tail of the resulting amplicon and subcloned into a mammalian expression vector. Neutrokine-alpha is also expressed in p-CMV-1 (Sigma Chemicals).

Purification of Recombinant Human Neutrokine-alpha.

Full-length cDNA encoding Neutrokine-alpha is subcloned into baculovirus expression vector pA2 and transfects Sf9 insect cells (Patel, VP, et al., J. Exp. Med. 185: 1163-72 (1997). )). Recombinant Neutrokine-alpha was purified from cell supernatants 92 hours post infection using a combination of anion exchange, size exclusion and hydrophobic interaction columns. Purified protein is prepared in buffer containing 0.15 M NaCl, 50 mM NaOAc (pH 6), sterile filtered and stored at 4 ° C. until needed. SDS-PAGE and RP-HPLC analysis showed that the purity of rNeutrokine-alpha was greater than 95%. Endotoxin concentrations are below detection limits in LAL assays (Associates of Cape Cod, Falmouth, Mass.). The final purified Neutrokine-alpha protein has the N-terminal sequence of Ala-Val-Gln-Gly-Pro. This was identical to the sequence of soluble Neutrokine-alpha from CHO cell line stably transfected with full length Neutrokine-alpha gene.

Monoclonal Antibody Production.

BALB / cAnNCR mice were immunized with a 50 μg suspension of HisTag-Neutrokine-alpha suspended in complete Freund's adjuvant followed by two doses of the antigen suspended in incomplete Freund's adjuvant. Hybridomas and monoclonal antibodies are described in Gefter, M. L. et al., Somatic. Cell Genet. 3: 231-36 (1977); Akerstrom, B., et al., J. Immunol. 135: 2589-92 (1985).

Cell line.

All human cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA).

FACS analysis.

Neutrokine-alpha expression is PE conjugated to mouse anti-human Neutrokine-alpha mAb 2E5 (IgG1) and then to IgG of mice on human cell lines, freshly isolated normal peripheral blood nucleated cells and in vitro cultured monocytes. Evaluated with (ab ') 2 goat antibody (CALTAG Laboratories, Burlingame, CA). Cells were analyzed by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) Using propidium iodide to remove dead cells. Neutrokine-alpha binding uses rneutrokine-alpha biotinylated with N-hydroxysuccinimidobiotin reagent (Pierce, Rockford, IL) followed by PE-conjugated streptavidin (Dako Corp, Glostrup, Denmark) Measure by

Chromosomal Mapping.

To determine the intrachromosomal position of the Neutrokine-alpha gene, Neutrokine-alpha specific primers (5 'primer: 5'-TGG TGT CTT TCT ACC AGG TGG-3' and 3 'primers: 5'-TTT CTT CTG GAC CCT GAA CGG-3 ') is used to screen monochromosomal somatic hybrid panels (Quantum Biotechnology, Canada) carrying each chromosome by PCR. The expected 233 bp PCR product is detected only in human chromosome 13 hybrids. Neutrokine-alpha produced SHGC on chromosome 13 using a 83 radiation hybrid (Research Genetics, St. Louis, MO) panel and the Stanford Human Genome Center database (http: //www.shgc.stanford.edu.RH/rhserver). Binding to the -36171 marker was observed. This map was duplicated with the cytogenetic map of human chromosome 13, resulting in human Neutrokine-alpha corresponding to chromosome band 13q34.

B lymphocyte proliferation assay.

Human tonsil B cells were purified by magnetic beads (MACS) consumption method of CD3-positive cells. The resulting cell population usually has 95% or more of B cells, as measured by the expression of CD19 and CD20. Various dilutions of human r Neutrokine-alpha or control protein recombinant human IL2 were collected in a total volume of 150 μl of culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / ml penicillin, 100 μg / ml streptomycin and ^10-5 is added to each well of a 96-well plate was added 10 ⁵ B cells were suspended in the dilution plate sorbitan blank RPMI 1640) containing (SAC) or wherein -IgM. Proliferation is quantified with ³ H-thymidine (6.7 Ci / mM) 20 h pulses (1 μCi / well) from 72 h after factor addition.

Histological Analysis.

Spleens were fixed in 10% neutral buffered formalin, impregnated with paraffin, cut to 5 μm, mounted on glass slides and stained with hematoxylin and eosin or immunohistochemistry for CD45R (B220) Detected by enzyme labeled indirect method (Hilbert, DM, et al., Eur. J. Immunol. 23: 2412-18 (1993)).

Neutrokine-alpha cell surface expression Cell line Cell morphology Neutrokine-α cell surface expression Monocyte system U-937 Lymphoma, Histiocyte / Macrophage + BL-60 Leukemia, acute promyeloid + K-562 Leukemia, Chronic Myeloid + THP-1 Leukemia, acute monocytes + T-system Jurkat Leukemia, T Lymphocytes - SUP-T13 Leukemia, T Lymphocytes - MOLT-4 Leukemia, T Lymphocytes - B-system Daudi Burkitt's disease, lymphocyte - Namalwa Bucket's disease, lymphocyte - Raji Bucket's disease, lymphocyte - Reh Leukemia, lymphocyte - ARH-77 Leukemia, plasma cells - IM9 Myeloma - RPMI 8226 Myeloma -

Example 7 Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation

Generation of a functional humoral immune response requires soluble cognate signaling between B lineage cells and their microenvironment. The signal can impart a positive stimulus to allow B lineage cells to sustain natural growth or a negative stimulus to direct B lineage cells to stop the current growth pathway. To date, numerous stimulatory and inhibitory signals have been shown to affect B cell responses including IL-2, IL-4, IL5, IL6, IL-7, IL-10, IL-13, IL14 and IL15. Interestingly, although these signals themselves are weak effectors, they can induce activation, proliferation, differentiation, homing, resistance and death of B cell individuals with various costimulatory proteins. One of the most studied of B cell costimulatory proteins is the TNF superfamily. In this family, CD40, CD27 and CD30, along with their respective ligands CD154, CD70 and CD153, have been shown to modulate various immune responses. Assays capable of detecting and / or observing the proliferation and differentiation of these B cell populations and their precursors are important tools for measuring the effects that various proteins can have on B cell populations in terms of proliferation and differentiation. Two assays designed to detect the differentiation, proliferation or inhibition of B cell individuals and their precursors are described below.

In vitro assay

Purified Neutrokine-alpha and / or Neutrokine-alphaSV proteins or truncated forms thereof were evaluated for their activity inducing activation, proliferation, differentiation or inhibition of B cell individuals and their precursors. The activity of purified Neutrophin-alpha and / or Neutrokine-alphaSV proteins on purified human tonsil B cells qualitatively measured in a dosage range of 0.1 to 10,000 ng / ml was stabilized with formalin as a priming agent. Evaluation was performed by standard B lymphocyte co-stimulation assays in which purified tonsil B cells were cultured in the presence of Aureus Cowan I (SAC) or immobilized anti-human IgM antibody. Secondary signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to induce B cell proliferation as measured by the triple hydrogenated thymidine incorporation rate. New synergists can be easily identified using this assay. This assay involves separating human amygdala B cells by magnetic beads (MACS) consumption of CD3 positive cells. The resulting cell population has at least 95% B cells as measured by the expression of CD45R (B220). Various dilutions of each sample, culture medium (10% of the total volume 150㎕ ^{FBS, 5x10 -5 M 2ME, 100U} / ㎖ penicillin, 10㎍ / ㎖ streptomycin and 10 ^-5 RPMI 1640 containing SAC of dilution 10 ⁵ B cells suspended in) were added to each well of a 96 well plate. Proliferation or inhibition was quantified with ³ H-thymidine (6.7 Ci / mM) 20 h pulses (1 μCi / well) from 72 hours after factor addition. Positive and negative controls are IL2 and medium, respectively.

Agonists (including Neutrokine-alpha and / or Neutrokine-alphaSV polypeptide fragments) increase the B cell proliferation compared to the cell proliferation observed when contacting the same B cell number with the same concentration of priming agent. Calculate. The antagonists described herein are Neutrokine-alpha soluble forms (e.g. Neutrokine- represented by SEQ ID NO: 2) that induce an increase in B cell proliferation activity in the absence of the same B cell number, the same concentration of priming agent and no antagonist. Decrease in B cell proliferation when compared to a control containing the same concentration of alpha polypeptides (71-285, 81-285, 112-285 or 134-285).

