AU2008201411A1 - Neutrokine-alpha and neutrokine-alpha splice variant - Google Patents

Description

19/03/2008 16:.05 61 2 92586999 4 062837999 N0.640 904 Regulation 3.2

AUSTRALIA

Patents Act 1990 (Cth)

IGT

COMPLETE SPECIFICATION FOR A STANDARD PATENT (ORIGINAL) Invention Title: Neutrokine-alpha and neutrokine-alpha splice variant The following statement is a full description of this invention, including the best method of performing it known to us: 203936956,1 COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/200e 16:05 61 2 92586999 4 062837999 NO.640 00 0 0 cIA Neutrokine-alpha and Neutrokmne-alpha Splice Variants s The present invention relates to a novel cytokine which has been designated Neutrokine-alpha ("Neutrokane-alpha"). In addition, an apparent splicing variantof O Neutrokine-alpha has been identified and designated Neutrokine-alphaSV In specific 00 embodiments, the present invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alphaSV polypeptides. In additional embodiments.

Neutrokine-alpha and Neutrokine-alphaSV polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.

is RelatedArt Human tumor necrosis factors (TNF-alpha) and (TNF-beta. or Iymphotoxn) are related members of a broad class of polypeptide mediators, which includes the interferons. Interleukrns and growth factors, collectively called cytokanes (Beutler. B.

and Cerami, Annu. Rev Immunol. 7:625-653 (1989)). Sequence analysis of cytokine receptors has defined several subfamilies of membrane proteins the ig superfamily the hematopoietn (cytokine receptor superfamily) and the tumor necrosis factor (TNF)/nerve growth factor (NGCF) receptor superfamily (for review of TNF superfamily see, Gruss and Dower. Blood 85(12) .3378-3404 (1995) and Aggarwal and Natarajan. Eur Cvrwokne Nent'.. 7(2).93-124 (1996)). The TNFINGF receptor superfamily contains at least 10 difference proteins. Gruss and Dower, supra.

Ligands for these receptors have been identified and belong to at least two cytokine superfamilies. Gruss and Dower supra.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/20oe 16:05 61 2 92586999 4 062837999 N0.640 106 o 00 Cl Tumor necrosis factor (a mixture of TNF-alpha and TNF-bcta) was onginally 3 discovered as a result of its ant-tumor activy however, now it is recognized as a plelotropic cytokne capable of numerous biological activities including apoptosis of some transformed cell lines, mediation of cell actvation and proliferation and also as S playmg mportant roles in immune regulation and inflammation.

_To date, known members of the TNF-ligand superfamily include TNF-alpha, TNF-beta (lymphotoxmn-alpha). LT-beta. OX40L, Fas ligand, CD30L, CD27L, and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, TNF-like Cl molecules with approximately 20% sequence homology in the extracellular domains 00 S1o (range. 12%-36%) and exist mainly as membrane-bound forms with the biologically 0 Cl active form being a tnmenc/multimenc complex. Soluble forms of the TNF ligand superfamily have only been identified so far for TNF LT-beta, and Fas ligand (for a general review see Gruss, H. and Dower, Blood, 85(12) .3378-3404 (1995)).

which is hereby incorporated by reference im us entirety These proteins are involved in regulation of cell proliferation, activation, and differentiaton, including control of cell survival or death by apoptosis or cytotoxicity (Armitage, Curr Opm. Immunol.

6:407 (1994) and Smith, Cell 75:959 (1994)).

Tumor necrosis factor-alpha (TNF-alpha: also termed cachectm; hereinafter "TNP') is secreted primarily by monocytes and macrophages in response to endotoxin or other stimuli as a soluble homotrimer of 17 kD protein subunits (Smith, R.A. et a., Biol. Chem. 262:6951-6954 (1987)). A membrane-bound 26 kD precursor form of TNF has also been described (Knegler, M. et at, Cell 53-45-53 (1988)).

Accumulating evidence indicates that TNF is a regulatory cytokine with pletotropic biological activities. These activities include: inhibmon of lipoprotein lipase synthesis ("cachectm activity) (Beutler, B. et al., Nature 316:552 (1985)), acuvation of polymorphonuclear leukocyces (Klebanoff, S.J. etal., J. Immunol.

136:4220 (1986); Perussia. et al., J. Immwwl. 138:763 (1987)), inhibiion of cell growth or stimulation of cell growth (Vilcek. J. er J. Exp. Med. 163:632 (1986); Sugarman, B. I st at, Sctence 230:93 Lach L.B. ei j. inamnug.

3o 138:2913 (1987)), cycotoxic action on certain transformed cell types (Lachman. L.B. ec COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/208 16:05 61 2 92586999 062837999 N0.640 907 00 3 0 0 al., supra; Darzynklewicz, Z. er at, Cane. Res. 44:83 (1984)), anuviral activity (Kohase. M. et at, Cell 45:659 (1986); Wong, G.H.W et al. Nature 323:819 (1986)).

Sstimulanon of bone resorpion (Bertolim, D.R. e al., Nature 319:516 (1986); Saklatvala, Nature 322:547 (1986)), stimulation of collagenase and prostaglandin E2 production (Dayer, er al., J Exp. Med. 162:2163 (1985)); and nmmunoregulatory actions, including activation of T cells (Yokota, S. et aL, J. Immunol. 140531 (1988)), B cells (Kehrl, J.H. et J. Exp. Med. 166:786 (1987)), monocytes (Philip. I et al..

Nature 323:86 (1986)), thymocytes (Ranges, G.E. et aL, J Exp. Med 167 1472 O (1988)), and stimulation of the cell-surface expression of major histocompatibility 00 0o complex (MHC) class I and class II molecules (Collins, T et al.. Proc. Natl. Acad. Sct.

0 USA 83'446 (1986); Pijol-Borrel. R. et Nature 326:304 (1987)).

TNF Is noted for its pro-mflanmaory actons which result n tissue injury such as induction of procoagulant activity on vascular endothelial cells (Pober, J.S et at, J.

Immunol 136:1680 (1986)), increased adherence of neutrophils and lymphocytes is (Pober, J.S. et a. J Immunol. 138:3319 (1987)), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166:1390 (1987)).

Recent evidence implicates TNF m the pathogenesis of many infections (Cerami, A. et at. Inrunol. Today 9:28 (1988)), immune disorders, neoplastc pathology in cachexia accompanying some malignancies (Oliff, A. et al., Cell 50:555 (1987)), and m autoimmune pathologies and graft-versus host pathology (Piguet, P -F et at. J. Exp. Med. 166:1280 (1987)). The assocation of TNF with cancer and infectious pathologies is often related to the host's catabolic state. A major problem in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results IS known as cachexia (Kern. K. A. et al. J. Parent.

Enter Nutr 12:286-298 (1988)). Cachexia Includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth. The cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer nornality nuimbr of studies have suggescu thai TNF ia an iiiportant mediator of the cachexia in cancer, infectious pathology and in other catabolic states.

TNF is thought to play a central role an the pathophysiologcal consequences of Gram-negative sepsis and endotoxac shock (Michic. H.R. er al.. Br J. Surg. 76:670-671 (1989); Debets, J. M. H. et Second Vienna Shock Forum. p.463-466 (1989); COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 D08 00 o 4.

Cl Simpson, S. Q. et Cnt. Care Clin. 5:27-47 (1989)), including fever, malaise, Sanorexia, and cachexia. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF (Kornbluth. S.K. et aL, J. mmunoww _137;2585-2591 (1986)) and other cytokines. Because TNF could immic many S biological effects of endtoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endoloxin-related illness. TNF and other monocyte-denved cytokines mediate the metabolic and neurohormonal responses to endotoxm (Michie, H.R. er N. Eng. J. Med 318:1481 1486 (1988)). Endotoxm CN administratlon to human volunteers produces acute illness with flu-like symptoms 00 including fever, tachycardia, increased metabolic rate and stress hormone release 0 C (Revhaug, A. et al, Arch. Surg. 123 162 170 (1988)). Elevated levels of circulating TNF have also been found in patients suffenng from Gram-negative sepsis (Waage. A.

et al.. Lancer 1.355-357 (1987); Hammerle, A.F et al. Second Vienna Shock Forum p.

715-718 (1989); Debets, J. M. H. et aL, Crl. Care Med. 17-489-497 (1989); Calandra, IS T e aL. J. Infec. Dis. 161:982-987 (1990)).

Passive immunotherapy directed at neutralizing TNF may have a beneficial effect in Gram-negauve sepsis and endotoxemia. based on the increased TNF production and elevated TNF levels m these pathology states, as discussed above.

Antibodies to a "modulator matenal which was characterized as cachecun (later found to be identical to TNF) were disclosed by Cerami et a. (EPO Patent Publication 0,212,489 March 4, 1987). Such antibodies were said to be useful in diagnostic immunoassays and m therapy of shock In bacterial infections. Rubin et al. (EPO Patent Publicaton 0,218,868, April 22, 1987) disclosed monoclonal antibodies to human TNF the hybndomas secreting such antibodies, methods of producing such antibodies.

and the use of such antibodies in immunoassay of TNF Yone et al. (EPO Patent Publication 0,288,088. October 26, 1988) disclosed anti-TNF antibodies. includina mAbs. and their utility m immunoassay diagnosis of pathologies, in particular Kawasaki's pathology and bactenal infection. The body fluids of patents with K-wasaki'k pathology (inrfani acute fcbrile mrucocutaneous lymph nude syndrome: Kawasaki, Allergy 16:178 (1967); Kawasaki, Shonica (Pediatrics) 26:935 (1985)) were said to contain elevated TNF levels which were related to progress of the pathology (Yone et al. supra).

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 D09 00 0 Other investigators have described mAbs specific for recombinant human TNF which had neutralizing activity i vitro (Lang, C-M. et al. Biochem. Biophys. Res.

SComm. 137:847-854 (1986); Meager, A. et at. Hvbndoma 6:305-311 (1987); Fendly et 0 Hybridoma 6:359-369 (1987); Bnngman, T S et at. Hvbndoma 6:489-507 (1987); Hirai, M. ei at, J. Immuno.o Meth. 96:57-62 (1987); Moller, A. et al. (Cytokne 2:162 169 (1990)). Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays (Fendly et supra; Hira et at, supra; Moller et al.

supra) and to assist m the purification of recombinant TNF (Bnngman et al., supra).

0 However, these studies do not provide a basis for producing TNF neutralizing o 10 antibodies that can be used for m vavo diagnostic or therapeutn uses in humans, due to unmunogeniciy lack of specificity and/or pharmaceutical suitability Neutralizing antsera or mAbs to TNF have been shown in mammals other than man to abrogate adverse physiologeial changes and prevent death after lethal challenge in experimental endotoxenua and bacteremia. This effect has been demonstrated, e.g., is in rodent lethality assays and in primate pathology model systems (Mathison. J.C. et at, J. Clin. Invest. 81 1925-1937 (1988); Beutler, B. et aL, Science 229:869-871 (1985); Traccy K- J. e al.. Nature 330-662-664 (1987); Shimamoxo, Y et a..

Immunol. Len. 17:311 318 (1988); Silva. A. T et at. J. Infect. Di. 162:421-427 (1990); Opal. S. M. et J. Infect. Dis. 161 1148-1152 (1990); Hinshaw L.B. et al.

Circ. Shock 30279-292 (1990)).

To dale, experience with anti-TNF mAb therapy in humans has been limited but shows beneficial therapeutic results, in arthitis and sepsis. See. Elliott, M. J.

e al., Baillieres Cln 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 Cnt. Care. Med. 21:S436-40 (1993); Tracey K. J. et al., Crit. Care Med.

21:S415-22 (1993).

Mammalian development is dependent on both the proliferation and differentiation of cells as well as programmed cell death which occurs through apoptusts ('vaiker, et at., meniauus nchiev Ex. Patia,. J 8 1998). rtpoposis plays a cntical role m the destruction of immune thymocytes that recognize self antgens.

Failure of this normal elimination process may play a role in autoimmune diseases (Gammon et at, Immunology Today 12:193 (1991)).

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2S08 16:05 61 2 92586999 4 062837999 N0.640 00 6 0 0c Itoh et at. (Cell 66:233 (1991)) described a cell surface antigen, Fas/CD95 that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in Sactivated T-cells, B-cells, neutrophils and in thymus. liver, heart and lung and ovary in Sadult mice (Watanabe-Fukunaga e aL. J. Immunol. 148:1274 (1992)) in addition to s activated T-cells, B-cells. neutrophils. In expenments where a monoclonal Ab is cross-linked to Fas, apoptosis is induced (Yonehara et J Exp. Med 169,1747 (1989); Trauth et al., Science 245:301 (1989)). In addition, there is an example where binding of a monoclonal Ab to Fas is stimulatory to T-cells under certain conditions C( (Alderson et al.. I Exp. Med 178:2231 (1993)).

CO

o 10 Fas antigen is a cell surface protein of relative MW of 45 Kd. Both human and O murme genes for Fas have been cloned by Watanabe-Fukunaga et al., (J Immuno 148:1274 (1992)) and Itoh et at (Cell 66:233 (1991)). The proteins encoded by these genes are both transmembrane proeins with structural homology to the Nerve Growth Factor/Tumor Necrosis Factor receptor superfamily which includes two TNF receptors, the low affinity Nerve Growth Factor receptor and CD40, CD27 CD30, and Recently the Fas ligand has been described (Suda et al, Cell 75-1169 (1993)).

The amuno acid sequence indicates that Fas ligand is a type nI transmembrane protein belonging to the TNF family Thus, the Fas ligand polypepnde comprises three main domains: a short intracellular domain at the amino terminal end and a longer extracellular domain at the carboxy terminal end. connected by a hydrophobic transmembrane domain. Fas ligand is expressed in splenocytes and thymocytes, consistent with T-cell mediated cyltooxicity The purifed Fas liand has a MW of kD Recently It has been demonstrated that Fas/Fas ligand interactons are required for apoptosis following the activaton of T-cells (Ju et aL. Nature 373-444 (1995); Brunner et Nature 373-441 (1995)). Activation of T-cells induces both proteins on the cell surface. Subsequent interaction between the ligand and receptor results m apoptosis of the cells. This suppots the possible regulatory iroi for apoptis indu:cu by Fas/Fas ligand interaction during normal immune responses.

Accordingly there is a need to provide cytokines similar to TNF that are involved in pathological conditions. Such novel cytokmes may be used to make novel COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 oil 00 0' antibodies or other antagonists that bind these TNF-like cytokines for diagnosis and therapy of disorders related to TNF-like cytokanes.

Summary of the Invention s In accordance with one embodiment of the present invention, there is provided a novel extracellular domain of a Neutrokine-alpha polypepude, and a novel extracellular domain of a Neutrokine-alphaSV polypeptlde, as well as biologically C-I active and diagnostically or therapeutically useful fragments, analogs and derivatives 0 0 thereof.

In accordance with another embodiment of the present invention, there are provided isolated nucleic acid molecules encoding human Neutrokme-alpha or Neutrokme-alphaSV including mRNAs, DNAs, cDNAs, genonuc DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments and derivatves thereof.

The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of. a polynuclcotide encoding a cytokine and an apparent splice vanant thereof that are structurally similar to TNF and related cytokines and have similar biological effects and activities. This cytokine is named Neurolune-alpha and the invention includes Neutrokine-alpha polypeptldes having at least a portion of the amino acid sequence in Figures IA and 1B (SEQ ID NO:2) or amino acid sequence encoded by the cDNA clone (HNEDU 15) deposited on October 22, 1996 assigned ATCC number 97768. The nucleotide sequence determined by sequencing the deposited Neutrokine-alpha clone, which is shown m Figures IA and 1B (SEQ ID NO- contains an open reading frame encoding a complete polypepude of 285 amino acid residues including an N-termmal methionine, a predicted ntracellular domain of about 46 armno acid residues, a predicted transmembrane domain of about 26 amino acids, a predicted extracellular domain of about 213 amino acids, and a deduced molecular we.ght for the complete protein of Labout 3 kDa. at ior uiier type ii transmembrane proteins, soluble forms of Neurokmne-alpha include all or a portion of the extracellular domain cleaved from the transmembrane domain and a polypepide comprising the complete Neutrokine-alpha polypeptide lacking the transmembrane domain. the extracellular domain linked to the mtracellular domain.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 P12 00 c, The apparent splice vanant of Neutrokine-alpha is named Neutrokine-alphaSV and the invenuon includes Neutrokne-alphaSV polypeptdes comprising, or alternatively consisting of, at least a portion of the ammo acid sequence in Figures Sand 5B (SEQ ID NO- 19) or ammino acid sequence encoded by the cDNA clone HDPMC52 deposited on December 10, 1998 and assigned ATCC number 203518. The nucleotde sequence determined by sequencinmg the deposited Neutrokme-alphaSV clone, wluch is shown in Figures SA and 58 (SEQ ID NO- 18), contains an open reading frame encoding a complete polypepude of 266 armno acid residues including C-i an N-terminal methionine, a predicted intracellular domain of about 46 anuno acid o 10 residues, a predicted transmembrane domain of about 26 ammno acids, a predicted o extracellular domain of about 194 ammo acids, and a deduced molecular weight for the complete protein of about 29 kDa. As for other type I1 transmembrane proteins, soluble forms of Neutrokmne-alphaSV include all or a portion of the extracellular domain cleaved from the transmembrane domain and a polypeptade comprsng the is complete Neutrokne-alphaSV polypeptide lacking the transnembrane domain, the extracellular domain linked to the intracellular domain.

Thus, one embodiment of the invention provides an isolated nucleic acid molecule comprising, or alternatvely consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: a nucleotide sequence encoding a full-length Neutrokine-alpha polypeptide having the complete amino acid sequence m Figures IA and 1B (SEQ ID NO:2) or as encoded by the cDNA clone contained in the deposit having ATCC accession number 97768. a nucleotide sequence encoding the predicted extracllular domain of the Neutrokine-alpha polypeptude having the amino acid sequence at positions 73 to 285 in Figures IA and 1B (SEQ ID NO:2) or as encoded by the clone contained in the deposit having ATCC accession number 97768: a nucleoude sequence encoding a fragment of the polypepude of having Neutrokine-alpha functional activity biological aciatvity); a nucleotude sequence encoding a polypepude comprising the Neutrrokirme-lpha ncraceiJu!ar domain (predicted to counaiitue anmnu aui residues irumi about I to about 46 in Figures IA and 1B (SEQ ID NO:2)) or as encoded by the clone contained in the deposit having ATCC accession number 97768: a nucleotude sequence encoding a polypeptide comprising the Neutrokmne-alpha transmembrane domain (predicted to constitute amino acid residues from about 47 to about 72 in COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 D13 00 0 o Figures IA and IB (SEQ ID NO:2) or as encoded by the cDNA clone contained in the deposit having ATCC accession number 97768; a nucleoude sequence encoding a Ssoluble Neuirokinc-alpha polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain; and a nucleoude sequence complementary to any of the nucleotide sequences in or above.

Furher embodiments of the invention include isolated nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide having a nuclcotde sequence at least 80%, 85% or 90% identical, and more preferably at least 95%, 96%, o 97%, 98% or 99% identical, to any of the nucleoude sequences in 00 Io or above, or a polynucleotide which hybridizes under stringent hybndi2ation 0 conditions to a polynucleoude in or above. This polynucleotide which hybridizes does not hybridize under singent hybridization conditions to a polynucleoude having a nucleotide sequence consisting of only A residues or of only T residues.

Is Another embodiment of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotlde sequence selected from the group consisting of: a nucleotide sequence encoding a full-length Neutrokine-alphaSV polypeptude having the complete amino acid sequence in Figures SA and 5B (SEQ ID NO' 19) or as encoded by the cDNA clone contained in the ATCC Deposit deposited on December 10, 1998 as ATCC Number 203518; a nucleotide sequence encoding the predicted extracellular domain of the Neutrokine-alphaSV polypepude having the amino acid sequence at positions 73 to 266 in Figures 1A and IB (SEQ ID NO:2) or as encoded by the cDNA clone contained in ATCC 203518 deposited on December 10, 1998: a nucleoude sequence encoding a polypeptide comprsmg the Neutrokine-alphaSV intracellular domain (predicted to constitute armno acid residues from about 1 to about 46 In Figures and 5B (SEQ ID NO' 19)) or as encoded by the cDNA clone contained in ATCC No. 203518 deposited on December 10, 1998; a nucleotide sequence encoding a nolvnentrii romprlnng the Neutrokne-alphaS" nri-asmeibiiic uuilzau .predicieo to constitute amino acid residues from about 47 to about 72 in Figures 5A and 5B (SEQ ID NO: 19) or as encoded by the cDNA clone contained in ATCC No. 203518 deposited on December 10, 1998; a nucleotide sequence encoding a soluble Neutrokine-alphaSV polypepude having the extracellular and intracellular domains but COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P14 0 do 0 c lacking the transmembrane domain; and a nucleoude sequence complemenary to c any of the nucleonde sequences in or above.

SFurther embodiments of the invention include isolated nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide having a nucleoude sequence at least 80%, 85% or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nuclcotide sequences in (e) or above, or a polynucleotide which hybridizes under strnngent hybndization conditions to a polynucleoude in or above. This polynucleotide Cl which hybndizes does not hybridize under stnngent hybndization conditions to a 00 polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.

In one embodiment, the apparent splice vanant of Ncutrokine-alpha compnsine, or alternatively consisting of, at least a portion of the ammno acid sequence from Gly- 142 to Leu-266 as shown in Figures 5A and 5B (SEQ ID NO' 19) or amino acid s1 sequence encoded by the cDNA clone HDPMC52 deposited on December 10, 1998 and assigned ATCC Deposit No. 203518.

In additional embodiments, the nucleic acid molecules of the invention comprise, or alternatively consist of, a polynucleotde which encodes the amino acid sequence of an epitope-beanng ponion of a Neutroline-alpha or Neutrokine-alphaSV polypeptide having an ammo acid sequence in or above. A further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which encodes the Q amno acid sequence of a Neutrokme-alpha or Neuiroknc-alphaSV polypepude having an ammno acid sequence which contains at least one armno acid addition, substituton.

and/or deletion but not more than 50 ammno acid additions, substitutions and/or deletions, even more preferably not more than 40 amino acid additions, substitutions, and/or deletions, still more preferably not more than 30 amino acid additions, substitutions, and/or deletions, and still even more preferably not more than 20 armno acid additions, substitutions. and/r deletions. Of course, in o;rdr of evci-iiiiretu aiig preference, it is highly preferable for a polynucleotide which encodes the amino acid sequence of a Neutrokne-alpha or Neutrokine-alphaSV polypeptide to have an ammno acid sequence which contains not more than 10, 9 8, 7 6, 5, 4, 3, 2 or I or 1 100, 1 COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/208 16:05 61 2 92586999 4 062837999 NO.640 915 0 O 1 25, 1-20. 1-15, 1 10, or 1 5 amino acid additions, substitutions and/or deletions.

Conservative subsumunons are preferable.

SThe present ivention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for producton of Neutrokine-alpha polypeptdes by recombinant techmques.

In accordance with a further embodiment of the present nvention, there is Sprovided a process for producing such polypeptldes by recombinant techniques 00 comprising culturing recombinant prokaryotzc and/or eukaryotic host cells, containing a SNcutrokme-alpha or Neutrokne-alphaSV nucleic acid sequence of the invention, under conditions promoting expression of said polypeptide and subsequent recovery of said polypeptide.

The invention further provides an isolated Neutrokine-alpha polypeptide I comprising, or alternatively consisting of, an ammo acid sequence selected from the group consstung of: the ammno acid sequence of the full-length Neutrokmne-alpha polypepude having the complete amino acid sequence shown m Figures IA and IB positions 1 285 of SEQ ID NO:2) or as encoded by the cDNA plasmid contained in the deposit having ATCC accession number 97768, the amino acid sequence of the full-length Neutrokne-alpha polypeptide having the complete amino acid sequence shown m SEQ ID NO;2 excepting the N-termmal methionne positions 2 to 285 of SEQ ID NO:2); a fragment of the polypeptide of having Neutroklne-alpha functional actvily biological activity): the amno acid sequence of the predicted extracellular domain of the Neutrokin-alpha polypeptde having the ammo acid sequence at positions 73 to 285 in Figures IA and IB (SEQ ID NO:2) or as encoded by the cDNA plasmnd contained in the deposit having ATCC accession number 97768, a nucleotide sequence encoding the Neutrokine-alpha polypeptide having the anmno acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2); the amino acid seouence of the Neutroknm-alpha zntrace!!ular doman (predicted to constitute armno acid residues from about I to about 46 in Figures IA and IB (SEQ ID NO:2)) or as encoded by the cDNA plasmid contained in the deposit having ATCC accession number 97768; the amino acid sequence of the Neutrokne-alpha transmembrane domain (predicted to constitute amino acid residues COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 D16 00 -1 2 0 from about 47 to about 72 mi Figures 1A and IB (SEQ ID NO:2)) or as encoded by the cDNA plasrnd contained in the deposit having ATCC accession number 97768; the Sarmno acid sequence of the soluble Neutrokme-alpha polypeptide having the Sextracellular and mtracellular domains but lacking the transmembrane domain, wherein each of these domains is defined above; and fragments of the polypeptde of or The polypepudes of the present invention also include polypepudes having an anuno acid sequence at least 80% identical, more preferably at least 85% or 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% Sidentical to those described in or above, as well as 0 10o polypepttdes having an amino acid sequence wih at least 80%. 85%. or 90% similarity and more preferably at least 95% similarity to those above. Additional embodiments of the invention relates to polypeptades which comprse, or alternatively consist of. the amnno acid sequence of an epltope-beanng portion of a Neutrokme-alpha polypeptide having an amino acid sequence described in or (i) above. Polypeptdes having the aruno acid sequence of an epntope-beanng portion of a Neutrokne-alpha polypeptide of the invention include portions of such polypeptides with at least 4, at least 5, at least 6. at least 7 at least 8, and preferably at least 9 at least 10, at least 11 at least 12, at least 13, at least 14, at least o, at least 20, at least at least 30, at least 40, at least 50, and more preferably at least about 30 anmno acids to about 50 armno acids, although epitope-beanng polypeptides of any length up to and including the enure amino acid sequence of a polypepude of the invention described above also are included in the invention.

(Highly preferred embodiments of the invention are directed to nucleic acid molecules comprsing, or alternatively consistig of a polynucleoude having a nucleotide sequence at least 80%, 85%, 90% identical and more preferably at least 96%, 97%, 98%. 99% or 100% identcal to a polynucleotide sequence encoding the Neutrokne-alpha polypeptide having the amino acid sequence at positions 134-283 in Figures IA and IB (SEQ ID NO:2). Preferred embodiments of the invention are dirr.ed to n.lcle. acad .e!cule-s compr.isig, or amtemnauvcy uinsIsung oi a polynucleonde having a nucleotide sequence at least 90% identical to a polynucleonde sequence encoding the Neutroktne-alpha polypeptide having the amino acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2). More preferred embodiments of the invention are directed to nucleic acid molecules comprising, or COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO0.640 P17 00 43 0 alternauvely consisting of a polynucleotide having a nuclcotde sequence at least identical to a polynucleotide sequence encoding the Neutrokine-alpha polypepude Shaving the amino acid sequence at positions 134-285 in Figures IA and IB (SEQ ID 0NO:2). More preferred embodiments of the mvention are directed to nucleic acid molecules comprismg. or alternatively consisting of a polynucleotde having a nucleotlde sequence at least 96% identical to a polynucleoude sequence encoding the Neutrolune-alpha polypeptide having the ammo acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2).

0 Additionally more preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatively consisting of a polynucleoude having a nucleonde sequence at least 97% to a polynucleotde sequence encoding the Neutrokine-alpha polypeptde having the amino acid sequence at positions 134-285 m Figures 1A and IB (SEQ ID NO:2). Additionally more preferred embodiments of the inventon are directed to nucleic acid molecules comprising, or alternatively consisting is of a polynucleotide having a nucleotide sequence at least 98% to a polynucleotide sequence encoding the Neutrolune-alpha polypepude having the amino acid sequence at posiions 134-285 in Figures 1A and IB (SEQ ID NO:2). Additionally more preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatively consisting of a polynucleotide having a nucleotide sequence at least 99% identical to a polynucleotide sequence encoding the Neutrokmealpha polypeptde having the ammo acid sequence at positions 134-283 in Figures 1A and IB (SEQ ID NO:2).