In vivo analysis

BALB / c mice are injected (intraperitoneally) twice daily with buffer, Neutrokine-alpha and / or Neutrokine-alphaSV protein or its truncated form 2 mg / kg. Mice subjected to this treatment for four consecutive days are killed and various tissues and serum are collected and used for analysis. Comparing H & E fragments from normal spleen and Neutrokine-alpha and / or Neutrokine-alphaSV protein treated spleen, red regions that may indicate periarterial lymphatic spread and / or activation of differentiation and proliferation of B cell individuals The results obtained by the activity of Neutrokine-alpha and / or Neutrokine-alphaSV proteins on spleen cells, such as a significant increase in nucleus cell fidelity, are confirmed. Immunohistochemical studies using the B cell marker, anti-CD45R (B220), show an increase in B cells in loosely defined B cell regions where all physical changes to the spleen cells, such as the de-organization of the spleen, infiltrate existing T cell regions. It is used to measure whether it is.

Flow cytometry for the spleen of mice treated with Neutrokine-alpha and / or Neutrokine-alphaSV protein revealed that ThB ⁺ , CD45R (Nutrokin-alpha / or Neutrokine-alphaSV protein was observed in control mice). B220) is used to determine if the percentage of pull B cells specifically increases.

Similarly, the expected result of increased mature B cells in vivo is the relative increase in serum Ig titer. Thus, serum IgM and IgA concentrations are compared between Neutrokine-alpha and / or Neutrokine-alphaSV protein treated mice and mice treated with buffer only.

Example 8 Effect of Neutrokine-alpha and Its Agonists on the Treatment of Graft-versus-Host Disease-related Atrophy and Dysplasia in Mice

Analysis of the usefulness of Neutrokine-alpha in treating, preventing and / or diagnosing graft-versus-host disease (GVHD) -associated lymphoid dysplasia / atrophy (BALB / c X C57BL / 6) F1 (CBF1) The mouse model is carried out using C57BL / 6 parents. This parent, used in the F1 mouse model, is well characterized and is a reproducible animal model of GVHD in bone marrow transplant patients known in the art (Gleichemann, et al., Immunol. Today 5: 324, 1984). . Soluble Neutrokine-alpha is believed to induce proliferation and differentiation of B lymphocytes and to treat lymphoid dysplasia and atrophy observed in the GVHD animal model (Piguet, et al., J. Exp. Med. 166: 1280 (1987); Hattori, et al., Blood 90: 542 (1997)).

Initiation of GVHD disease is induced by intravenous injection of (1 1 to 5x10 ⁸ spleen cells from C57BL / 6 mice (both mice obtained from Jackson Lab, Bar Harbor, Maine) to F1 mice (both mice obtained from Jackson Lab). do. Neutrokine from 6 to 8 mice after a parental injection of lymphoid dysplasia and atrophy (about 5 days), moderate (about 12 days) or severe (about 20 days) Alpha 0 1-5.0 mg / kg or buffer control is administered daily intraperitoneally, intramuscularly or intradermally. The effect of Neutrokine-alpha on lymphoid dysplasia and atrophy of the spleen is analyzed by FACS and histopathology at various time points (3-4 times) between 10 and 30 days. Briefly, splenocytes are prepared from normal CBF1, GVHD or Neutrokine-alpha treated mice, and fluorescein phycoerythrin-conjugated antiH-2Kb, biotin conjugated antiH2Kd and FITC conjugated antiCD4, anti Stain with CD8 or anti-B220 and then with chromium conjugated avidin. All such conjugated antibodies can be purchased from PharMingen (San Diego, Calif.). Cells are then analyzed by FACScan (Becton Dickinson, San Jose, Calif.). Recipient and donor lymphocytes were identified as H-2Kb + Kd + and H-2Kb + Kd− cells, respectively. The cell number of CD4 + T, CD8 + T and B220 + B cells of recipient or donor origin is calculated from the total number of recovered splenocytes and the proportion of each subpopulation is determined by three color analysis. Histological evaluation of relative tissue damage in other GVHD related organs (liver, skin and intestine) can be performed after killing the animal.

Finally, Neutrokine-alpha and buffered animals are subjected to clinical assessments every other day to assess poor health, body weight and lethality.

Neutrokine-alpha agonists and antagonists can be investigated in an acute GVHD mouse model.

Example 9 Isolation of Antibody Fragments Induced Against Neutrokine-alphapolypeptides from the scFvs Library

Natural V-genes isolated from human PBLs are constructed into large libraries of antibody fragments that contain responsiveness to Neutrokine-alpha and Neutrokine-alphaSV with or without exposure of the donor (herein See US Pat. No. 5,885,793, incorporated herein by reference.

Library rescue

The library of scFvs is constructed from RNA of human PBLs as described in WO92 / 01047 (incorporated herein by reference). About 10 ⁹ E. carrying phagemid to select for antibody fragments that phage express. E. coli was inoculated in 50 ml of 2xTY (2xTY-AMP-GLU) containing 1% glucose and 100 μg / ml ampicillin and grown to OD 0.8 under shaking. 5 ml of this culture was inoculated into 50 ml of 2xTY-AMP-GLU, 2x10 ⁸ TU of Delta gene 3 helper (M13 delta gene III, WO92 / 01047) was added, and the culture was 45 at 37 ° C. without shaking. Incubate for minutes, then incubate for 45 minutes at 37 ° C under shaking. The culture was centrifuged at 4000 rpm for 10 minutes and the pellet was resuspended in 2 liters of 2x TY containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and grown overnight. Phage are prepared as described in WO92 / 01047.

M13 delta gene III is prepared as follows. The M13 delta gene III helper phage does not encode the gene III protein. Thus, phage (medium) expressing antibody fragments exhibit greater binding potency against the antigen. Infectious M13 delta gene III particles are prepared by growing helper phage in cells carrying a pUC19 derivative that supplies wild type Gene III protein during phage morphology. The culture was incubated for 1 hour at 37 ° C. without shaking, followed by another 1 hour at 37 ° C. under shaking. Spin down cells (IEC-Centra 8, 4000 revs / min, 10 minutes) and resuspend in 300 ml of 2xTY liquid medium (2xTY-AMP-KAN) containing 100 μg / ml ampicillin and 25 μg / ml kanamycin And grow under shaking at 37 ° C. overnight. Phage particles were purified and concentrated from the culture via two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give about 10 ¹³ transduction unit / ml (ampicillin resistant clone) concentration is obtained.

Library Panning

Immune tubes (Nunc) are coated overnight in PBS to which 4 ml of the polypeptide of the invention at 100 μg / ml or 10 μg / ml is added. The tube is blocked with 2% Marvel-PBS at 37 ° C. for 2 hours and then washed three times with PBS. Add about 10 ¹³ TU of phage to this tube and incubate for 30 minutes at room temperature while rotating on the up-and-down turntable and leave for another 1.5 hours. The tube is washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage is eluted by addition of 1 ml of 100 mM triethylamine and spun for 15 minutes on an upper and lower turntable, after which the solution is neutralized immediately with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. The eluted phages were incubated with bacteria for 30 minutes at 37 ° C. Infect 10 ml of E. coli TG1. This teeth. Coli is then plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is screened with delta gene 3 helper phage as described above to produce phage for subsequent screening steps. This process is repeated with a total of four affinity tablets, in which the tube wash is increased 20 times with PBS, 0.1% Tween-20, 20 times with 3rd and 4th PBS.

Properties of the binder

Phage eluted from the 3rd and 4th screening were used. E. coli HB2151 was infected and soluble scFv was produced from a single colony for analysis (Marks, et al., 1991). ELISA was performed on microtiter plates coated with 10 pg / ml of the polypeptide of the invention contained in 50 mM bicarbonate pH 9.6. Clones that are positive in ELISA are further characterized by PCR fingerprinting (eg WO92 / 01047) and subsequent sequencing.

Example 10 Neutralization of Neutrokine-alpha / Neutrokine-alpha Receptor Interactions with Anti-Neutrokine-Alfamonoclonal Antibodies

A monoclonal antibody to Neutrokine-alpha protein was prepared according to the following method. Briefly, mice were percutaneously injected (anterior part of the back) of an emulsion in which 50 μg of His-tagged Neutrokine-alpha protein prepared by the method of Example 2 in 100 μl of PBS was emulsified in 100 μl of complete Freund's adjuvant. . 25 μg Neutrokine-alpha in unsafe Freund's adjuvant was injected three more times percutaneously at two week intervals. Finally, animals were rested for 1 month prior to intraperitoneal administration of 25 μg Neutrokine-alpha in PBS. Four days later mice were killed and splenocytes were harvested for fusion.

The “fusion” method is a method in which the manufacturer modified a previously disclosed method (Gefter, ML, et al. Somatic Cell Genet 3: 231-36 (1977); Boehringer Mannheim, PEG 1500 (Cat. No. 783641), product description). According to the PEG1500 (Boehringer Mannheim), 2x10E7 P3X63Ag8.653 plasmacytoma cells and spleen cells derived from the spleen were performed.

After fusion, cells were resuspended in 400 ml of HAT medium supplemented with 20% FBS and 4% hybridoma supplement (Boehringer Mannheim) and distributed to 96 well plates at a density of 200 μl / well. Seven days after fusion, 100 μl of medium was aspirated off and replaced with 100 μl of fresh medium. After 14 days of fusion, hybridomas are selected for antibody production.

Hybridoma supernatants were screened for binding to Neutrokine-alpha proteins immobilized on plates using ELISA. Plates are coated with Neutrokine-alpha by overnight incubation of Neutrokine-alpha in PBS at a concentration of 2 μg per ml overnight. Hybridoma supernatants are diluted 1:10 with PBS, injected into each well of Neutrokine-alpha-coated plates and incubated overnight at 4 ° C. The next day, the plates are washed three times with PBS containing 0.1% Tween-20 and developed using the anti-mouse IgG ABC system (Vector Laboratories). The color reaction was stopped by adding 25 ml / well of 2M H ₂ SO ₄ . The plate is read at 450 nm.

Hybridoma supernatants are examined for Ig isotype using iso strips. Cloning is performed by limiting dilution of HT medium. About 3 × 10 E6 cells in 0.9 ml of HBSS were injected into pristane pretreated mice. After 7-9 days, ascites fluid is collected with a 19 g syringe. All antibodies are purified by Protein G affinity chromatography using the Acta FPLC system (Pharmacia).

After the first and two consecutive transdermal injections, all three mice developed a strong immune response and serum titers were 10E-7 as measured by ELISA on Neutrokine-alpha coated plates.

In one experiment, splenocytes from positive mice are used to generate more than 1000 native hybridomas. 917 of these are selected to produce antineutrokine-alpha antibodies. Screening is performed using a 1: 1 diluted supernatant to detect all positive clones. Of the 917 hybridomas selected, 76 were found to be positive, of which 17 were observed to be IgG producers. After affinity testing and cloning, nine were selected for further propagation and purification.

All purified monoclonal antibodies can bind to various forms of Neutrokine-alpha (including proteins produced in the baculovirus system (Example 2) and His-tagged proteins) in Western blot analysis and ELISA. Six of the nine clones could also bind Neutrokine-alpha present on the surface of THP-1 cells. However, none of the antibodies tested can capture Neutrokine-alpha from solution.

High-affinity anti-neutrokine-alphamonoclonal antibodies that recognize Neutrokine-alpha expressed on the cell surface but not Neutrokine-alpha in solution are useful for in vivo neutralization studies and in vitro monocyte and B cell analysis. Can be. This antibody is also useful for sensitive detection of Neutrokine-alpha in Western blots.

In another experiment, splenocytes from positive mice are used to produce more than 1000 native hybridomas. 729 of the native hybridomas are selected to produce anti-neutrokine-alpha antibodies. Screening is performed under stringent conditions using supernatant diluted 1:10 to collect only higher affinity clones. Of the 729 hybridomas selected, 23 were positive, including 16 IgM producers and 7 IgG producers (4 of which provided strong IgM baseline). In this experiment, the iotype distribution of IgG was biased only to the IgG2 subclass. Three of the seven IgG hybridomas produced antibodies of the IgG2a subclass, two producing antibodies of IgG2b, while the other two were IgG1 producers.

All positive hybridoma-derived supernatants produced in the second experiment are tested for activity inhibiting the proliferation of Neutrokine-alpha mediators of B cells. In a first screening experiment, two hybridomas producing IgG neutralizing antibodies were detected (antibodies 16C9 and 12C5). In another experiment, the IgG neutralizing activity of this hybridoma (ie 16C9 and 12C5) is confirmed, and further strong neutralizing supernatants from hybridomas 15C10 and 4A6 are identified.

Three clones were then propagated in vivo (15C10 clones were propagated in a hollow fiber system) and antibodies were purified by affinity chromatography. All three of these clones can bind Neutrokine-alpha on the surface of THP-1 cells and can also bind (ie, capture) Neutrokine-alpha in solution.

Specifically, an experiment is conducted using the antineutrokine-alphamonoclonal antibody described in the second experiment to determine whether the antibody neutralizes Neutrokine-alpha / Neutrokine-alpha receptor binding. Briefly, Neutrokine-alpha proteins were biotinylated using EZ-link T NHS-biotin reagent (Pierce, Rockford, IL). Biotinylated Neutrokine-alpha is used to identify cell surface proteins that bind Neutrokine-alpha. Preliminary results confirm that Neutrokine-alpha binds to receptors on B lymphoid cells.

The addition of anti-Neutrokine-alpha antibodies produced in the second experiment described above neutralized the binding of Neutrokine-alpha to Neutrokine-alpha receptors. In a specific embodiment, the antineutrokine-alpha antibody 15C10 neutralizes the binding of Neutrokine-alpha to the Neutrokine-alpha receptor.

In other words, the anti-neutrokine-alpha monoclonal antibody produced in the second experiment described above (in particular, antibody 15C10) recognizes and binds both membrane-bound Neutrokine-alpha protein and soluble Neutrokine-alpha protein and in vitro. Neutrakine-alpha / Neutrokine-alpha receptor neutralizes binding.

It is apparent that the present invention can be implemented in a manner different from that specifically described in the detailed description and examples above. Various modifications of the invention are possible in light of the above teachings and therefore fall within the scope of the appended claims.

The entire contents of all publications (patents, patent applications, technical magazines, experiments, books or other documents) cited herein are incorporated by reference.

Also in computerized form and in written form in co-pending applications 09 / 005,874 (1998.1.12), US60 / 036,100 (1997.1.14) and PCT / US96 / 17957 (October 25, 1996) filed with this specification. A list of all sequences submitted is hereby incorporated by reference.                 

Information about deposited microorganisms

(PCT Rule 13bis)

A. The information described below relates to the microorganisms mentioned in line 21 of page 7 of the specification.  B. Deposit Confirmation Additional deposits are indicated in the next chapter.                                      Name of Depositary American Type Culture Collection  Depositary address (including postal code and country)                                                                                                                            United States Virginia 20110-2209 Manassas University Boulevard 10801                                                                                Deposited: October 22, 1996 Accession number: 97768  C. Additional Information (blank if not applied) The above information is included in the next section.                                       D. The designated country to which the information provided applies (if the information does not apply to all specified countries)                                       In connection with the designation of the European European patent, the sample of deposited microorganisms may be collected by the person requesting the sample until the statement of the grant of the European patent is published or until the date the application is rejected or withdrawn or until it is due to be withdrawn. It will be useful only by distributing the sample to the nominated expert (Rule 28 (4) EPC)  E. Separate provision of information (leave blank if not applied)                                       The information listed below will then be submitted to the International Bureau (specify the general nature of the information, eg "Accession Number of the Deposit").                                                                                                                          For repair office For International Bureau  □ You have received this document along with your international application.                                                                                 □ This document was received by the International Bureau on a dated basis.  Official                                       Official

 Form PCT / RO / 134 (July 1992)                 

ATCC Deposit No. 97768

Canada

Applicants will only distribute samples of biological deposits referred to herein to independent experts nominated by the Commissioner until the Canadian patent is granted on the basis of the application, or if the application is rejected or abandoned and can no longer be recovered or withdrawn. The applicant must, therefore, inform the International Bureau in writing before the technical preparation for publication of the International Patent Office is completed.

Norway

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Norwegian Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provisions. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and drawn up by the Norwegian Patent Office or, in individual cases, by an applicant.