The present invention also encompasses the above polynucleoide sequences fused to a heterologous polynucleotide sequence. Polypepudes encoded by these 2s polynucleotides and nucleic acid molecules are also encompassed by the invention.

The invention further provides an isolated Neutrokme-alphaSV polypepude comprising or alternatively consisting of, an amino acid sequence selected from the group consisting of: the amno acid sequence of the full-length Neutrokine-alphaSV polypeptide having iic uinmpee ani"uu aseu quence Siuowu iI Figures J an positions I 266 of SEQ ID NO-19) or as encoded by the cDNA clone contained in ATCC No. 203518 deposited on December 10. 1998; the amino acid sequence of the full-length Neutrokine-alphaSV polypepttde having the complete amino acid sequence shown in SEQ ID NO: 19 excepting the N-terminal methionine positions COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 A 11111111 19/03/2008 16:05 61 2 92F69 sqqq c2oorQo NO. 640 18 00 o 14 CN 2 to 266 of SEQ ID NO: 19); the ammo acid sequence of the predicted extracellular domain of the Neutrolane-alphaSV polypeptde having the amuno acid sequence at Spositions 73 to 266 in Figures 5A and 5B (SEQ ID NO-19) or as encoded by the cDNA clone contained in ATCC No. 203518 deposited on December 10, 1998; the anuno acid sequence of the Neutrokme-alphaSV ntracellular domain (predicted to constitute ammo acid residues from about I to about 46 in Figures SA and 5B (SEQ ID NO- 19)) or as encoded by the eDNA clone contained m ATCC No. 203518 deposited on December 10, 1998; the amino acid sequence of the Neutrokmne-alphaSV C transmembrane domain (predicted to constitute amno acid residues from about 47 to 0 10 about 72 in Figures 5A and 5B (SEQ ID NO: 19)) or as encoded by the cDNA clone 0 contained in ATCC No. 203518 deposited on December 10, 1998, the amino acid sequence of the soluble Neutroklne-alphaSV polypepude having the extracellular and intracellular domains but lacking the transmembrane domain, wherein each of these domains is defined above; and fragments of the polypeptide of is or The polypeptides of the present invention also include polypepudes having an amno acid sequence at least 80% identical, more preferably at least 85% or identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those described m or above, as well as polypeptides having an amino acid sequence with at least 80%. 85%. or 90% similarity and more preferably at least 95% similarity to those above. Additional embodiments of the invention relates to polypeptides which comprise, or alternatively consist of, the ammo acid sequence of an epttope-beanng portion of a Neutrokme-alphaSV polypeptide having an amino acid sequence described m or above. Peptdes or polypepudes having the amino acid sequence of an epitope-barmng portion of a Neutrokme-alphaSV polypeptide of the invention include portions of such polypepudes with at least 4, at least 5, at least 6, at least 7 at least 8, and preferably at least 9 at least 10. at least II at least 12, at least 13, at least 14, at least 15, as least 20, at least 25, at least 30. at least at least 50, and more preferably at least about 30 ammno acids to about 50 ammo acids, atIhough nstp,-beatnr polyp pd of any kiigti up tu w14u nicudi; itih entire amino acid sequence of a polypeptide of the invention described above also are included m the invention.

Certain non-exclusive embodiments of the invention relate to a polypeptide which has the amino acid sequence of an epitope-beanng portion of a Neutrokne-atpha COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 51 3 5~oao -x flcflfl~nnn 00N.640 0 0 or Neutrokme-alphaSV polypeptide having an amino acid sequence described in or above. In other embodiments, the invention provides San isolated antibody that binds specifically uniquely) to a Neutrokine-alpha or Neutrokine-alphaSVpolypeptide having an amino acid sequence described m S or above.

The invention further provides methods for isolating antibodies that bmd specifically uniquely) to a Neutrokine-alpha or Neutrokme-alphaSV polypepude having an anuno acid sequence as described herein. Such antibodies are useful 0 diagnostically or therapeutically as described below 00 10 The invention also provides for pharmaceutical compositions comprising Ssoluble Neutrokne-alpha and/or Neutrokine-alphaSV polypeptdes, particularly human Neutrokine-alpha and/or Neutrokine-alphaSV polypepudes, and/or anti-Neutrokmealpha antibodies and/or anti-Neutrokine-alphaSV antibodies which may be employed, for instance, to treat, prevent, prognose and/or diagnose tumor and tumor metataasis, is infections by bacteria, viruses and other parasites, immunodeficiencies, mflammatory diseases, lymphadenopathy autoimmune diseases, graft versus host disease, stimulate peripheral tolerance, destroy some transformed cell lines, mediate cell activation.

survival and proliferation, to mediate immune regulation and inflammatory responses, and to enhance or inhibit immune responses.

In certain embodiments, soluble Neutrokine-alpha and/or Neutrokme-alphaSV polypeptides of the invention, or agonitsi thereof, are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency severe combined immunodeficiency (SCID)*X linked, SCID-autosomal, adenosine deammase deficiency (ADA deficiency), X-linked agammaglobulinemza (XLA), Bruton s disease, congenital agammaglobulinerma, X-linked infantile agammaglobulinemia, acquired agammaglobulinerma. adult onset agammaglobulinema, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy unspecified hypogammaglobulinemia, agammagirob.inia, coimoni "arb!c imTmundefiiercy 'ID) tacquired), Wiskot-Aldnch Syndrome (WAS), X-linked immunodeficiency with hyper IgM. non X-linked immunodeficiency with hyper IgM, selective IgA deficiency IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficieney with thymoma, Ig heavy chain deletions, kappa chain COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/200e 16:05 61 2 92586999 4 062837999 NO.G40 920 00 o -1( C deficiency B cell lymphoproliferauve disorder (BLPD), selective IgM Immnunodeficiency recessive agammaglobulinemia (Swiss type), reticular dysgenesas, neonatal neutropemnia, severe congenital leukopena, thymic alymphoplasia-aplasia or dysplasia with ammunodeficiency ataxza-telangleciasia. short limbed dwarfism. X linked lymrnphoproliferauve syndrome (XLPi, Nezelof syndrome-combmined immunodeficiency with Igs, punne nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined smunodeficiency or conditions associated with an immunodeficiency c In a specific embodiment, Neutrokine-alpha and/or Neutrokne-alphaSV 00 O i polypeptides or polynucleotdes of the Invention, or agonists thereof, is administered to 0 treat, prevent, prognose and/or diagnose common variable immunodeficiency In a specific embodiment, Neutrokine-alpha and/or Neutrokmne-alphiiSV polypeptides or polynucleotides of the invention, or agonisrs thereof. is admrnimstered to treat, prevent, prognose and/or diagnose X-linked agamnmaglobulinemla.

In another specifeic embodiment, Neutrokine-alpha and/or Neutrokmne-alphaSV polypeptides or polynucleoudes of the inventon, or agonists thereof, is administered to treat, prevent, prognose andf/or diagnose severe combmined immunodeficiency (SCID).

In another specific embodiment. Neutrokane-alpha and/or Neutrokine-alphaSV polypeptides or polynucleoudes of the invention, or agonists thereof, as adnimstered to treat, prevent, prognose and/or diagnose Wiskott-Aldnch syndrome.

In another specific embodiment, Neutrokine-alpha and/or Neutrokine-alphaSV polypeptdes or polynucleoides of the invention, or agonists thereof, is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.

In another embodiment, Neutrokine-alpha antagonists and/or Neuttrokine alphaSV antagonists an anti-Neutrokine-alpha antibody), are administered to treat, prevent, prognose and/or diagnose an autoimmune disease rheumatoid arthnts, systemic lupus erhythematosus, idiopathic thrombocylopenta purpura.

autoummune hemolytic anemia, autoimmune neonatal thrombocytopenia, au!o!mmufotytopen:a hemolyac anemia, antipspholipiu synldromii, ureniIus, allergte encephalomyclitus, myocarditis, relapsing polychondnus. rheurnmautic heart disease, glomerulonephntus IgA nephropathy). Multiple Selerosis, Neuritis, Uveitis Ophthalmia, Polyendocnnopaties Purpura Henloch-Scoenkin purpura), Reiter's Disease. Stiff-Man Syndrome, Aurolmmune Pulmonary Inflammation, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 D21 00 Guillasn-Barre Syndrome, insulin dependent diabetes mellitis, and autounmune inflammatory eye, autoimmune thyroidits. hypothyroidism Hashamoto's t hyroldits, Goodpasture s syndrome, Pemphigus, Receptor autonmmunties such as, for example. Graves Disease Myasthenia Gravis, and insulin resistance, autoimmune hemolytic anenua, autoimmune thrombocytopenic purpura schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositsldermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility glomerulonephntts such as primary glomerulonephrns and IgA Snephropathy bullous pemphigod, Sjogren's syndrome, diabetes millitus, and to adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic O fibrosis), chronic active hepatits, primary biliary cirrhosis, other endocrine gland Ci i failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urucana, atopic dermatitis, asthma, inflammatory myopathics, and other inflammatory granulamatous, degenerative, and atrophic disorders) or conditions associated with an autotmmune is disease. In a specific preferred embodiment, rheumatoid anhrits is treated, prevented, prognosed and/or diagnosed using anti-Neutrokme-alpha antibodies and/or anti- Neutrokine-alphaSV antibodies and/or other antagonist of the invention. In another specific preferred embodiment, systemic lupus crythemosus is treated, prevented, prognosed, and/or diagnosed using anu-Neutrokne-alpha antibodies and/or anti- Neutrokne-alphaSV and/or other antagonist of the invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, prognosed, and/or diagnosed using anti-Neutrokne-alpha antibodies and/or anti- Neutrokane-alphaSV and/or other antagomnst of the invention. In another specific preferred embodiment IgA nephropathy is treated, prevented, prognosed and/or diagnosed using anu-Neutrokme-alpha antibodies and/or ant-Neutrokine-alphaSV and/or other antagonist of the invention. In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, prognosed and/or diagnosed using anti- Neuroknme-alph antibodies and/or ant:-Neutroktne-alphS" antibodies.

The inventon further provides compositions comprising a Neutrokine-alpha or Neutrokme-alphaSV polynucleotide, a Neutrokme-alpha or Neutrokine-alphaSV polypepude, and/or an anu-Neutrokme-alpha antibody or ant-Neutrokme-alphaSV antibody for administration to cells i vitro, to cells ex vivo. and to cells in iw, or to a COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 022 00 0 l multicellular organism. In preferred embodiments, the compositions of the invention c comprise a Neutrokme-alpha and/or Neutrokie-alphaSV polynucleotide for expression Sof a Neutrokine-alpha and/or Neutrokzne-alphaSV polypepude in a host organism for Streatment of disease. In a most preferred embodiment, the compositions of the invention compnse a Neutrolune-alpha and/or Neutrokine-alphaSV polynucleotide for expression of a Neutrokme-alpha and/or Nemrokmne-alphaSV polypepude in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency Particularly preferred in this regard is expression in a human 0C patient for treatment of a dysfunction associated with aberrant endogenous actvity of a 00 o 10 Neutrokmine-alpha or Neutrokine-alphaSV gene expression to enhance the normal 0 B-cell function by expanding B-cell numbers or increasing B cell lifespan).

K The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by Neutrokine-alpha and/or Neutroklne-alphaSV which involves contacting cells which express Neutrokne-alpha and/or Neutrokine-alphaSV with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby an increased cellular response over the standard indicates that the compound is an agonst and a decreased cellular response over the standard indicates that the compound is an antagonist.

In another embodiment, a method for identifying Neutrokine-alpha and/or Neutrolkne-alphaSV receptors is provided, as well as a screening assay for agonists and antagonists using such receptors. This assay ivolves determining the effect a candidate compound has on Neutrokme-alpha and/or Neutrokme-alphaSV binding to the Neutrokine-alpha and/or NeutrokmncalphaSV receptor. In particular, the method involves contacting a Neutrokine-alpha and/or Neutrokine-alphaSV receptor with a Neutrokine-alpha and/or Neutrokmne-alphaSV polypeptide of the invention and a candidate compound and determining whether Neutrokine-alpha and/or Neutrokine-alohaSV nnlvnentde hinding to the Neutrok.ne alpha ndor Neutrokine-alphaSV receptor is increased or decreased due to the presence of the candidate compound. The antagonists may be employed to prevent septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthrtis, cachexia (wasting or malnutrition), immune system COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/208 16:05 61 2 92586999 4 062837999 N0.640 023 0< funcion, lymphoma, and autommmune disorders rheumatoid arthritis and systemic lupus erythematosus).

The present nventors have discovered that Neutrokne-alpha s expressed not O only m cells of monocyttc lineage, but also m kidney lung, penpheral leukocyte, bone s marrow T cell lymphoma, B cell lymphoma, activated T cells, stomach cancer, smooth muscle, macrophages. and cord blood tissue. The present inventors have further discovered that Neutrolne-alphaSV appears to be expressed highly only in primary dendnuc cells. For a number of disorders of these tissues and cells, such as tumor and C tumor metastass, infection of bacteria, viruses and other parasites, immunodeficiencies 0 0 10 chronic variable immunodeficlency), septic shock, inflammation, cerebral o malara, activauon of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis, autoimmune diseases rheumatoid arthnts and systemic lupus erythematosus) and cachexia (wastng or malnutrition). It is believed that significantly higher or lower levels of Neutrokine-alpha and/or Neutrolne-alphaSV gene expression IS can be detected in certam utssues bone marrow) or bodily fluids serum, plasma, unne, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a standard" Neutrokine-alpha and/or Neutrokme-alphaSV gene expression level, the Neutrokine-alpha and/or Neutrokme-alphaSV expression level m tissue or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful dunng diagnosis of a disorder, which involves: assaying Neutrokne-alpha and/or Neutrokne-alphaSV gene expression level m cells or body fluid of an individual; comparing the Neutrokme-alpha and/or Neutrokme-alphaSV gene expression level with a standard Neutrokine-alpha and/or i Neutrokme-alphaSV gene expression level, whereby an increase or decrease in the assayed Neutrokme-alpha and/or Neutrokine-alphaSV gene expression level compared to the standard expression level Is indicative of a disorder.