Australia

Applicant hereby declares that the distribution of microbial samples may be made to specialized recipients without understanding of the present invention prior to the approval of the patent or prior to the termination, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulation).

Finland

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the application is published or unpublished and final measured by the National Board of Patents and Regulations.

England

Applicant hereby requests that the sample distribution of microorganisms be made only to experts. This application must be submitted by the applicant to the International Bureau before the technical preparation for international publication of the application is completed.

Denmark

Applicants hereby request that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Danish Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Danish Patent Office or, in individual cases, a person recognized by the applicant.

Sweden

Applicants hereby request that the distribution of samples be made only to experts in the field until the present application is published or unpublished by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using Form PCT / RO / 134, copied in Annex Z of Book 1 of the PCT Applicant's Guide) before the expiration of 16 months from the priority date. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Swedish Patent Office or, in an individual case, recognized by the applicant.

Netherlands

Applicant hereby requests that microorganisms only distribute their samples to experts as provided in Patent Rule 31F (1) until the date of approval of the Dutch patent or the date of rejection, withdrawal or extinction of the application. This application must be submitted by the applicant to the Dutch Industrial Property Office before the earliest of the time points at which the application is available by a third party under Section 22C or Section 25 of the Dutch Patent Provisions.                 

Information about deposited microorganisms

(PCT Rule 13bis)

 A. The information described below relates to the microorganisms mentioned in line 5 of page 8 of the specification.  B. Deposit Confirmation Additional deposits are indicated in the next chapter.                                      Name of Depositary American Type Culture Collection  Depositary address (including postal code and country)                                                                                                                            United States Virginia 20110-2209 Manassas University Boulevard 10801                                                                                Deposited Date: December 10, 1998 Accession number: 203518  C. Additional Information (blank if not applied) The above information is included in the next section.                                       D. The designated country to which the information provided applies (if the information does not apply to all specified countries)                                       In connection with the designation of the European European patent, the sample of deposited microorganisms may be collected by the person requesting the sample until the statement of the grant of the European patent is published or until the date the application is rejected or withdrawn or until it is due to be withdrawn. It will be useful only by distributing the sample to the nominated expert (Rule 28 (4) EPC)  E. Separate provision of information (leave blank if not applied)                                       The information listed below will then be submitted to the International Bureau (specify the general nature of the information, eg "Accession Number of the Deposit").                                                                                                                          For repair office For International Bureau  □ You have received this document along with your international application.                                                                                 □ This document was received by the International Bureau on a dated basis.  Official                                       Official

 Form PCT / RO / 134 (July 1992)                 

ATCC Deposit No. 203518

Canada

Applicants will only distribute samples of biological deposits referred to herein to independent experts nominated by the Commissioner until the Canadian patent is granted on the basis of the application, or if the application is rejected or abandoned and can no longer be recovered or withdrawn. The applicant must, therefore, inform the International Bureau in writing before the technical preparation for publication of the International Patent Office is completed.

Norway

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Norwegian Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provisions. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and drawn up by the Norwegian Patent Office or, in individual cases, by an applicant.

Australia

Applicant hereby declares that the distribution of microbial samples may only be made to specialized recipients prior to patent approval or prior to the loss, rejection or withdrawal of the application without an understanding of the present invention (Rule 3.25 (3) of the Australian Patent Regulation).

Finland

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the application is published or unpublished and final measured by the National Board of Patents and Regulations.

England

Applicant hereby requests that the sample distribution of microorganisms be made only to experts. This application must be submitted by the applicant to the International Bureau before the technical preparation for international publication of the application is completed.

Denmark

Applicants hereby request that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Danish Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Danish Patent Office or, in individual cases, recognized by the applicant.

Sweden

Applicants hereby request that the distribution of samples be made only to experts in the field until the present application is published or unpublished by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using Form PCT / RO / 134, copied in Annex Z of Book 1 of the PCT Applicant's Guide) before the expiration of 16 months from the priority date. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Swedish Patent Office or, in an individual case, recognized by the applicant.

Netherlands

Applicant hereby requests that microorganisms only distribute their samples to experts as provided in Patent Rule 31F (1) until the date of approval of the Dutch patent or the date of rejection, withdrawal or extinction of the application. This application must be submitted by the applicant to the Dutch Industrial Property Office before the earliest of the time points at which the application is available by a third party under Section 22C or Section 25 of the Dutch Patent Provisions.                 

Information about deposited microorganisms

(PCT Rule 13bis)

 A. The information described below relates to the microorganisms mentioned in line 8 of page 229 of the specification.  B. Deposit Confirmation Additional deposits are indicated in the next chapter.                                      Name of Depositary American Type Culture Collection  Depositary address (including postal code and country)                                                                                                                            United States Virginia 20110-2209 Manassas University Boulevard 10801                                                                                Deposition Date: January 7, 2000 Accession number: PTA-1158  C. Additional Information (blank if not applied) The above information is included in the next section.                                       D. The designated country to which the information provided applies (if the information does not apply to all specified countries)                                       In connection with the designation of the European European patent, the sample of deposited microorganisms may be collected by the person requesting the sample until the statement of the grant of the European patent is published or until the date the application is rejected or withdrawn or until it is due to be withdrawn. It will be useful only by distributing the sample to the nominated expert (Rule 28 (4) EPC)  E. Separate provision of information (leave blank if not applied)                                       The information listed below will then be submitted to the International Bureau (specify the general nature of the information, eg "Accession Number of the Deposit").                                                                                                                          For repair office For International Bureau  □ You have received this document along with your international application.                                                                                 □ This document was received by the International Bureau on a dated basis.  Official                                       Official

 Form PCT / RO / 134 (July 1992)                 

ATCC Deposit No. PTA-1158

Canada

Applicants will only distribute samples of biological deposits referred to herein to independent experts nominated by the Commissioner until the Canadian patent is granted on the basis of the application, or if the application is rejected or abandoned and can no longer be recovered or withdrawn. The applicant must, therefore, inform the International Bureau in writing before the technical preparation for publication of the International Patent Office is completed.

Norway

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Norwegian Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provisions. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and drawn up by the Norwegian Patent Office or, in individual cases, by an applicant.

Australia

Applicant hereby declares that the distribution of microbial samples may be made to specialized recipients without understanding of the present invention prior to the approval of the patent or prior to the termination, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulation).

Finland

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the application is published or unpublished and final measured by the National Board of Patents and Regulations.

England

Applicant hereby requests that the sample distribution of microorganisms be made only to experts. This application must be submitted by the applicant to the International Bureau before the technical preparation for international publication of the application is completed.

Denmark

Applicants hereby request that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Danish Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Danish Patent Office or, in individual cases, a person recognized by the applicant.

Sweden

Applicants hereby request that the distribution of samples be made only to experts in the field until the present application is published or unpublished by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using Form PCT / RO / 134, copied in Annex Z of Book 1 of the PCT Applicant's Guide) before the expiration of 16 months from the priority date. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Swedish Patent Office or, in an individual case, recognized by the applicant.

Netherlands

Applicant hereby requests that microorganisms only distribute their samples to experts as provided in Patent Rule 31F (1) until the date of approval of the Dutch patent or the date of rejection, withdrawal or extinction of the application. This application must be submitted by the applicant to the Dutch Industrial Property Office before the earliest of the time points at which the application is available by a third party under Section 22C or Section 25 of the Dutch Patent Provisions.                 

Information about deposited microorganisms

(PCT Rule 13bis)

 A. The information described below relates to the microorganisms mentioned in line 8 of page 229 of the specification.  B. Deposit Confirmation Additional deposits are indicated in the next chapter.                                      Name of Depositary American Type Culture Collection  Depositary address (including postal code and country)                                                                                                                            United States Virginia 20110-2209 Manassas University Boulevard 10801                                                                                Deposition Date: January 7, 2000 Accession number: PTA-1159  C. Additional Information (blank if not applied) The above information is included in the next section.                                       D. The designated country to which the information provided applies (if the information does not apply to all specified countries)                                       In connection with the designation of the European European patent, the sample of deposited microorganisms may be collected by the person requesting the sample until the statement of the grant of the European patent is published or until the date the application is rejected or withdrawn or until it is due to be withdrawn. It will be useful only by distributing the sample to the nominated expert (Rule 28 (4) EPC)  E. Separate provision of information (leave blank if not applied)                                       The information listed below will then be submitted to the International Bureau (specify the general nature of the information, eg "Accession Number of the Deposit").                                                                                                                          For repair office For International Bureau  □ You have received this document along with your international application.                                                                                 □ This document was received by the International Bureau on a dated basis.  Official                                       Official

 Form PCT / RO / 134 (July 1992)                 

ATCC Deposit No. PTA-1159

Canada

Applicants will only distribute samples of biological deposits referred to herein to independent experts nominated by the Commissioner until the Canadian patent is granted on the basis of the application, or if the application is rejected or abandoned and can no longer be recovered or withdrawn. The applicant must, therefore, inform the International Bureau in writing before the technical preparation for publication of the International Patent Office is completed.

Norway

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Norwegian Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provisions. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and drawn up by the Norwegian Patent Office or, in individual cases, by an applicant.

Australia

Applicant hereby declares that the distribution of microbial samples may be made to specialized recipients without understanding of the present invention prior to the approval of the patent or prior to the termination, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulation).

Finland

Applicant hereby requests that the distribution of the sample be made only to experts in the field until the application is published or unpublished and final measured by the National Board of Patents and Regulations.

England

Applicant hereby requests that the sample distribution of microorganisms be made only to experts. This application must be submitted by the applicant to the International Bureau before the technical preparation for international publication of the application is completed.

Denmark

Applicants hereby request that the distribution of the sample be made only to experts in the field until the present application is published or unpublished by the Danish Patent Office. This application must be submitted to the Norwegian Patent Office before the time when the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Danish Patent Office or, in individual cases, a person recognized by the applicant.

Sweden

Applicants hereby request that the distribution of samples be made only to experts in the field until the present application is published or unpublished by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using Form PCT / RO / 134, copied in Annex Z of Book 1 of the PCT Applicant's Guide) before the expiration of 16 months from the priority date. If such application is submitted by the applicant, it is granted that the expert can use it when a third party applies for the distribution of the sample. The expert may be a person on the list of experts recognized and prepared by the Swedish Patent Office or, in an individual case, recognized by the applicant.

Netherlands

Applicant hereby requests that microorganisms only distribute their samples to experts as provided in Patent Rule 31F (1) until the date of approval of the Dutch patent or the date of rejection, withdrawal or extinction of the application. This application must be submitted by the applicant to the Dutch Industrial Property Office before the earliest of the time points at which the application is available by a third party under Section 22C or Section 25 of the Dutch Patent Provisions.