An additional embodiment of the invention is related to a method for treatng an individual in need of an increased or constitutive level of Neutrokine-alpha and/or Neutrokine-alDhaSV activi t v in t hl ompsng s mrrnad cstering o uch an individual a composition comprsing a therapeutically effective amount of an isolated Neutrokine-alpha and/or Neutrolnce-alphaSV polypeptide of the mvention or an agonist thereof.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008i 1F Cl ct A a~ann r r~~ a joo 7 Wo-r- NO.640 [24 00 o zo 0 0 Pc A still further embodiment of the invention is related to a method for treaing an c individual in need of a decreased level of Neutrokne-alpha and/or Neutrokne-alphaSV Sactivity in the body comprisng, admnistering to such an individual a composition O comprising a therapeutcally effective amount of an Neutrokie-alpha and/or Neutrokne-alphaSV antagonist. Preferred antagonists for use m the present invention are Neutrokmne-alpha-specific and/or Neutrokine-alphaSV-specific antibodies.

Bref Descnpton of the Figures 00 (N The following drawmgs are illustrative of embodiments of the mvention and are O not meant to limnt the scope of the invention as encompassed by the claims.

N 10 Figures IA and IB shows the nucleoide (SEQ ID NO I1) and deduced armno acid (SEQ ID NO:2) sequences of Neutrokne-alpha. Anuno acids i to 46 represent the predicted intracellular domain, amino acids 47 to 72 the predicted transmembrane domain (the double-underlined sequence), and amino acids 73 to 285, the predicted extracellular domain (the remaining sequence). Potential asparagmne-linked glycosylation sites are marked in Figures IA and 18 with a bolded asparagme symbol in the Neutrokine-alpha amino acid sequence and a bolded pound sign above the first nucleotide encoding that asparagine residue in the Neutrokine-alpha nucleotnde sequence. Potential N-linked glycosylation sequences are found at the following locations in the Neutrokine-alpha amino acid sequence: N-124 through Q-127 (N-124.

S-125, S-126. Q-127) and N-242 through C 245 (N-242, N-243 S-244, C 245).

Regions of high identity between Neutrokine-alpha, Neutrokine-alphaSV TNF-alpha, TNF-beta, LT-beta, and the closely related Fas Ligand (an alignment of these sequences is presented in Figure 2) are underlined in Figures 1A and IB. These regions are not limiting and are labeled as Conserved Domain CD-II, CD-IIL CD-IV CD-V CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI in Figures 1A and IB.

Figures 2A and 2B show the regions of identity between the amio acid sequiences of Neutrokine-alpha (SE Q I N:2) anu reutrokmic-alpiaSV (SEQ ID NO and TNF-alpha ("TNFalpha in Figures 2A and 2B GenBank No. Z 5026; SEQ ID NO:3). TNF-beta ("TNFbeta n Figures 2A and 2B GenBank No. Z15026: SEQ ID NO:4), Lymphotoxm-beta ("LTbeta in Figures 2A and 2B. GenBank No.

L] 1016; SEQ ID NO:5), and FAS ligand ("FASL in Figures 2A and 2B; GenBank No.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/288 16:05 61 2 92586999 4 062937999 N0.640 00 21 0 UI1821 SEQ ID NO:6), determned by the "McgAlign routine which is part of the k computer program called "DNA*STAR. Residues that match the consensus are shaded.

SFigure 3 shows an analysis of the Neutrokne-alpha amino acid sequence.

s Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity' amphipathuc regions; flexible regions; antigemc index and surface probability are shown, as predicted for the armno acid sequence of SEQ ID NO:2 using the default parameters of the recited computer programs. In the Antigenic Index Jameson-Wolf' graph. the O indicate location of the highly antgenie regions of Neutrokne-alpha regions from o00 0 which epnope-bearng peptides of the invention may be obtained. Antgenic Spolypeptides include from about Phe 115 to about Leu-147 from about lie 150 to about Tyr 163, from about Ser-171 to about Phe-194. from about Glu-223 to about Tyr-246, and from about Ser-27 I to about Phe-278. of the ammno acid sequence of SEQ ID NO:2.

I~ The data presented in Figure 3 are also represented in tabular form in Table I.

The columns are labeled with the headings "Res" "Positon and Roman Numerals I-XIV The column headings refer to the following features of the amino acid sequence presented m Figure 3, and Table 1: "Res amino acid residue of SEQ ID NO:2 and Figures IA and IB. "Position positon of the corresponding residue withm SEQ ID NO:2 and Figures IA and IB; I: Alpha. Regions Garnier-Robson; II: Alpha, Regions Chou-Fasman; Ill: Beta, Regions Garmer-Robson; IV Beta, Regions Chou-Fasman: V Turn, Regions Garier-Robson; VI: Turn, Regions Chou-Fasman; VII: Coil.

Regions Garner-Robson; VIII: Hydrophilicity Plot Kyte-Doolittle; IX.

Hydrophobicty Plot Hopp-Woods; X. Alpha, Amphapathic Regions Eiscnberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus-Schulz;

XII:

Anugenc Index Jameson-Wolf; and XIV Surface Probability Plot Emma.

Figures 4A, 4B, and 4C show the alignment of the Neutrokmne-alpha nucleotde sequence determined from the human cDNA deposited in ATCC No. 97768 with related human cDNA clones of the invention which have been designated HSOAD55 (SEQ ID NO;7). HSLAH84 (SEQ ID NO:8) and HLTBM08 (SEQ ID NO:9).

Figures 5A and SB shows the nucleotide (SEQ ID NO- 18) and deduced aurno acid (SEQ ID NO- 19) sequences of the Neuirolone-alphaSV protein. Amino acids 1 to COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0. 640 D26 00 0 C' 46 represent the predicted inracellular domain, anmno acids 47 to 72 the predicted ct transmembrane domain (the double-underlined sequence), and ammno acids 73 to 266.

Sthe predicted extracellular domain (the remaining sequence). Potential asparaginc-linked glycosylation sites are marked m Figures 5A and 5B with a bolded s asparagine symbol in the Neutrokine-alphaSV amino acid sequence and a bolded pound sign above the first nucleotide encoding that asparagne residue in the Neutrokine-alphaSV nucleotde sequence. Potential N-linked glycosylauon sequences are found at the following locations in the Neutrokme-alphaSV amino acid sequence: CN N-124 through Q-127 (N-124. S-125, S-126. Q-127) and N-223 through C 226 (N-223, 00 o to N-224, S-225, C 226). Antigenic polypeptides include from about Pro-32 to about 0 Leu-47 from about Giu-t 16 to about Ser-143, from about Phe 153 to about Tyr-173, from about Pro-218 to about Tyr-227 from about Ala-232 to about Gin-241 from about 11e-244 to about Ala-249- and from about Ser- 2 52 to about Val-257 of the amino acid sequence of SEQ ID NO:19 Regions of high identity between Neutrokme-alpha, Neutrokine-alphaSV TNF-alpha. TNF-beta. LT-beta, and the closely related Fas Ligand (an aligment of these sequences is presented in Figure 2) are underlined in Figures IA and IB. These conserved regions (of Neutrokine-alpha and Neutrokme-alphaSV) are labeled as Conserved Domain CD-II, CD-III, CD-V CD-VI, CD-VI. CD-VIII. CD-IX.

CD-X, and CD-XI in Figures 5A and 5B. Neutrokme-alphaSV does not contain the sequence of CD-IV described in the legend of Figures IA and IB.

An additional alignment of the Neutrokmne-alpha polypeptide sequence (SEQ ID NO:2) with APRIL, TNF alpha, and LT alpha is presented in Figure 7A. In Figure 7A, beta sheet regions are indicated as described below in the Figure 7A legend.

Figure 6 shows an analysis of the Neutrokine-alphaSV ammo acid sequence.

Alpha, beta, turn and coil regions; hydrophilicity and hydropbobictty- amphipathic regions; flexible regions; anugenic index and surface probability are shown, as predicted for the amino acid sequence of SEQ ID NO' 19 using the default parameters of the rected computer ,programs., The.c iO.cnofl h. ighly antigenic regions of the Neutrokme-alpha protein. regions from which epitope-beanng peptides of the invention may be obtained is indicated in the Antigenc Index Jameson-Wolf" graph.

Antigemc polypeptides include, but are not limned to, a polypepude comprising amino acid residues from about Pro-32 to about Leu-47 from about Glu- 116 to about Ser-143, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 P27 00 23 00 Cl from about Phe-153 to about Tyr-173, from about Pro-218 to about Tyr-227 from Sabout Ser 252 to about Thr-258, from about Ala-232 to about Gln-241 from about SIle-244 to about Ala-249- and from about Ser 252 to about Val-257 of the ammo acid sequence of SEQ ID NO: 19 s The data shown m Figure 6 can be easily represented in tabular format similar to the data shown in Table I. Such a tablular representation of the exact data disclosed n Figure 6 can be generated using the MegAlign component of the DNA*STAR computer sequence analysis package set on default parameters. This is the identical CS program that was used to generate Figures 3 and 6 of the present applicaton.

So Figure 7A. The amino-acid sequence of Neutrokine-alpha and alignment of its Spredicted ligand-bmding domain with those of APRIL. TNF-alpha, and LT-alpha (specifically amino acid residues 115-250 of the human APRIL polypeptide (SEQ ID ATCC Accession No. AF046888), amno acid residues 88-233 of TNF alpha (SEQ ID NO:3; GenBank Accession No. Z15026), and LT alpha ((also designated TNF-beta) amino acid residues 62 205 of SEQ ID NO:4; GenBank Accession No.

Z15026)). The predicted membrane-spanning region of Neutrokme-alpha is indicated and the site of cleavage of Neutrokne-alpha is depicted with an arrow Sequences overlaid with lines (A thru H) represent predicted beta-pleated sheet regions.

Figure 7B. Expression of Neutrokme-alpha mRNA. Northern hybridization analysis was performed using the Neutrokme-alpha orf as a probe on blots of poly RNA (Clonetech) from a spectrum of human tissue types and a selection of cancer cell lines. A 2.6 kb Neutroknc-alpha mRNA was detected at high levels in placenta, heart, lung, fetal liver, thymus, and pancreas. The 2.6 kb Neutrokme-alpha mRNA was also detected m HL-60 and K562 cell lines.

Figures 8A and 8B. Neutrokme-alpha expression increases following activation of human monocytes by IFN-gamma. Figure 8A. Flow cytomeinc analysis of Neutrokme-alpa protein expression on in virro cultured monocytes. Purified monocytes were cultured for 3 days in presence or absence of IFN-gamma (100 U/ml).

Cells were then stainrl with a NmutArokie-a!pha-spec:fic mA* (2E f scii lines, or an isotype-matched control (1gGI) (dashed lines). Comparable results were obtained with monocytes purified from three different donors in three independent experiments.

Figure SB. Neutrokinc-alpha-specific TaqMan primers were prepared and used to assess the relative Neutrokine-alpha mRNA expression levels in unstimulated and IFN- COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P28 00 gamma (100 U/mL) treated monocytes. Nucleotide sequences of the TaqMan pnmers t are as follows: Probe: 5'-CCA CCA GCT CCA GGA GAA GGC AAC TC 3' (SEQ G ID NO:24); 5' amplification pnrimer: 5'-ACC GCG GGA CTG AAA ATC T 3' (SEQ ID NO:25); and 3' amplification pnmer 5'-CAC GCT TAT TTC TGC TGT s TCT GA-3' (SEQ ID NO:26).

Figures 9A and 9B. Neutrokine-alpha is a potent B lymphocyte stimulator.

Figure 9A. The biological activity of Neutrokine-alpha was assessed in a standard Bo lymphocyte co-stimulation assay utilizing Staphyloococcus aureus cowan 1 SAC as the 0 C pnimng agent. SAC alone yielded background counts of 1427 316. Values arc 00 0 !o reported as mean standard deviatun of triplicate wells. Similar results were c" r obtained using recombinant Neutrokme-alpha purified from stable CHO transfectants and transiently transfected HEK 293T cells. Figure 9B. Proliferation of tonsillar B cells with Neutrokine-alpha and co-sumulation with ant-lgM. The bioassay was performed as described for SAC with the excepton that individual wells were precoated with goat anti-human IgM antibody at 10 micrograms/mL in PBS.

Figures 10A and 10B. Neutrolkne-alpha receptor expression among normal human penpheral blood mononuclear cells and tumor cell lines. Figure 10A. Human peripheral blood nucleated cells were obtained from normal volunteers and isolated by density gradient centnfugaton. Cells were stained with botmnylated Neutrokine-alpha followed by PE-conjugated streptavidin and FITC or PerCP coupled mAbs specific for CD3. CD20, CD14, CD56, and CD66b. Cells were analyzed on a Becton Dickmson FACScan using the CellQuest software. Data represent one of four independent expenments. Figure 10B. Neutrokine-alpha binding to histocytc cell line U-937 and the myeloma line IM-9 2S Figures 11A, 11B and 1IC. In vivo effects of Neutrokme-alpha administration in BALBIcAnNCR nuce. Figure IlA. Formalin-fixed spleens were paraffin embedded and 5 micrometer sections stained with hematoxylin and eosin (upper panels). The lower panels arc secuons taken from the same animals stained with anti- CD45R(B2201 mAb and developed with hrsPr.adish-perid;c coupled rabbit anti-rat Ig (mouse adsorbed) and the substrate diarmnobenzidine teirahydrochlonde (DAB).

Slides were counter-stained with Mayer's hematoxylin. CD45R(B220) expressing cells appear brown. Figure 11B. Flow cytometnc analyses of normal (left panel) and Neutrokne-alpha-treated (nght panel) stained with PE-CD45R(B220) and FITC ThB COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/200e 16:05 61 2 92586999 4 062837999 NO.640 U29 00 r y (Ly6D). Figure 11C. Serum IgM. IgG, and IgA levels in normal and Neutrokinealpha treated mrce.

Detailed Description The present invenuon provides isolated nucleic acid molecules comprising a s polynucleotide encoding a Neutronke-alpha polypepudes having the armno acid sequences shown in Figures IA and IB (SEQ ID NO:2), which was determined by sequencing a cDNA clone. The nucleoude sequence shown m Figures IA and 1B C (SEQ ID NO: was obtained by sequencing the HNEDU 15 clone, which was o deposited on October 22, 1996 at the American Type Culture Collection, 10801 S1to University Boulevard, Manassas, Virginia 20110-2209 and assigned ATCC Accession No. 97768. The deposited clone is contained in the pBluescnpt SK( plasmiad (Stratagene, La Jolla, CA).