<110> Human Genome Sciences, Inc. <120> Neutrokine-alpha and Neutrokine-alpha Splice Variant <130> PF343PCT2 <140> Unassigned <141> 2000-02-22 <150> 60 / 122,388 <151> 1999-03-02 <150> 60 / 124,097 <151> 1999-03-12 <150> 60 / 126,599 <151> 1999-03-26 <150> 60 / 127,598 <151> 1999-04-02 <150> 60 / 130,412 <151> 1999-04-16 <150> 60 / 130,696 <151> 1999-04-23 <150> 60 / 131,278 <151> 1999-04-27 <150> 09 / 255,794 <151> 1999-02-23 <150> 60 / 131,673 <151> 1999-04-29 <150> 60 / 136,784 <151> 1999-05-28 <150> 60 / 142,659 <151> 1999-07-06 <150> 60 / 145,824 <151> 1999-07-27 <150> 60 / 167,239 <151> 1999-11-24 <150> 60 / 168,624 <151> 1999-12-03 <150> 60 / 171,108 <151> 1999-12-16 <150> 60 / 171,626 <151> 1999-12-23 <150> 60 / 176,015 <151> 2000-01-14 <160> 38 <170> KOPATIN 1.71 <210> 1 <211> 1100 <212> DNA <213> Homo sapiens <220> <221> CDS (147) .. (1001) <400> 1 aaattcagga taactctcct gaggggtgag ccaagccctg ccatgtagtg cacgcaggac 60 atcaacaaac acagataaca ggaaatgatc cattccctgt ggtcacttat tctaaaggcc 120 ccaaccttca aagttcaagt agtgat atg gat gac tcc aca gaa agg gag cag 173                              Met Asp Asp Ser Thr Glu Arg Glu Gln                                1 5 tca cgc ctt act tct tgc ctt aag aaa aga gaa gaa atg aaa ctg aag 221 Ser Arg Leu Thr Ser Cys Leu Lys Lys Arg Glu Glu Met Lys Leu Lys  10 15 20 25 gag tgt gtt tcc atc ctc cca cgg aag gaa agc ccc tct gtc cga tcc 269 Glu Cys Val Ser Ile Leu Pro Arg Lys Glu Ser Pro Ser Val Arg Ser                  30 35 40 tcc aaa gac gga aag ctg ctg gct gca acc ttg ctg ctg gca ctg ctg 317 Ser Lys Asp Gly Lys Leu Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu              45 50 55 tct tgc tgc ctc acg gtg gtg tct ttc tac cag gtg gcc gcc ctg caa 365 Ser Cys Cys Leu Thr Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln          60 65 70 ggg gac ctg gcc agc ctc cgg gca gag ctg cag ggc cac cac gcg gag 413 Gly Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu      75 80 85 aag ctg cca gca gga gca gga gcc ccc aag gcc ggc ctg gag gaa gct 461 Lys Leu Pro Ala Gly Ala Gly Ala Pro Lys Ala Gly Leu Glu Glu Ala  90 95 100 105 cca gct gtc acc gcg gga ctg aaa atc ttt gaa cca cca gct cca gga 509 Pro Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro Gly                 110 115 120 gaa ggc aac tcc agt cag aac agc aga aat aag cgt gcc gtt cag ggt 557 Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val Gln Gly             125 130 135 cca gaa gaa aca gtc act caa gac tgc ttg caa ctg att gca gac agt 605 Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser         140 145 150 gaa aca cca act ata caa aaa gga tct tac aca ttt gtt cca tgg ctt 653 Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu     155 160 165 ctc agc ttt aaa agg gga agt gcc cta gaa gaa aaa gag aat aaa ata 701 Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile 170 175 180 185 ttg gtc aaa gaa act ggt tac ttt ttt ata tat ggt cag gtt tta tat 749 Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr                 190 195 200 act gat aag acc tac gcc atg gga cat cta att cag agg aag aag gtc 797 Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val             205 210 215 cat gtc ttt ggg gat gaa ttg agt ctg gtg act ttg ttt cga tgt att 845 His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile         220 225 230 caa aat atg cct gaa aca cta ccc aat aat tcc tgc tat tca gct ggc 893 Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly     235 240 245 att gca aaa ctg gaa gaa gga gat gaa ctc caa ctt gca ata cca aga 941 Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg 250 255 260 265 gaa aat gca caa ata tca ctg gat gga gat gtc aca ttt ttt ggt gca 989 Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala                 270 275 280 ttg aaa ctg ctg tgacctactt acaccatgtc tgtagctatt ttcctccctt 1041 Leu Lys Leu Leu             285 tctctgtacc tctaagaaga aagaatctaa ctgaaaatac caaaaaaaaa aaaaaaaaa 1100 <210> 2 <211> 285 <212> PRT <213> Homo sapiens <400> 2 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu   1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro              20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu          35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val      50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg  65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly                  85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu             100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn         115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln     130 135 140 Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys 145 150 155 160 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser                 165 170 175 Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr             180 185 190 Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met         195 200 205 Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu     210 215 220 Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu 225 230 235 240 Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly                 245 250 255 Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu             260 265 270 Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu         275 280 285 <210> 3 <211> 233 <212> PRT <213> Homo sapiens <400> 3 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala   1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe              20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe          35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro      50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser  65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro                  85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu             100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser         115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly     130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro                 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu             180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu         195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly     210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 <210> 4 <211> 205 <212> PRT <213> Homo sapiens <400> 4 Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Arg Gly Thr Thr   1 5 10 15 Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala              20 25 30 Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala          35 40 45 Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala      50 55 60 Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg  65 70 75 80 Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn                  85 90 95 Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln             100 105 110 Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro         115 120 125 Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe     130 135 140 His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln 145 150 155 160 Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr                 165 170 175 Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val             180 185 190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu         195 200 205 <210> 5 <211> 244 <212> PRT <213> Homo sapiens <400> 5 Met Gly Ala Leu Gly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg   1 5 10 15 Gly Ser Leu Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu              20 25 30 Leu Leu Ala Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro          35 40 45 Gln Asp Gln Gly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln      50 55 60 Ala Gln Gln Gly Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu  65 70 75 80 Thr Asp Leu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro                  85 90 95 Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe             100 105 110 Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro         115 120 125 Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg     130 135 140 Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg 145 150 155 160 Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu                 165 170 175 Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala             180 185 190 Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly         195 200 205 Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser     210 215 220 His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala 225 230 235 240 Val Met Val Gly <210> 6 <211> 281 <212> PRT <213> Homo sapiens <220> <223> Description of Combined DNA / RNA Molecule: n equals       a, t, g, or c <400> 6 Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp   1 5 10 15 Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys              20 25 30 Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro          35 40 45 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro      50 55 60 Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly  65 70 75 80 Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly                  85 90 95 Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala             100 105 110 Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu         115 120 125 Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg     130 135 140 Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu 145 150 155 160 Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr                 165 170 175 Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr             180 185 190 Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser         195 200 205 His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met     210 215 220 Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala 225 230 235 240 Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His                 245 250 255 Leu 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         275 280 <210> 7 <211> 337 <212> DNA <213> Homo sapiens <220> <223> Description of Combined DNA / RNA Molecule: n equals       a, t, g, or c <220> <221> misc_feature <222> (3) N equals a, t, g, or c <220> <221> misc_feature <222> (58) N equals a, t, g, or c <220> <221> misc_feature (222) (67) .. (71) N equals a, t, g, or c <220> <221> misc_feature <222> (212) N equals a, t, g, or c <220> <221> misc_feature <222> (255) N equals a, t, g or c <220> <221> misc_feature <222> (297) N equals a, t, g or c <220> <221> misc_feature <222> (300) N equals a, t, g, or c <220> <221> misc_feature <222> (320) N equals a, t, g, or c <220> <221> misc_feature <222> (335) N equals a, t, g, or c <400> 7 ggntaactct cctgaggggt gagccaagcc ctgccatgta gtgcacgcag gacatcanca 60 aacacannnn ncaggaaata atccattccc tgtggtcact tattctaaag gccccaacct 120 tcaaagttca agtagtgata tggatgactc cacagaaagg gagcagtcac gccttacttc 180 ttgccttaag aaaagagaag aaatgaaact gnaaggagtg tgtttccatc ctcccacgga 240 aggaaagccc ctctntccga tcctccaaag acggaaagct gctggctgca accttgntgn 300 tggcattgtg ttcttgctgn ctcaaggtgg tgttntt 337 <210> 8 <211> 509 <212> DNA <213> Homo sapiens <220> <223> Description of Combined DNA / RNA Molecule: n equals       a, t, g, or c <220> <221> misc_feature <222> (10) N equals a, t, g, or c <220> <221> misc_feature <222> (13) N equals a, t, g, or c <220> <221> misc_feature <222> (209) N equals a, t, g, or c <220> <221> misc_feature <222> (315) N equals a, t, g, or c <220> <221> misc_feature <222> (322) N equals a, t, g, or c <220> <221> misc_feature <222> (325) N equals a, t, g, or c <220> <221> misc_feature <222> (334) N equals a, t, g, or c <220> <221> misc_feature <222> (343) N equals a, t, g, or c <220> <221> misc_feature <222> (347) N equals a, t, g, or c <220> <221> misc_feature <222> (351) N equals a, t, g, or c <220> <221> misc_feature <222> (356) N equals a, t, g, or c <220> <221> misc_feature (409) .. (410) N equals a, t, g, or c <220> <221> misc_feature <222> (416) N equals a, t, g, or c <220> <221> misc_feature <222> (422) N equals a, t, g, or c <220> <221> misc_feature <222> (424) N equals a, t, g, or c <220> <221> misc_feature (426) (426) .. (427) N equals a, t, g, or c <220> <221> misc_feature <222> (429) N equals a, t, g, or c <220> <221> misc_feature <222> (431) N equals a, t, g, or c <220> <221> misc_feature <222> (433) N equals a, t, g, or c <220> <221> misc_feature (438) (438) N equals a, t, g, or c <220> <221> misc_feature (222) (443) .. (444) N equals a, t, g, or c <220> <221> misc_feature <222> (446) .. (447) N equals a, t, g, or c <220> <221> misc_feature <222> (449) .. (450) N equals a, t, g, or c <220> <221> misc_feature <452> (452) .. (453) N equals a, t, g, or c <220> <221> misc_feature <222> (458) N equals a, t, g, or c <220> <221> misc_feature <222> (461) .. (462) N equals a, t, g, or c <220> <221> misc_feature <222> (466) N equals a, t, g, or c <220> <221> misc_feature <222> (469) N equals a, t, g, or c <220> <221> misc_feature (471) .. (472) N equals a, t, g, or c <220> <221> misc_feature <222> (474) N equals a, t, g, or c <220> <221> misc_feature <222> (478) .. (481) N equals a, t, g, or c <220> <221> misc_feature <222> (496) N equals a, t, g, or c <220> <221> misc_feature <222> (498) N equals a, t, g, or c <220> <221> misc_feature <222> (504) N equals a, t, g, or c <400> 8 aattcggcan agnaaactgg ttactttttt atatatggtc aggttttata tactgataag 60 acctacgcca tgggacatct agttcagagg aagaaggtcc atgtctttgg ggatgaattg 120 agtctggtga ctttgtttcg atgtattcaa aatatgcctg aaacactacc caataattcc 180 tgctattcag ctggcattgc aaaactggna ggaaggagat gaactccaac ttgcaatacc 240 aggggaaaat gcacaattat cactgggatg gagatgttca cattttttgg gtgccattga 300 aactgctgtg acctncttac ancangtgct gttngctatt ttncctncct nttctntggt 360 aacctcttag gaaggaagga ttcttaactg ggaaataacc caaaaaaann ttaaangggt 420 angngnnana ngnggggnng ttnncnngnn gnnttttngg nntatnttnt nntngggnnn 480 ngtaaaaatg gggccnangg gggnttttt 509 <210> 9 <211> 497 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (168) N equals a, t, g, or c <220> <221> misc_feature <222> (213) N equals a, t, g, or c <220> <221> misc_feature <222> (288) N equals a, t, g, or c <220> <221> misc_feature <222> (325) N equals a, t, g, or c <220> <221> misc_feature <222> (346) N equals a, t, g, or c <220> <221> misc_feature <222> (406) N equals a, t, g, or c <220> <221> misc_feature <222> (415) N equals a, t, g, or c <220> <221> misc_feature <222> (419) N equals a, t, g, or c <220> <221> misc_feature <222> (437) N equals a, t, g, or c <220> <221> misc_feature <222> (442) N equals a, t, g, or c <220> <221> misc_feature <222> (467) N equals a, t, g, or c <220> <221> misc_feature <222> (473) N equals a, t, g, or c <220> <221> misc_feature <222> (476) N equals a, t, g, or c <220> <221> misc_feature <222> (481) N equals a, t, g, or c <220> <221> misc_feature (222) (483) .. (484) N equals a, t, g, or c <220> <221> misc_feature <222> (494) N equals a, t, g, or c <400> 9 aattcggcac gagcaaggcc ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa 60 tctttgaacc accagctcca ggagaaggca actccagtca gaacagcaga aataagcgtg 120 ccgttcaggg tccagaagaa acagtcactc aagactgctt gcaactgntt gcagacagtg 180 aaacaccaac tatacaaaaa ggctcccttc tgntgccaca tttgggccaa ggaatggaga 240 gatttcttcg tctggaaaca ttttgccaaa ctcttcagat actctttnct ctctgggaat 300 caaaggaaaa tctctactta gattnacaca tttgttccca tgggtntctt aagttttaaa 360 aggggagtgc