The present invention also provides isolated nucleic acid molecules comprising a polynucleoude encoding Neutrokine-alphaSV polypepudes having the amino acid is sequences shown in Figures SA and 5B (SEQ ID NO' 19), which was determined by sequencing a cDNA clone. The nucleoude sequence shown m Figures 5A and (SEQ ID NO: 18) was obtained by sequencing the HDPMC52 clone, which was deposited on December 10, 1998 at the American Type Culture Collection, and assigned ATCC Accession No. 203518. The deposited clone is contained in the pBluescnpt SK( plasmid (Stratagene, La Jolla, CA).

The Neutroksne-alpha and Neutrokinc-alpha polypeptides of the present inventon share sequence homology with the translanon products of the human mRNAs for TNF-alpha, TNF-bca, LTbeta, Fas ligand, APRIL, and LTalpha. (See, Figures 2A.

2B. and 7A). As noted above, TNF-alpha is thought to be an important cytokine that plays a role in cytooxicty necrosis, apoptosis, costimulaton, proliferation, lymph node formation, immunoglobulin class switch, differentiation, antlviral activity and regulation of adhesion molecules and other cytokmes and growth factors.

Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc., Foster City CA), and all amino acid COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92~9giqqq aciQ27roQQ NO. 640 00 sequences of polypepudes encoded by DNA molecules determined herei were Spredicted by translation of a DNA sequence determmed as above. Therefore, as is known m the art for any DNA sequence determined by this automated approach, any Snucleotide sequence determined herein may contain some errors. Nucleotide sequences S determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotade sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known m the art.

00 As is also known in the an, a single inserton or deletion in a determined nucleotide Sto sequence compared to the actual sequence will cause a frame shift m translation of the CNl nucleotlde sequence such that the predicted amino acid sequence encoded by a determined nueleotide sequence will be completely different from the ammo acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion, By nucleotde sequence of a nucleic acid molecule or polynucleonde is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleondes, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides G, C and where each thymidine deoxyribonucleoude in the specified deoxyribonuclcotide sequence is replaced by the ribonucleotlde undine Using the information provided herein, such as the nucleorde sequence in Figures IA and 1B, a nucleic acid molecule of the present invention encoding a Neutrokme-alpha polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as staning material.

Illustrative of the invention, the nucleic acid molecule described in Figures IA and IB (SEQ ID NO-1) was discovered in a cDNA library derived from neutrophils.

Expressed sequence lags corresponding to a portion of the Neutrokine-alpha cDNA were also found in kidney lung, peripheral leukocyte, bone marrow T cell lymphoma, B cell lymphoma, activated T cells, stomach cancer, smooth muscle, macrophages, and cord blood tissue. In addiinn, using the nucleotds I for-nt;ao provided in igures and 5B, a nucleic aid molecule of the present invention encoding a Neutrokine-alphaSV polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in Figures COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 lIGG 05 GI D 1 C0Cq p -v oocorf NO.640 P31 00 0 A and 5B (SEQ ID NO-18) was discovered in a eDNA library derived from primary dendnutc cells.

SThe Neutrokine-alpha plasmid HNEDU 15 deposited as ATCC Accession No.

97768 contains an open reading frame encoding a protein of about 285 ammino acid residues, a predicted ntracellular domain of about 46 amino acids (amuno acid residues from about I to about 46 m Figures IA and IB (SEQ ID a predicted transmembrane domain of about 26 anuno acids (underlined amino acid residues from about 47 to about 72 in Figures IA and IB (SEQ ID a predicted extracellular o domain of about 213 amino acids (ammo acid residues from about 73 to about 285 in Sto Figures IA and IB (SEQ ID and a deduced molecular weight of about 31 kDa.

SThe Neutrokme-alpha polypepude shown in Figures IA and IB (SEQ ID NO:2) is about 20% similar and about 10 identical to human TNF-alpha, which can be accessed on GenBank as Accession No. 339764.

The Neutrokine-alphaSV plasmid HDPMC52, deposited as ATCC Accession s1 No. 203518, contains a predicted open reading frame encoding a protein of about 266 amnno acid residues, a predicted mtracellular domain of about 46 amino acids (amino acid residues from about 1 to about 46 in Figures 5A and 5B (SEQ ID NO:19)), a predicted transmembrane domain of about 26 amino acids (underlined amino acid residues from about 47 to about 72 in Figures 5A and 5B (SEQ ID NO:19)), a predicted extracellular domain of about 194 anuno acids (amino acid residues from about 73 to about 266 in Figures 5A and 5B (SEQ ID NO and a deduced molecular weight of about 29 kDa. The Neutrokme-alphaSV polypeptide shown m Figures 5A and (SEQ ID NO 19) is about 33.9% similar and about 22.0% identical to human TNF-alpha which can be accessed on GenBank as Accession No. 339764.

AS one of ordinary skill would appreciate, due to the possibilities of sequencing errors discussed above, the actual complete Neutrokine-alpha and/or Neutrokne-alphaSV polypeptides encoded by the deposited cDNAs, which comprise about 285 and 266 amino acids, respectively may be somewhat shorter In particular, the deterrnmd N, eutrck..:e- iph .d Ne rk n.-aphI. coding sequerces co0tj;ai a common second methionme codon which may serve as an alternative start codon for translation of the open reading frame, at nucleotide positions 210-212 m Figures 1A and IB (SEQ ID NO: 1) and at nucleotide positions 64-66 in Fiures 5A and 5B (SEQ ID NO:18). More generally the actual open reading frame may be anywhere in the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 932 00 o 24 range of ±20 amino acids, more likely in the range of ±10 ammo acids, of that predicted C from either the first or second methionine codon from the N-terminus shown in Figures I A and IB (SEQ ID NO:1) and in Figures SA and 5B (SEQ ID NO:18). It will further _be appreciated that, the polypeptide domains described herein have been predicted by computer analysis, and accordingly that depending on the analytical cntena used for identifying various functional domains, the exact address" of the extracellular, mtracellular and transmembrane domains of the Neutrokne-alpha and SNeutrokine-alphaSV polypepudes may differ slightly For example, the exact location C of the Neutrokine-alpha and Neutrokne-alphaSV extracellular domains in Figures IA 00 o to and IB (SEQ ID NO:2) and Figures 5A and 5B (SEQ ID NO:19) may vary slightly C- the address may shift" by about 1 to about 20 residues, more likely about 1 to about 3 residues) depending on the critena used to define the domain. In this case, the ends of the transmembrane domains and the beginning of the extracellular domains were predicted on the basis of the identificaton of the hydrophobic ammo acid is sequence in the above indicated positions, as shown in Figures 3 and 6 and in Table I.

In any event, as discussed further below the invention further provides polypeptides having vanous residues deleted from the N-terminus and/or C-terminus of the complete polypeptides, including polypeptides lacking one or more armno acids from the N-termn of the extracellular domains described herein, which constitute soluble forms of the extracellular domains of the Neutrokine-alpha and Neutrokine-alphaSV polypeptides.

As indicated, nuclec acid molecules and polynucleotides of the present inmvention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

By "isolated" nucleic acid moleculc(s) is intended a nucleic acid molecule (DNA or RNA). which hal s been -,moved from nat'.r cnvironm-m.. For example.

recombinant DNA molecules contained in a vector are considered tsolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or punfied (parually or substantially) DNA molecules in solution. Isolated RNA molecules COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P33 00 2A 0 0 include in vvwo or m vitro RNA transcnpts of the DNA molecules of the present invention. However, a nucleic acid contained in a clone that is a member of a library a genomic orcDNA library) that has not been isolated from other members of the O' library n the form of a homogeneous solution containing the clone and other members of the library) or a chromosome isolated or removed from a cell or a cell lysate a chromosome spread" as in a karyotype), is not "isolated" for the purposes of this invention. As discussed further herein, isolated nucleic acid molecules according to the present invention may be produced naturally recombinantly or C synthetically 0 10 Isolated nucleic acid molecules of the present invention include DNA molecules Scompnsing, or alternatively consisting of, an open reading frame (ORF) with an inmrlaon codon at positions 147-149 of the nucleotde sequence shown in Figures A and IB (SEQ ID NO-1). In addition, Isolated nucleic acid molecules of the invention include DNA molecules which comprise, or alternatvely consist of, a sequence substantially different from those described above; but which due to the degeneracy of the genetic code, still encodes the Neutroklne-alpha protein. Of course, the genetic code is well known in the art. Thus, it would be rounne for one skilled in the an to generate the degenerate variants described above. In another embodiment, the invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a sequence encoding the Neutrokne-alpha polypeptide having an amino acid sequence encoded by the eDNA contained in the plasmid having ATCC accession number 97768. Preferably this nucleic acid molecule compnses, or alternatively consists of a sequence encoding the extracellular domain the mature or soluble polypeptde sequence of the polypeptLde encoded by the cDNA contained in the plasmid having ATCC accession number 97768.

Isolated nucleic acid molecules of the present invention also include DNA molecules compnsing an open reading frame (ORF) with an initiation codon at positions 1 3 of the nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO: 18), In addition, isolated nucleic acid molecules of the irveriion include DiN^n niuacculc which comprise, or alternatvely consist of, a sequence substantially different from those described above, but which due to the degeneracy of the genetic code, still encodes the Neutrokmne-alphaSV polypepude. Of course, the genetic code is well known in the an. Thus, it would be routine for one skilled in the art to generate the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 062837999 NO.640 P34 0 0 c degenerate variants described above. In another embodiment, the invention provides C isolated nucleic acid molecules comprising, or alternatively consisting of, a sequence Sencoding the Neutroknc-alphaSV polypeptide having an anuno acid encoded by the cDNA contained in the plasmid having ATCC accession number 203518. Preferably s this nucleic acid molecule comprises, or alternatively consists of, a sequence encoding the extracellular domain or the mature soluble polypeptde sequence of the polypeptide encoded by the eDNA contained m the plasmid having ATCC accession number 203518.

The invenuon further provides an isolated nuclec acid molecule comprisng, or alternatively consistmg of. the nucleotide sequence shown m Figures 1A and 1B (SEQ ID NO: 1) or the nucleotide sequence of the Neutrokine-alpha cDNA contained in the plasmid having ATCC accession number 97768, or a nucleic acid molecule having a sequence complementary to one of the above sequences. In addition, the invention provides an isolated nucleic acid molecule comprising, or alternatively consisung of.

the nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO-18) or the nucleotide sequence of the Neutrokine-alpha SV cDNA contained in the plasnd having ATCC accession number 203518, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, have uses which include, but are not lirmted to, as probes for gene mapping by n stmu hybridization with chromosomes, and for detectng expression of the Neutrokine-alpha and Neutrokine-alphaSV in human issue, for instance, by Northern or Western blot analysis.

In one embodiment, the polynucleoudes of the inventon comprse, or alternatively consist of, the sequence shown in SEQ ID NO:22. The sequence provided as SEQ ID NO:22 was constructed from several overlapping mouse EST sequences obtained from GenBank (Al182472, AA422749 AA254047 and AI122485). The EST sequences were aligned to generate the Neutrokme-alpha-like polynucleotide sequence provided as SEQ ID NO:22. The ammo acid sequence resulting from the translation of SEQ ID NO:22 's prowded -as SEQ ID NO:23. -ragmcnts. variants, and denvauivs of the sequences provided as SEQ ID NO:22 and SEQ ID NO:23 are also encompassed by the invention.

In another embodiment, the polynucleondes of the invention compnse, or alternatively consist of, the sequence shown in SEQ ID NO:27 and/or a sequence COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/20088 16:05 61 2 92586999 4 062837999 N0.640 00 0 31 O encoding the amino acid sequence disclosed in SEQ ID NO:28. fragments, variants, and derivatives thereof. These polynucleotdes are also encompassed by the invention.

For example, certain embodiments of the invention are directed to polynucleotdes comprising, or alternatively consisting of, a sequence encoding a polypeptide sequence that is at least 80%, 85%. 90%. 92%. 95%. 96%. 97%, 98%, or 99% identical to amuno acids 68-219 of SEQ ID NO:28. The amino acid sequence resulting from the translation of SEQ ID NO:27 is provided as SEQ ID NO:28. Polypeptuds comprising, or altrnatively consisting of, the amino acid sequence ofSEQ ID NO:28, and o fragments, variants, and derivatives of the sequence provided as SEQ ID NO:28 are 00 o1 also encompassed by the invention. For example, certain embodiments of the inventon 0 0 are directed to polypeptides comprising, or alternatively consistmg of, a polypepude Cl sequence that is at least 80%. 85%, 90%, 92%, 95%, 96%, 97%. 98%. or 99% identical to ammo acids 68-219 of SEQ ID NO:28. A nucleic acid molecule having the sequence provided as SEQ ID NO:27 was obtained by RT-PCR from cyanomologous s1 monkey Macaca irus) PBMC using two degenerate primers. Briefly total RNA was prepared from cyanomologous monkey PBMC by using Tnzol (available from Life Technologies, Inc., Rockville. MD) according to the manufacturer's protocol.

Then a single stranded cDNA was synthesized from the cyanomologous monkey PBMC preparation using standard methods with an oligo-dT primer Neutrokine alpha-specific pnmers were designed based on the conserved region between the mouse and human Neutrokmnealpha molecules (SEQ ID NOs:22 and 1 respectively). A cyanomologous monkey Neutroklne-alpha nucleic acid molecule was then generated by PCR using the eDNA template in combination with the following two degenerate oligonucleotlde primers. 5' pnmer- 5' TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO:35) and 3' prime 5'-GTI ACA GCA GTT TIA IIG CAC C 3' (SEQ ID NO:36). In the sequence of the degenerate pnmers (SEQ ID NOs:35 and 36), "1" represents deoxymosne or dideoxyinosine.

In another embodiment, the polynucleotudes of the invention comprise, or alternattvelv cons.it of. rhe KMewqnr. shnwn in SEQ ID KO:29 and/or a sequence encoding the amino acid sequence disclosed m SEQ ID NO:30, fragments, variants, and derivatives thereof. These polynucleotides are also encompassed by the invention.