ccttaggagg aaaaggggat aaatattggc caaggnactg gttantttnt 420 aaatatggtc aggtttntat anctggtagg cctcgccatg ggcattnatt canggngagg 480 ncnntctttt gggntga 497 <210> 10 <211> 27 <212> DNA <213> Homo sapiens <220> <223> Description of Combined DNA / RNA Molecule: x =       deoxyinosine <400> 10 gtgggatcca gcctccgggc agagctg 27 <210> 11 <211> 33 <212> DNA <213> Homo sapiens <400> 11 gtgaagcttt tattacagca gtttcaatgc acc 33 <210> 12 <211> 26 <212> DNA <213> Homo sapiens <400> 12 gtgtcatgag cctccgggca gagctg 26 <210> 13 <211> 33 <212> DNA <213> Homo sapiens <400> 13 gtgaagcttt tattacagca gtttcaatgc acc 33 <210> 14 <211> 28 <212> DNA <213> Homo sapiens <400> 14 gtgggatccc cgggcagagc tgcagggc 28 <210> 15 <211> 33 <212> DNA <213> Homo sapiens <400> 15 gtgggatcct tattacagca gtttcaatgc acc 33 <210> 16 <211> 129 <212> DNA <213> Homo sapiens <400> 16 gcgggatccg ccaccatgaa ctccttctcc acaagcgcct tcggtccagt tgccttctcc 60 ctggggctgc tcctggtgtt gcctgctgcc ttccctgccc cagttgtgag acaaggggac 120 ctggccagc 129 <210> 17 <211> 30 <212> DNA <213> Homo sapiens <400> 17 gtgggatcct tacagcagtt tcaatgcacc 30 <210> 18 <211> 903 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (798) <400> 18 atg gat gac tcc aca gaa agg gag cag tca cgc ctt act tct tgc ctt 48 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu   1 5 10 15 aag aaa aga gaa gaa atg aaa ctg aag gag tgt gtt tcc atc ctc cca 96 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro              20 25 30 cgg aag gaa agc ccc tct gtc cga tcc tcc aaa gac gga aag ctg ctg 144 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu          35 40 45 gct gca acc ttg ctg ctg gca ctg ctg tct tgc tgc ctc acg gtg gtg 192 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val      50 55 60 tct ttc tac cag gtg gcc gcc ctg caa ggg gac ctg gcc agc ctc cgg 240 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg  65 70 75 80 gca gag ctg cag ggc cac cac gcg gag aag ctg cca gca gga gca gga 288 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly                  85 90 95 gcc ccc aag gcc ggc ctg gag gaa gct cca gct gtc acc gcg gga ctg 336 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu             100 105 110 aaa atc ttt gaa cca cca gct cca gga gaa ggc aac tcc agt cag aac 384 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn         115 120 125 agc aga aat aag cgt gcc gtt cag ggt cca gaa gaa aca gga tct tac 432 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr     130 135 140 aca ttt gtt cca tgg ctt ctc agc ttt aaa agg gga agt gcc cta gaa 480 Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu 145 150 155 160 gaa aaa gag aat aaa ata ttg gtc aaa gaa act ggt tac ttt ttt ata 528 Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile                 165 170 175 tat ggt cag gtt tta tat act gat aag acc tac gcc atg gga cat cta 576 Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu             180 185 190 att cag agg aag aag gtc cat gtc ttt ggg gat gaa ttg agt ctg gtg 624 Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val         195 200 205 act ttg ttt cga tgt att caa aat atg cct gaa aca cta ccc aat aat 672 Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn     210 215 220 tcc tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa ctc 720 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu 225 230 235 240 caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga gat 768 Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp                 245 250 255 gtc aca ttt ttt ggt gca ttg aaa ctg ctg tgacctactt acaccatgtc 818 Val Thr Phe Phe Gly Ala Leu Lys Leu Leu             260 265 tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaa ctgaaaatac 878 caaaaaaaaa aaaaaaaaaa aaaaa 903 <210> 19 <211> 266 <212> PRT <213> Homo sapiens <400> 19 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu   1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro              20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu          35 40 45 Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val      50 55 60 Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg  65 70 75 80 Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly                  85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu             100 105 110 Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn         115 120 125 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr     130 135 140 Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu 145 150 155 160 Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile                 165 170 175 Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu             180 185 190 Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val         195 200 205 Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn     210 215 220 Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu 225 230 235 240 Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp                 245 250 255 Val Thr Phe Phe Gly Ala Leu Lys Leu Leu             260 265 <210> 20 <211> 136 <212> PRT <213> Homo sapiens <400> 20 His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp   1 5 10 15 Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg              20 25 30 Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val          35 40 45 Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met      50 55 60 Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe  65 70 75 80 Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser                  85 90 95 Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser             100 105 110 Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly         115 120 125 Thr Phe Leu Gly Phe Val Lys Leu     130 135 <210> 21 <211> 462 <212> DNA <213> Homo sapiens <400> 21 atggctgttc agggtccgga agaaaccgtt actcaggact gccttcagct gatcgcagac 60 tctgaaactc cgaccatcca gaaaggttct tacacctttg ttccttggct gctttctttc 120 aaacgtggtt ctgccctgga agagaaagaa aacaaaatcc tggttaaaga aactggttac 180 ttctttatct acggtcaggt tctttacact gataagacct acgccatggg tcacctgatt 240 cagcgtaaga aagttcacgt tttcggtgac gagctgtctc tggttactct gtttcgctgc 300 attcagaaca tgccggaaac tcttcctaac aactcctgct actctgctgg catcgcaaaa 360 ctggaagagg gtgatgaact gcagctggca attcctcgtg aaaacgcaca aatttctctg 420 gacggtgatg taaccttctt tggtgcactg aaacttctgt aa 462 <210> 22 <211> 1040 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (468) <400> 22 cgc gtg gta gac ctc tca gct cct cct gca cca tgc ctg cct gga tgc 48 Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys   1 5 10 15 cgc cat tct caa cat gat gat aat gga atg aac ctc aga aac aga act 96 Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr              20 25 30 tac aca ttt gtt cca tgg ctt ctc agc ttt aaa aga gga aat gcc ttg 144 Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu          35 40 45 gag gag aaa gag aac aaa ata gtg gtg agg caa aca ggc tat ttc ttc 192 Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe      50 55 60 atc tac agc cag gtt cta tac acg gac ccc atc ttt gct atg ggt cat 240 Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His  65 70 75 80 gtc atc cag agg aag aaa gta cac gtc ttt ggg gac gag ctg agc ctg 288 Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu                  85 90 95 gtg acc ctg ttc cga tgt att cag aat atg ccc aaa aca ctg ccc aac 336 Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn             100 105 110 aat tcc tgc tac tcg gct ggc atc gcg agg ctg gaa gaa gga gat gag 384 Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu         115 120 125 att cag ctt gca att cct cgg gag aat gca cag att tca cgc aac gga 432 Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly     130 135 140 gac gac acc ttc ttt ggt gcc cta aaa ctg ctg taa ctcacttgct 478 Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 145 150 155 ggagtgcgtg atccccttcc ctcgtcttct ctgtacctcc gagggagaaa cagacgactg 538 gaaaaactaa aagatgggga aagccgtcag cgaaagtttt ctcgtgaccc gttgaatctg 598 atccaaacca ggaaatataa cagacagcca caaccgaagt gtgccatgtg agttatgaga 658 aacggagccc gcgctcagaa agaccggatg aggaagaccg ttttctccag tcctttgcca 718 acacgcaccg caaccttgct ttttgccttg ggtgacacat gttcagaatg cagggagatt 778 tccttgtttt gcgatttgcc atgagaagag ggcccacaac tgcaggtcac tgaagcattc 838 acgctaagtc tcaggattta ctctcccttc tcatgctaag tacacacacg ctcttttcca 898 ggtaatacta tgggatacta tggaaaggtt gtttgttttt aaatctagaa gtcttgaact 958 ggcaatagac aaaaatcctt ataaattcaa gtgtaaaata aacttaatta aaaaggttta 1018 agtgtgaaaa aaaaaaaaaa aa 1040 <210> 23 <211> 155 <212> PRT <213> Homo sapiens <400> 23 Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys   1 5 10 15 Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr              20 25 30 Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu          35 40 45 Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe      50 55 60 Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His  65 70 75 80 Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu                  85 90 95 Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn             100 105 110 Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu         115 120 125 Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly     130 135 140 Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu 145 150 155 <210> 24 <211> 26 <212> DNA <213> Homo sapiens <400> 24 ccaccagctc caggagaagg caactc 26 <210> 25 <211> 19 <212> DNA <213> Homo sapiens <400> 25 accgcgggac tgaaaatct 19 <210> 26 <211> 23 <212> DNA <213> Homo sapiens <400> 26 cacgcttatt tctgctgttc tga 23 <210> 27 <211> 657 <212> DNA <213> Homo sapiens <400> 27 taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagggccac 60 cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120 gctgtcaccg caggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180 cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240 ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300 ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360 gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420 gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480 gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540 tcagctggca ttgcaaaact ggaagaagga gatgaacttc aacttgcaat accacgagaa 600 aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657 <210> 28 <211> 219 <212> PRT <213> Homo sapiens <400> 28 Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu   1 5 10 15 Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro              20 25 30 Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile          35 40 45 Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg      50 55 60 Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys  65 70 75 80 Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser                  85 90 95 Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu             100 105 110 Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe         115 120 125 Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His     130 135 140 Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu 145 150 155 160 Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn                 165 170 175 Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu             180 185 190 Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly         195 200 205 Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu     210 215 <210> 29 <211> 657 <212> DNA <213> Homo sapiens <400> 29 taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagagccac 60 cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120 gctgtcaccg cgggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180 cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240 ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300 ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360 gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420 gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480 gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540 tcagctggca ttgcaaaact ggaagaaggg gatgaacttc aacttgcaat accacgagaa 600 aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657 <210> 30 <211> 219 <212> PRT <213> Homo sapiens <400> 30 Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu   1 5 10 15 Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro              20 25 30 Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile          35 40 45 Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg      50 55 60 Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys  65 70 75 80 Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser                  85 90 95 Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu             100 105 110 Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe         115 120 125 Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His     130 135 140 Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu 145 150 155 160 Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn                 165 170 175 Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu             180 185 190 Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly         195 200 205 Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu     210 215 <210> 31 <211> 38 <212> DNA <213> Homo sapiens <400> 31 ggtcgccgtt tctaacgcgg ccgttcaggg tccagaag 38 <210> 32 <211> 49 <212> DNA <213> Homo sapiens <400> 32 ctggttcggc ccaaggtacc aagcttgtac cttagatctt ttctagatc 49 <210> 33 <211> 21 <212> DNA <213> Homo sapiens <400> 33 ctggtagttc ttcggagtgt g 21 <210> 34 <211> 19 <212> DNA <213> Homo sapiens <400> 34 cgcgttagaa acggcgacc 19 <210> 35 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (7) N equals deoxyinosine <220> <221> misc_feature <222> (12) N equals deoxyinosine <220> <221> misc_feature <222> (16) N equals deoxyinosine <400> 35 taccagntgg cngccntgca ag 22 <210> 36 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (3) N equals deoxyinosine <220> <221> misc_feature <222> (14) N equals deoxyinosine <220> <221> misc_feature <222> (16) .. (17) N equals deoxyinosine <400> 36 gtnacagcag tttnanngca cc 22 <210> 37 <211> 866 <212> DNA <213> Mus musculus <400> 37 atggatgagt ctgcaaagac cctgccacca ccgtgcctct gtttttgctc cgagaaagga 60 gaagatatga aagtgggata tgatcccatc actccgcaga aggaggaggg tgcctggttt 120 gggatctgca gggatggaag gctgctggct gctaccctcc tgctggccct gttgtccagc 180 agtttcacag cgatgtcctt gtaccagttg gctgccttgc aagcagacct gatgaacctg 240 cgcatggagc tgcagagcta ccgaggttca gcaacaccag ccgccgcggg tgctccagag 300 ttgaccgctg gagtcaaact cctgacaccg gcagctcctc gaccccacaa ctccagccgc 360 ggccacagga acagacgcgc cttccaggga ccagaggaaa cagaacaaga tgtagacctc 420 tcagctcctc ctgcaccatg cctgcctgga tgccgccatt ctcaacatga tgataatgga 480 atgaacctca gaaacatcat tcaagactgt ctgcagctga ttgcagacag cgacacgccg 540 gccttggagg agaaagagaa caaaatagtg gtgaggcaaa caggctattt cttcatctac 600 agccaggttc tatacacgga ccccatcttt gctatgggtc atgtcatcca gaggaagaaa 660 gtacacgtct ttggggacga gctgagcctg gtgaccctgt tccgatgtat tcagaatatg 720 cccaaaacac tgcccaacaa ttcctgctac tcggctggca tcgcgaggct ggaagaagga 780 gatgagattc agcttgcaat tcctcgggag aatgcacaga tttcacgcaa cggagacgac 840 accttctttg gtgccctaaa actgct 866 <210> 38 <211> 177 <212> DNA <213> Mus musculus <400> 38 mdsaktcccs kgdmkvgydt kgawgcrdgr aatassstam syaaadmnrm syrgsataaa 60 gatagvktaa rhnssrghrn rragtdvdsa acgcrhshdd ngmnrndcad sdtaknkvvr 120 tgyysvytda mghvrkkvhv gdsvtrcnmk tnnscysaga rgdarnasrn gddtgak 177