For example, certain embodiments of the invention arc directed to polynucleotides comprising, or alternatively consistmg of, a sequence encoding a polypepode sequence COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2C008a 1F cl flfcnrnnn 2-o uj e -rPooC 1 4 cubtjar NO.640 D36 00 that is at least 80%, 85%. 90%, 92% 95%, 96%, 97%, 98%, or 99% identical to amino acids 68-219 of SEQ ID NO:30. The amino acid sequence resulting from the translation of SEQ ID NO:29 is provided as SEQ ID NO:30. Polypeptides comprising, or alternatively consisting of, the amto acid sequence of SEQ ID NO:30, and fragments, variants, and denvatives of the sequences provided as SEQ ID NO:29 and SSEQ ID NO:30 are also encompassed by the inventon. For example, certain embodiments of the invention are directed to polypeptides comprising, or alternatively consisting of, a polypepude sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 0 97%, 98%, or 99% identical to amino acids 68-219 of SEQ ID NO:30. A nucleic acid 00 0 in molecule having the sequence provided as SEQ ID NO:29 was obtained by RT-PCR from rhesus monkey PBMC using two degenerate primers. Briefly total RNA was prepared from rhesus monkey PBMC by using Tnzol (available from Life Technologies, Inc., Rockville. MD) according to the manufacturer's protocol. Then a single stranded cDNA was synthesized from the rhesus monkey PBMC preparation is using standard methods with an oligo-dT primer. Neutrokine-alpha-specific pnmers were designed based on the conserved region between the mouse and human Neutrokmne-alpha molecules (SEQ ID NOs:22 and 1 respectively). A rhesus monkey Neuurokme-alpha nucleic acid molecule was then generated by PCR using the cDNA template m combination with the following two degenerate oligonucleotde pnnmers. pnmer- TAC CAG ITG GCI GCC ITO CAA G-3' (SEQ ID NO:35) and 3' prmer- ACA GCA GTT TIA IIG CAC C 3' (SEQ ID NO:36). In the sequence of the degenerate pnmers (SEQ ID NOs:35 and 36), represents deoxymosme or dideoxymosine.

The invention also provides nucleic acid molecules having nucleotlde sequences related to extensive portions of SEQ ID NO- I and SEQ ID NO-18 which have been determined from the following related cDNA clones: HSOAD55 (SEQ ID NO:7), HSLAH84 (SEQ ID NO:S). and HLTBM08 (SEQ ID NO:9).

The present invention is further directed to nucleic acid molecules encoding portions of the nucleotide seauences descrihed here n, as we!! as to fragm.nts of the isolated nucleic acid molecules described herein. In one embodiment, the invention provides a polynucleotide having a nucleotde sequence representing the pormon of SEQ ID NO'1 which consists of the nucleotides at positions 1 1001 of SEQ ID NO: In another embodiment, the invention provides a polynucleotide having a nucleoude COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 037 00 0 13 c sequence representing the porton of SEQ ID NO: 18 which consists of posmons I 798 t of SEQ ID NO 18.

SThe present invention is further directed to fragments of the nucleic acid ON molecules polynucleoudes) described herem. By a fragment of a nucleic acid molecule having, for example, the nucleotde sequence of the cDNA contained m the plasmid having ATCC accession number 97768, a nucleotide sequence encoding the polypepude sequence encoded by the cDNA contained in the plasmid having ATCC o accession number 97768, the nucleoude sequence of SEQ ID NO' I a nucleoude C sequence encoding the polypeplde sequence of SEQ ID NO:2, the nucleotide sequence of the cDNA contained in the plasmid having ATCC accession number 203518, a 0 Cl nucleotide sequence encoding the polypeptide sequence encoded by the cDNA contained in the plasmsd having ATCC accession number 203518, the nucleotide sequence of SEQ ID NO: 18, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO:20, or the complementary strand thereto, is intended fragments at least 15 nt, and more preferably at least 20 nt or at least 25 nt, still more preferably at least nt, and even more preferably at least 40, 50, 100, 150, 200, 250, 300, 325. 350. 375.

400.450. or 500 nt m length. These fragments have numerous uses which include, but are not lirmted to, diagnostic probes and pnmers as discussed herein. Of course, larger fragments, such as those of 501 1500 nt i length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotde sequences of the cDNA contained in the plasmid having ATCC accession number 97768, the nuclcotide sequence of SEQ ID NO:1. the nucleoude sequences of the cDNA contaned in the plasmid having ATCC accession number 203518. and the nucleotide sequence of SEQ ID NO-18. Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, eptiope-bearing portions of the Neutrokine-alpha and/or Neutrokme-alphaSV polypeptlde as identified in Figures IA and 1B (SEQ ID NO:2) and in Figures 5A and 5B (SEQ ID NO: 19), respectively and described in more detail below nolypeptdes encoded by these .poynucl.eoude fragwmems are also encompassed by the invention.

Also by a fragment of a nucleic acid molecule having, for example, the nucleoutde 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 a COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 938 O4 3 Cl nucleotde sequence encoding the polypeptide sequence of SEQ ID NO:23. a nucleoude sequence encoding the polypeptde sequence of SEQ ID NO:28. a Snucleotde sequence encoding the polypepude sequence of SEQ ID NO:30. or the Scomplementary strands thereof, is intended fragments at least 15 nt, and more preferably at least 20 nt or at least 25 nt, still more preferably at least 30 nt, and even Smore preferably at least 40,50, 100, 150. 200,250, 300, 325. 350, 375.400,450, or 500 n in length. These fragments have numerous uses which include, but are not limted to, diagnostic probes and pnmers as discussed herein. Of course, larger C fragments, such as those of 501 1500 nt in length are also useful according to the present mvention as are fragments corresponding to most, if not all, of the nucleotde /sequence of SEQ ID NO:21 the nucleotide sequence of SEQ ID NO:22, the nucleottde sequence of SEQ ID NO:27 the nucleotde sequence of SEQ ID NO:29 a nucleotde esequence o S I N the nuceoud e sequence of SEQ ]D NO:23. a nucleoude sequence encoding the polypeptde sequence of SEQ ID NO:28, a nucleoude sequence encoding the polypepude sequence of SEQ ID NO:30, or the complementary strands thereof.

Polypeptides encoded by these polynucleotide fragments are also encompassed by the invention.

Representative examples of Neutrolne-alpha polynucleotide fragments of the invention include, for example, fragments that compnse, or alternatively consist of. a sequence from about nucleoude I to 50, 51 to 100. 101 to 146, 147 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 to 700,701 to 750, 751 to 800, 800 to 850, 851 to 900, 901 to 950, 951 to S1000, 1001 to 1050, and/or 1051 to 1082 of SEQ ID NO I or the complementary strand thereto, or the cDNA contained in the plasmid having ATCC accession number 97768. In this context about" includes the particularly recited ranges, and ranges that are larger or smaller by several 3. 2, or 1) nuclcoudes, at either terminus or at both termini.

Representative examples of Neutrokme-alphaSV polynucleoude fragments of the unventson include, for ex.amp!, fra-,,nts that comprise, or ailtativily conit a sequence from about nucleoude I 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 to 700, 701 to 750, 751 to 800, 800 to 850, and/or 851 to 900 of SEQ ID NO 18, or the complementary strand thereto, or the cDNA contained in the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 P39 00 o 3S N plasmid having ATCC accession number 203518. In this context "about" includes the particularly recited ranges, and ranges that are larger or smaller by several 4, 3, 2, or 1) nuclcotides, at either terminus or at both terminl.

O In certain preferred embodiments, polynucleotide of the invention comprise, or s alternatively consist of. nucleotide residues 571-627 580-627 590-627 600-627 610-627 571-620.580-620, 590-620, 600-620, 571-610,580-610,590-610, 571-600, 580-600, and/or 571 590 of SEQ ID NO-1 In certain other preferred embodiments, polynucleotides of the invention C' comprise, or alternatively consist of nucleotide residues 1-879 25-879 50-879 00 Sto 75-879 100-879 125-879 150-879 175-879 200-879 225-879 250-879 275-879 0' 300-879 325-879 350-879 375-879 400-879 425-879 450-879 475-879 500-879 525-879 550-879 575-879 600-879 625-879 650-879 675-879 700-879 725-879 750-879 775-879 800-879 825-879 850-879 1-850, 25-850, 50-850, 75-850, 100-850, 125-850, 150-850, 175-850, 200-850,225-850,250-850, 275-850, 300-850, 325-850, 350-850, 375-850, 400-850,425-850, 450-850, 475-850, 500-850, 525-850, 550-850,575-850,600-850, 625-850. 650-850,675-850,700-850, 725-850,750-850, 775-850. 800-850, 825-850, 1-825, 25-825, 50-825, 75-825, 100-825, 125-825.

150-825, 175-825. 200-825, 225-825. 250-825,275-825, 300-825. 325-825,350-825, 375-825, 400-825,425-825, 450-825, 475-825. 500-825, 525-825. 550-825, 575-825, 600-825,625-825.650-825, 675-825. 700-825.725-825. 750-825. 775-825,800-825, 1-800, 25-800, 50-800, 75-800, 100-800, 125-800, 150-800, 175-800, 200-800.

225-800, 250-800, 275-800, 300-800, 325-800, 350-800, 375-800. 400-800, 425-800, 450-800,475-800,500-800, 525-800, 550-800, 575-800. 600-800, 625-800,650-800, 675-800,700-800, 723-800, 750-800.775-800. 1 775,25-775,50-775.75-775, 100-775. 125-775, 150-775, 175-775, 200-775, 225-775, 250-775, 275-775, 300-775, 325-775,350-775, 375-775,400-775, 425-775, 450-775, 475-775, 500-775,525-775, 550-775, 575-775, 600-775. 625-775, 650-775, 675-775. 700-775, 725-775, 750-775.

1 750, 25-750, 50-750,75-750, 100-750, 125-750, 150-750, 175-750, 200-750, 225-750. 250-750. 275-750. 1An-7F0 125-750, 350-'50, 400-750. 45-750, 450-750,475-750.500-750,525-750, 550-750,575-750, 600-750, 625-750.650-750.

675-750, 700-750, 725-750, 1 725, 25-725, 50-723, 75-725, 100-725 125-725, 150-725. 175-725, 200-725. 225-725, 250-725. 275-725. 300-725, 325-725, 350-723.

375-725, 400-725. 423-72. 450-725, 475-725, 500-725, 525-725, 550-725, 575-725.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO. 640 940 00 O 33 c 600-725, 625-725, 650-725, 675-725, 700-725. 1 700, 25-700, 50-700, 75-700.

100-700, 125-700, 150-700, 175-700, 200-700 225-700, 250-700, 275-700. 300-700, 325-700,35000375-700, 37-, 400-700, 425-700, 450-700, 475-700, 500-700, 525-700, 550-700,575-700,600-700,625-700, 650-700, 675-700. 1-675. 25-675, 50-675, s 75-675, 100-675. 125-675, 150-675. 175-675,200-675, 225-675 250-675,275-675.

300-675,325-675,350-675,375-675,400-675, 425-675,450-675,475-675,500-675, 525-675, 550-675, 575-675, 600-675, 625.675. 650-675, 1-650, 25-650, 50-650, 75-650, 100-650, 125-650, 150-650, 175-650, 200-650, 225-650, 250-650, 275-650.

00 300-650, 325-650, 350-650,375-650,400-650,425-650. 450-650. 475-650, 500-650, o to 525-650.550-650, 575-650, 600-650, 625-650, 1-625, 25-625, 50-625, 75-625, NC 100-625, 125-625, 150-625. 175-625, 200-625, 225-625 250-625, 275-625, 300-623 325-625, 350-625, 375-625. 400-625, 425-625, 450-625.475-625. 500-625. 525-625 550-625,575-625, 600-625, 1-600, 25-600. 50-600,75-600, 100-600, 125-600, 150-600,175-600, 200-600.225-600, 250-600,275-600, 300-600. 325-600.350-600.

375-600,400-600,425-600,450-600,475-600, 500-600, 525-600. 550-600. 575-600.

1 575, 25-575. 50-575. 75-575, 100-575, 125-575, 150-575. 175-575, 200.575, 225-575 250-575.275-575,300-575,325-575,350-575, 375-575 400-575,425-575.

450-575, 475-575,500-575, 525-575, 550-575, 1 550. 25-550, 50-550, 75-550, 100-550, 125-550, 150-550, 175-550, 200-550. 225-550. 250-550, 275-550, 300-550, 325-550, 350-550, 375-550, 400-550, 425-550,450-550, 475-550 500-550, 525-550, 1 525, 25-525, 50-525, 75-525, 100-525, 125-525, 150-525. 175-525, 200-525 225-525, 250-525, 275-525, 300-525 325-525, 350-525, 375-525, 400-525,425-525.

450-525, 475-525 500-525, 1 500, 25-500,50-500, 75-500 100-500. 125-500, 150-500, 175-500,200-500,225-500,250-500, 275-500. 300-500,325-500.350-500, 375-500, 400-500. 425-500, 450-500. 475-500, 1-475, 25475, 50-475, 75475, 100-475, 125-475, 150475, 175-475, 200-475, 225475, 250-475, 275-475, 300-475, 325-475,350-475 375-475,400-475,425-475. 450-475, 1-450. 25-450, 50-450, 75-450, 100-450, 125-450, 150-450, 175-450, 200-450. 225-450,250450. 275-450, 300450.325-450. 350-451 37-450 ann-450,42545,1, 21-425. 25425,50425.

75-425, 100-425, 125-425, 150-425, 175-425, 200-425, 225-425, 250-425, 275-42:.

300-415, 325-425, 350-425, 375-425,400-425, 1400. 25-400, 50-400,75-400, 100-400. 125-400, I50-400. 175-400. 200-400. 225-400. 250-400. 275-400, 300-400.

325400, 350-400, 375400, I 375 25-375, 50-375, 75-375, 100-375, 125-375, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 941 00 o 37 0 150-375, 175-375, 200-375, 225-375, 250-375, 275-375, 300-375, 325-375, 350-375, 1 350, 25-350, 50-350, 75-350. 100-350, 125-350, 150-350, 175-350, 200-350, S225-350, 250-350, 275-350. 300-350, 325-350, 1 325.25-325, 50-325. 75-325, 100-325, 125-325. 150-325, 175-325, 200-325, 225-325, 250-325, 275-325, 300-325 1 300,25-300,50-300. 75-300, 100-300, 125-300, 150-300, 175-300, 200-300, 225-300, 250-300,275-300, I 275,25-275,50-275,75-275, 100-275, 125-275, 150-275, 175-275, 200-275, 225-275.250-275. 1 250,25-250. 50-250, 75-250, 100-250, 125-250, 150-250. 175-250, 200-250, 225-250. 1 225, 25-225, 50-225, C 75-225, 100-225, 125-225, 150-225, 175-225,200-225, I 200,25-200, 50-200, 75-200, S1o 100-200, 125-200, 150-200, 175-200, 1-175.25-175,50-175, 75-175, 100-175, 125-175, 150-175. 1 150, 25-150, 50-150 75-150, 100-150. 125-150, 125. 25-12., 50-125,75-125, 100-125.1 100, 25-100,50-100,75-100,1 75.25-75.50-75.1 25-50. and/or 1 25 of SEQ ID NO- 18.

In certain additional preferred embodiments, polynucleoldes of the invention comprse, or alternatively consist of nucleotide residues 400-627 425-627 450-627 475-627 500-627 525-627 550-627 575-627 600-627 400-600,425-600,450-600.

475-600, 500-600, 525-600, 550-600, 575-600,400.575, 425-575,450-575,475-575 500-575, 525-575. 550-575, 400-550,425-550,450-550,475-550,500-550, 525-550, 400-500, 425-500.450-500, 475-500,400-475,425-475,450-475,400-450,425-450, 571-800, 600-800.625-800, 650-800. 675-800,700-800, 725-800,750-800,775-800, 571 775, 600-775, 625-775, 650-775, 675-775, 700-775. 725-775,750-775,571 750, 600-750, 625-750, 650-750, 675-750, 700-750,725-750, 571 725.600-725,625-725, 650-725, 675-725, 700-725, 571 700, 600-700, 625-700, 650-700,675-700, 571-673 (i 600-675, 625-675, 650-675, 571-650, 600-650, 625-650. 571-625. 600-625, and/or 571-600 of SEQ ID NO: In additional preferred embodiments, polynucleoudes of the invention compise, or alternatvely consist of nucleotide residues 147-500, 147-450. 147-400.

147-350, 200-500,200-450,200-400, 200-350. 250-500. 250-450. 250-400. 250-350, S300-500, 300-450. -400, 300-350, 3 50 7 50, 350-70, 350-o0 350-o00, 350-350, 3o 400-750,400-700.400-650, 400-600,400-550, 425-750,425-700, 425-650, 425-600, 425-550. 450-1020, 450-1001 450-950,450-900 450-850. 450-800. 450-775.

500-1001 500-950.500-900,500-850,500-800.500-775,550-1001 550-950, 550-900, 550-850, 550-800. 550-775, 600-1001 600-950, 600-900, 600-850. 600-800.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P42 00 C" 600-775, 650-1001. 650-950. 650-900, 650-850, 650-800, 650-775. 700-1001.

t 700-950,700-900,700-850, 700-800, 700-775,825-1082, 850-1082, 875-1082, S900-1082.925-1082, 950-1082, 975-1082, 1000-1082, 1025-1082, and/or 1050-1082 of SEQ ID NO:I SPreferably the polynucleotide fragments of the invention encode a polypeptide which demonstrates a Neutrokne-alpha and/or Neutrokmn-alphaSV functional activity By a polypeptlde demonstrating "functional activity" is meant, a polypeptide capable of displaying one or more known functional acuvities associated with a full-length and/or secreted Neutrokme-alpha polypeptide and/or Neutrokme-alphaSV polypeptide. Such 00 functional activities include, but are not limited to, biological activity ability to C stimulate B cell proliferation, survival, differentiation, and/or activation), antigencity [ability to bind (or compete with a Neutrokine-alpha and/or Neutrokme-alphaSV polypeptide for binding) to an ants-Neutrokine-alpha and/or anti-Ncutrolkne-alphaSV antibody], immunogencity (ability to generate antibody which binds to a Neutrokmes1 alpha and/or Neutrokine-alphaSV polypeptide), ability to form mulumers with Neutrokme-alpha and/or Neutrokne-alphaSV polypepttdes of the invention, and ability to bind to a receptor or ligand for a Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide.

In additional specific embodiments, the polynucleotide fragments of the invention encode a polypeptbde comprising, or alternatively consisting of the predicted intracellular domain (amino acids I to 46 of SEQ ID NO:2), the predicted transmembrane domain (anuno acids 47 to 72 of SEQ ID NO:2), the predicted extracellular domain (anmno acids 73 to 285 of SEQ ID NO:2), or the predicted TNF conserved domain (amino acids 191 to 284 of SEQ ID NO:2) of Neutrokme-alpha. In additional embodiments, the polynucleotide fragments of the invention encode a polypeptide comprising, or alternatively consisting of any combination of 1.2. 3, or all 4 of the above recited domains. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

in audiuonal specific emoodimenis. tme polynucicotue tragments or me invention encode a polypeptde comprising, or atemnatively consisting of the predicted intracellular domain (amino acids I to 46 of SEQ ID NO-19), the predicted transmembrane domain (amino acids 47 i 72 of SEQ ID NO- 19), the predicted extracellular domain (ammo acids 73 to 266 of SEQ ID NO-19). or the predicted TNF COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92IaR9fqqq a2o o i NO.640 ;43 00 o 3 o conserved domain (ammno acids 172 to 265 of SEQ ID NO-19) of Neutrokne-alphaS

V

In additional embodiments, the polynucleotide fragments of the invention encode a polypeptide comprising, or alternatively consisting of any combination of 1, 2. 3. or all 4 of the above recited domains. Polypeptides encoded by these polynucleondes are also encompassed by the invenuon.

In another embodiment, polynucleotide fragments of the invention comprise, or alternatively consist of, polynucleotudes which encode an ammo acid sequence selected from residues Met- to Lys- 13, Leu- 114 to Thr-141, Ile 142 to Lys-160, Gly-161 to 0 GIn-198. Val-199 to Ala-248, and Gly-250 to Leu-285 of SEQ ID NO:2. Moreover, 00 to polynucleotides that encode any combination of two, three, four, five or more of these O amno acid sequences are also encompassed by the invention. Polypeptides encoded by these polynucleotides are also encompassed by the inventon.

In another embodiment, polynucleotide fragments of the invention comprise, or alternatively consist of, polynucleotldes which encode an armno acid sequence selected is from residues Met-I to Lys 113, Leu-114 to Thr 141 Gly-142 to Gln-179 Val-180 to Ala-229 and Gly-230 to Leu-266 of SEQ ID NO'19 Moreover. polynucleotides that encode any combination of two. three, four five or more of these amuno acid sequences are also encompassed by the Invention. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In another embodiment, polynucleoude fragments of the inventon comprise, or alternatively consist of, polynucleotides which encode an ammo acid sequence selected from residues Met-I to Lys-106, Leu-107 to Thr-134, Glu-135 to Asn-165. lie 167 to Lys-184, Gly 185 to Gln-224, Val-225 to Ala-272, and Gly-273 to Leu-309 of SEQ ID NO:23. Moreover, polynucleotides that encode any combination of two. three, four, five or more of these amino acid sequences are also encompassed by the invention.

Polypepudes encoded by these polynucleoudes are also encompassed by the invention.

In another embodiment, polynucleotide fragments of the invention comprise, or alternatively constst of, polynucleotides which encode an amno acid sequence selected from residues Tvr I in Lvy-47 Leu-48 to rThr 75, "ic o .ys-94, Giy-95 to Gin-13:, Val-133 to Ala- 82. and Gly-183 to Ala-219 of SEQ ID NO:28. Moreover.

polynucleotides that encode any combinaton of two, three, four, five or more of these amnno acid sequences are also encompassed by the invention. Polypeptdes encoded by these polynucleotides are also encompassed by the invention.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 044 00 4 00 CI In another embodiment, polynucleoide fragments of the invention comprnse or alternatively consist of, polynucleotides which encode an amnmo acid sequence selected from residues Tyr-1 to Lys-47 Leu-48 to Thr-75. le-76 to Lys-94, Gly-95 to Gln-132.

Val-133 to Ala-182, and Gly-183 to Ala-219 of SEQ ID NO:30. Moreover, polynucleotdes that encode any combination of two, three, four, five or more of these amino acid sequences are also encompassed by the invention. Polypeptidcs encoded by these polynucleotides are also encompassed by the invention.

In another embodiment, the polynucleotdes of the Invention compnse, or CI alternatively consist of, the sequence shown m SEQ ID NO:21. The sequence shown 00 lI o as SEQ ID NO;21 encodes a polypeptde consisung of an initiatng methionine residue 0 r linked to residues Ala-134 through Leu-285 of the Neutrokine-alpha polypeptide sequence shown as SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In certain additional preferred embodiments, polynucleotides of the invention is comprise, or alternatively consist of nucleotide residues 1-459 15-459 30-459 45-459 60-459 75459 90-459 105-459 120-459 135-459 150-459 165-459 180-459 195-459 210-459 225-459 240-459 255-459 270-459 285-459 300-459 315-459 330-459 345-459 360-459 375-459 390-459 405-459 420-459 435-459 450-459 1-450, 15-45030-450,45-450, 60-450, 75-450. 90-450, 105-450, 120-450, 135-450, 150-450. 165-450, 180-450, 195-450, 210-450. 225-450. 240-450.255-450, 270-450, 285-450, 300-450, 315.450.330-450, 34>-450. 360-450,375-450. 390-450, 405-450, 420-450.435-450, 1-435. 15-435, 30-435, 45-435. 60-435, 75-435, 90-435, 105435, 120-435, 135-435, 150-435, 16:-435, 180-435, 195-435. 210-435, 225435, 240-435, 255-435, 270-435, 285-435, 300-435, 315-435, 330-435, 345-435, 360-435, 375-435.390-435,405-435,420-435 1-420, 15-420,30-420.45-420, 60-420, 75-420, 90-420, 105-420, 120-420, 135-420, 150-420, 165420, 180-420. 195-420. 210-420, 225-420, 240-420, 255-420, 270420, 285-420, 300-420, 315-420, 330-420, 345-420, 360-420, 375420, 390-420. 405420, 1-405. 15-405, 30-405,45-405, 60-405. 75-405, 90-405, '0 405, 1210-4 505, 65-405, 80-450, 495-5, 2'-405, 225-405. 240-405, 255405. 270-405, 285-405, 300-405, 315-405. 330-405, 345-405, 360-405, 373-405, 390-405, 1-390. 15-390, 30-390,45-390, 60-390. 75-390. 90-390, 105-390, 120-390, 135-390, 150-390, 165-390, 180-390, 195-390, 210-390,225-390, 240-390. 255-390, 270-390, 285-390, 300-390. 315-390, 330-390, 345-390. 360-390.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 00 4 0 375-390. I 375, 15-375, 30-375, 45-375, 60-375. 75-375, 90-375, 105-375, 120-375, 135-375, 150-375, 165-375, 180-375, 195-375, 210-375, 225-375. 240375, 255-375, 270-375, 285-375,300-375. 315-375, 330-375, 345-375, 360-375, 1 360. 15-360, 30-360,45-360, 60-360, 75-360,90-360, 105-360. 120-360. 135-360, 150-360, 165-360, 180-360, 195-360. 210-360,225-360, 240-360, 255-360,270-360,285-360, 300-360, 315-360, 330-360, 345-360, 1 345, 15-345, 30-345, 45-345, 60-345, 75-345, 4 90-345, 105-345, 120-345, 135-345, 150-345, 165-345. 180-345, 195-345,210-345.

S225-345, 240-345, 255-345, 270-345, 285-345, 300-345, 315-345, 330-345, I 330.

o 15-330, 30-330, 45-330.60-330.75-330,90-330. 105-330, 120-330, 135-330, 150-330, 00 10 165-330, 180-330, 195-330. 210-330, 225-330, 240-330, 255-330, 270-330, 285-330, S300-330, 315-330, 1 315 15-315, 30-315,45-315, 60-315, 75-315, 90-315, 105-315, (N 120-315, 135-315,150-315, 165-315, 180-315. 195-31 5 210-315,225-315,240-315. i) 255-315,270-315,285-315, 300-315, 1 300, 15-300, 30-300,45-300, 60-300, 75-300, 90-300, 105-300, 120-300, 135-300, 150-300, 165-300, 180-300, 193-300,210-300, 225-300, 240-300, 255-300, 270-300, 285-300. 1 285, 15-285, 30-285,45-285, 60-285.

75-285, 90-285, 105-285, 120-285, 135-285, 150-285, 165-285, 180-285, 195-285, 210-285. 225-285, 240-285, 255-285, 270-285, I 270, 15-270, 30-270.45-270, 60-270.

75-270, 90-270, 105-270, 120-270, 135-270. 150-270, 165-270, 180-270, 195-270, 210-270, 225-270,240-270, 255-270, 1 255, 15-255, 30-255,45-255,60-255,75-255, 90-255, 105-255, 120-255.135-255, 150-255, 165-255. 180-255. 195-255,210-255, 225-255. 240-255, 1-240, 15-240, 30-240.45-240, 60-240,75-240.90-240, 105-240.

120-240, 135-240, 150-240, 165-240, 180-240, 195-240,210-240. 225-240, 1 225, 15-225, 30-225, 45-225, 60-225, 75-225, 90-225, 105-225, 120-225, 135-225, 150-225. 165-225, 180-225, 195-225, 210-225, I 210, 15-210, 30-210, 45-210, 60-210, 75-210, 90-210, 105-210, 120-210, 135-210, 150-210. 165-210, 180-210, 195-210,1 195.

15-195, 30-195, 45-195, 60-195. 75-195, 90-195, 105-195, 120-195, 133-195. 150-193.

165-195, 180-195, 1 180, 15-180, 30-180,45-180, 60-180, 75-180.180 9 80, 105-180.

120-180, 135-180, 150-180, 165-180, 1-165, 15-165, 30-165,45-165, 60-165, 75-165, gQ-165 105-165. 120-65, 1 35-15.1.50,-65, 1 150, 15-!50, 30150. 45-150, 60-150, 75-150,90-150, 105-150, 120-150. 135-150, 1 135, 15-135, 30-135,45-.135, 60-135, 75-135, 90-135, 105-135, 120-135, 1 120, 15-120,30-120,45-120, 60-120,75-120., 90-120, 105-120, 1-105, 15-105. 30-105,45-105. 60-105,75-105,90-105, 1-90, 15-90, 30-90, 45-90,60-90.75-90, I1 75. 15-75, 30-75, 45-75, 60-75, 1-60. 15-60, 30-60, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO0.640 946 0 C 45-60, 1,45. 15-45. 3045, 1 30, and/or 15-30 of SEQ ID NO:21 Polypeptides cencoded by these polynucleotides are also encompassed by the invention.