Claims (49)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Hybridoma of ATCC Accession No. PTA-1159 or PTA-1158. The antibody secreted by the hybridoma of ATCC accession No. PTA-1159 or PTA-1158. An antibody or portion thereof comprising the VH and VL domains of the antibody of claim 27 and specifically binding to the membrane-binding and / or soluble forms of the protein of SEQ ID NO: 2. An antibody or portion thereof comprising both the VH CDR and VL CDR domains of the antibody of claim 27 and which specifically binds to the membrane-binding and / or soluble forms of the protein of SEQ ID NO: 2. The antibody or portion thereof of claim 28 or 29, which is a monoclonal antibody. The method of claim 28 or 29, (a) chimeric antibodies; (b) humanized antibodies; (c) single chain antibodies; (d) Fab fragments; And (e) an antibody or portion thereof selected from the group consisting of F (ab ') 2 fragments. 30. The labeled antibody or portion thereof of any of claims 27-29. 33. The method of claim 32, wherein the label is (a) an enzyme label; (b) radioisotopes; (c) fluorescent labels; And (d) an antibody or portion thereof selected from the group consisting of biotin. The method of claim 33, wherein the label is (a) ¹²⁵ I; (b) ¹²¹ I; (c) ¹³¹ I; (d) ¹¹² In; And (e) an antibody or portion thereof that is a radioisotope selected from the group consisting of ⁹⁹ Tc. The antibody or portion thereof of any one of claims 27-29 conjugated to a therapeutic or cytotoxic agent. 30. An isolated cell that produces an antibody according to claim 27. A hybridoma producing the monoclonal antibody of claim 30. (a) contacting a biological sample with an antibody according to any one of claims 27-29 or a portion thereof; (b) detecting Neutrokine-alpha protein in a biological sample, including detecting Neutrokine-alpha protein. (a) An isolated protein comprising a Neutrokine-alpha protein fused to a toxin protein, comprising the amino acid sequence of amino acid residues 134 to 285 of SEQ ID NO: 2. 40. The method of claim 39, wherein the toxin protein is thymidine kinase, endonuclease, RNAse, alpha toxin, lysine, abrin, Pseudomonas exotoxin A, diphtheria toxin, saponin, momordine, gelonin, coniferous Protein that is an antiviral protein, alpha-sarsine or cholera toxin. delete A pharmaceutical composition for treating B cell cancer, comprising the antibody of any one of claims 27-29 or a portion thereof.  The pharmaceutical composition of claim 42, wherein the B cell cancer is selected from the group consisting of non-Hodgkin's lymphoma, multiple myeloma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and plasmacytoma. 30. A pharmaceutical composition for treating an autoimmune disease, comprising the antibody of any one of claims 27-29 or a portion thereof. 45. The pharmaceutical composition of claim 44, wherein the autoimmune disease is rheumatoid arthritis. 45. The pharmaceutical composition of claim 44, wherein the autoimmune disease is systemic lupus erythematosus. 45. The pharmaceutical composition of claim 44, wherein the autoimmune disease is multiple sclerosis, myasthenia gravis, Sjogren's syndrome, type I diabetes, idiopathic thrombocytopenic purpura, Guillain-Barre syndrome, Hashimoto's thyroiditis or Graves' disease. A diagnostic composition for diagnosing immunodeficiency, comprising the antibody of claim 27 or a portion thereof. 49. The method of claim 48, wherein the immunodeficiency is (a) common variable immunodeficiency syndrome (CVID); (b) acquired immunodeficiency syndrome (AIDS); (c) severe combined immunodeficiency (SCID); And (d) a diagnostic composition selected from the group consisting of hypomagglobulinemia.

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