SAccordingly specific embodiments of the invention are directed to O polynucleotdes encoding polypeptldes which comprise, or alternatively consist of, the amnno acid sequence of beta pleated sheet region A, A B, B' C, D E, F G, or H Sdisclosed in Figure 7A and described in Example 6. Additional embodiments of the nvention are directed to polynucleotides encoding Neutrokine-alpha polypepudes which comprise, or alteratively consist of, any combmation of 1 2, 3, 4. 5, 6, 7 8, 9 or C(N all 10 of beta pleated sheet regions A-H disclosed in Figure 7A and described in Example 6. Additional preferred embodiments of the invention are directed to 0' polypepuides which compnse, or alternatively consist of, the Neutrokine-alpha aminmo acid sequence of beta pleated sheet region A, A B, B' C, D, E, F G, or H disclosed in Figure 7A and described in Example 6. Additional embodiments of the invention are directed Neutrokne-alpha polypeptides which comprise, or alternatively consist of, any combinaion of 1 2, 3,4, 5, 6, 7 8, 9 or all 10 of beta pleated sheet regions A through H disclosed in Figure 7A and described m Example 6.

In certain other preferred embodiments, polynucleotdes of the invention comprise, or alternatively consist of, nucleotide residues 34-57 118-123, 133-141, 151 159 175-216, 232 255,280-315,328-357 370-393. and/or 430-456 of SEQ ID NO:21 Polypeptides encoded by these polynucleoudes are also encompassed by the invention, These polynucleotldc and polypepude fragments correspond to the predicted beta-pleated sheet regions shown in Figure 7A. In certain embodiments.

polynucleotides of the invention comprise, or alternatively consist of, a polynucleoude sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotde sequence encoding one, two, three, four, five, six, seven, eight, nine or ten of the betapleated sheet regions described above. The present invention also encompasses the above polynucleotide sequences fused to a helerologous polynucleoude sequence.

Polypeptldes encoded by these polynucleotlde sequences are also encompassed by the im"nton. In another embodiment, the invnion provides an isolated nucleic aciu molecule comprsing a polynucleotide which hybridizes under stnngent hybridization conditions to one, two. three, four, five, six, seven, eight, nine or ten of the beta-pleated sheet polynucleotides of the invention described above. The meaning of the phrase "stringent conditions" as used herein is described mfra.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2o08 16:05 61 2 92586999 4 062837999 N0.640 047 0 0 In further preferred embodiments, polynucleotides of the invention comprise, or alternatvcly consist of, nucleoude residues 576-599 660-665, 675-683. 693-701 717-758, 774-803, 822-857 870-899 912-935, and/or 972-998 of SEQ ID NO:1.

Polypepudes encoded by these polynucleotide fragments are also encompassed by the s invention. These polynucleotide and polypeptde fragments correspond to the predicted beta-pleated sheet regions shown i Figure 7A.

In additional preferred embodiments, polynucleoudes of the nvenuon comprise, or alternatively consist of, nucleotlde residues 457-462, 472-480, 490-498, S514-555. 571-600. 619-654, 667-696, 699-732, and/or 769-795 of SEQ ID NO:18.

00 to Polypeptzdes encoded by these polynucleotde fragments are also encompassed by the Sinvention. These polynucleoude and polypeptide fragments correspond to the predicted beta-pleated sheet regions shown in Figure 7A.

In yet further preferred embodiments, polynucleotides of the invention comprise, or alternauvely consist of. nucleotide residues 124-129 139-147 157-165, is 181 222, 238-267 286-321, 334-363, 376-399 and/or 436-462 of SEQ ID NO:22.

Polypeptides encoded by these polynucleoide fragments are also encompassed by the invention. These polynucleottde and polypeptide fragments correspond to.the predicted beta-pleated sheet regions shown in Figure 7A. Polypeptdes comprising, or alternatively consisting of the amino acid sequence of any combination of one, two, three, four, five, six, seven, eight, nine, ten, or all of these regions are encompassed by the ivention.

The relative positions of several introntexon boundaries were determnned for the mouse Neutrokme-alpha (SEQ ID NO:22 and SEQ ID NO;23) based on sequence analysts of mouse genonuc DNA. The apparent second exon from the 5' end of the mouse Neutrokne-alpha genomc clone (preliminarily designated "Exon consists of Tyr-187 to Gln-222 of the sequence shown in SEQ ID NO:23. The apparent third exon from the 5' end of the mouse Neutrokine-alpha genomic clone (preliminarily designated "Exon comprses Val-223 to Gly-273 of the sequence shown in SEQ ID NO:23.

Thus. m one embodiment, the invention pro-ides po.ynuico dcs encoding polypeptdes comprising, or alternatively consisting of, the amino acid sequence of residues Tyr-187 to Gln-222 of SEQ ID NO:23. The present inventon is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleoude sequence at least 80%, 85%, 90%, 95%. 96%, 97%, 98% or 99% identical to the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P48 0 44 Spolynucleoude sequence encoding the mouse Neutrokne-alpha polypeptdes described above. The present invention also encompasses the above polynucleoude sequences Sfused to a heterologous polynucleotde sequence. Polypeptdes encoded by these nucleic acids and/or polynucleoude sequences are also encompassed by the mventon.

In another embodiment, the invention provides polynucleoudes encoding polypepldes comprising, or alternatively consisung of, the amino acid sequence of residues Val-223 to Gly-273 of SEQ ID NO:23. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide 0 sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the 00 10 polynucleode sequence encoding the mouse Neatrokne-alpha polypepudes described o above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleoude sequence. Polypepudes encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention.

Moreover, the relative positions of the corresponding mtron/exon boundaries is were determined for human Neutrokme-alpha (SEQ ID NO- I and SEQ ID NO:2) based on an alignment of the sequences of mouse and human Neutrokmne-alpha polypeptudes.

The apparent second exon from the 5' end of human Neutrokine-alpha (also preliminarily designated "Exon consists of. Tyr-163 to Gin-198 of the sequence shown in SEQ ID NO:2. The apparent third exon from the 5' end of human Neutrokmne-alpha (also prelirmnarily designated "Exon consists of. Val-199 to Gly- 249 of the sequence shown in SEQ ID NO:2.

Thus, m one embodiment, the invention provides polynucleotdes encoding polypeptides comprising, or altematively conssting of, the anuno acid sequence of residues Tyr-163 to Gin-198 of SEQ ID NO:2. The present mvention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotde sequence at least 80%, 85%. 90%. 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Neutrokme-alpha polypeptides described above.

The present invention also encompasses the above polynucleotide sequences fused to a heterclogcus polynucleoude sequence. "olypepudes nuucu oy lcsc uuJeiu acius and/or polynucleoude sequences are also encompassed by the invention.

In another embodiment, the mvention provides polynucleotides encoding polypepudes comprising, or alternatively consisting of, the amno acid sequence of residues Val-199 to Gly-249 of SEQ ID NO:2. The present invention is also directed to COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2088 ct o~Eoconn r 1 0 2 1 C CoO l l 7 ar NO.640 P49 00 4r o nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%. 90%, 95%. 96%, 97%, 98% or 99% dentical to the Spolynucleonde sequence encoding the Neutrokme-alpha polypepudes described above.

SThe present invention also encompasses the above polynucleotide sequences fused to a s heterologous polynucleoude sequence. Polypeptides encoded by these nucleic acids and/or polynucleoude sequences are also encompassed by the invention.

The functional activity of Neutrolone-alpha and/or Neutrolone-alphaSV polypeptides, and fragmensn variants denvatves, and analogs thereof, can be assayed Sby various methods as described herein and as are well known in the art.

00 10 For example, m one embodiment where one is assaying for the ability to bind or Scompete with full-length Neutrokane-alpha and/or Neutrokne-alphaSV polypeptlde for binding to ant-Neutrokne-alpha and/or anu-Neutrokane-alphaSV antibody or banding to Neutrokme-alpha receptor(s) and/or Neutrokinc-alphaSV receptor(i) on B cells, vanous immunoassays known in the an can be used, including but not limited to, Is competitive and non-competitive assay systems using techniques such as radiolmmunoassays, ELISA (enzyme linked immunosorbent assay), sandwich immunoassays, immunoradiometrc assays, gel diffusion precipitation reactions, immunodiffusion assays, m situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, preciptation reactions, agglutination assays gel agglutination assays, hemagglutnation assays). complement fixauon assays, immunofluorescencc assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody In another embodiment, the pnmary antibody is detected by detectng binding of a secondary antibody or reagent to the pnmary antibody In a further embodiment, the secondary antibody as labeled. Many means are known m the art for detecting binding in an immunoassay and are wthin the scope of the present invention.

In another embodiment, where a Neutrokne-alpha and/or Neutroklne-alphaSV liga-l i dtinfmd, or the ability of a pulypptiuu fragment, variant or denvaive of the invention to mulimnenze is being evaluated, binding can be assayed. by means well-known in the art. such as, for example, reducing and non-reducing gel chromatography protein affinity chromatography and affinity blotting. See generally Phizicky et al., 1995, Microbiol. Rev 5994-123. In another embodiment, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/-8328Vi 1F l;lc. CI 7 ocrunnn rrrc.n~~~,, 3- 20- I C 4 oD8a NO.640 950 00 49 0 Sphysiological correlates of Neutrokine-alpha and/or Neutromne-alphaSV binding to its substrates (signal transduction) can be assayed.

SIn addition, assays described herein (see Examples 6 and 7) and otherwise known in the an may routinely be applied to measure the ability of Neutrokme-alpha Sand/or Nutrokine-alphaSV polypeptides and fragments, variants denvatives and analogs thereof to elicit Neutrokine-alpha and/or Neutroklne-alphaSV related biological activity to stimulate, or alternatively to inhibit (in the case of Neutrokmne-alpha and/or Neutrokine-alphaSV antagonists) B cell proliferation, o differentiation and/or activation and/or to extend B cell survival n vtro or m vvo).

00 10 Other methods will be known to the skilled artisan and are withm the scope of 0 the invention.

In additional embodiments, the polyoucleotdes of the invention encode polypeptides comprsing, or alternatively consisting of, functional attributes of Neurokine-alpha and Neutrokme-alphaSV Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, alpha-helix and alpha-helix forming regions ("alpha-regions"). beta-sheet and beta-sheet forming regions ("beta-regions"), tum and turn-forming regions ("lum-regions"), coil and coil-forming regions ("coil-regions"). hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenc index regions of Neutroknc-alpha and Neurrokine-alphaSV polypeptdes.

It is believed one or more of the beta pleated sheet regions of Neutrokme-alpha disclosed in Figure 7A is imporant for dimerizanon and also for interactions between Neutrokne-alpha and us ligands.

Cenam preferred regions in this regard are set out in Figure 3 (Table The data presented in Figure 3 and that presented m Table I, merely present a different format of the same results obtained when the amino acid sequence ofSEQ ID NO:2 is analyzed using the default parameters of the DNA*STAR computer algorithm.

hec abovc-imcniiond prcL 1 Cerrd egiorns set our; in Figure 3 and in 'able include, but are not limited to, regions of the aforementioned types identified by analysts of the amino acid sequence set out in Figures IA and IB. As set out in Figure 3 and m Table I, such preferred regions include Gamier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 925e6999 4 062937999 N0.640 951 00 o and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emmi Ssurface-forming regions and Jameson-Wolf regions of high antgenc index. Among Shighly preferred polynucleoudes in this regard are those that encode polypepudes s comprising, or alternatively consisting of, regions of Neutrokine-alpha and/or Neutrokme-alphaSV that combine several structural features, such as several 1, 2, 3 or 4) of the features set out above. Polypepudes encoded by the polynucleoudes are also encompassed by the invention.

SAdditionally the data presented in columns VIII, IX, XIlI, and XIV of Table I 00 10 can routinely be used to determine regions of Neulrokine-alpha which exhibit a high degree of potential for anttgenicty (column VIII of Table I represents hydrophilicity according to Kyte-Doolittle; column IX of Table I represents hydrophoblcity according to Hopp-Woods; column XIII of Table I represents anigenc index according to Jameson-Wolf; and column XIV of Table I represents surface probability according to is Emini). Regions of high antgeniciy are determined from the data presented m columns VIII, IX. XIII, and/or IV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide m an environment an which antigen recognition may occur in the process of initiation of an immune response. The data presented in Figure 6 can also routinely be presented in a similar tabular format by simply examining the amino acid sequence disclosed m Figure 6 (SEQ ID NO:19) using the modules and algorithms of the DNA*STAR set on default parameters. As above, the ammo acid sequence presented in Figure 6 can also be used to determine regions of Neutrokme-alpha which exhibit a high degree of potential for antigencity whether presented as a Figure (as in Figure 6) or a table (as in Table 1).

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/200e 16:05 6 2 92899 4 062837999 NO.G40 PD52 48 Table I Rn Poesson I 1I III IV V VI VI1 Vill Ix x XI xnl xxII xIv 00

C

S Met Asp Asp Ser Thr Gln Art inu Gin

S"

IS Art Len Thr

SO

Cys Le Lys LYs Arg Gin Glu Met Lys Len Lys Gin Cys Val Scr Ie Leu Pro Arg Lys in sey ro Ser Val Aug Snr Set Lys Asp Gly so Lys Leu Lau Ala Ala Tb Lau Len Ala Le Lou 073 -0.71 1.12 -0.66 1.62 -1.09 2.01 -1.5: 2.40 -2.13 2.20 -1.73 281 -134 2.00 -1.73 1.90 -1.3 2.00 -1,04 1.33 -0,66 (X41 -0.09 046 0.20 030 -0.19 0.91 -0.19 0.80 -0.87 1.61 -1.36 1.32 -14 1.6S7 -1.70 1.52 -A-39 2.38 -1.70 233 -1.70 1.62 -1.70 0.66 -1.13 0.36 -0.49 -0.53 -0.71 -4.74 -003 -1.00 -03 -0-08 0.40 -0.08 0.40 -08 -0.17 C 0.29 -0.81 0.93 -0.81 0.93 *1.07 C 0.97 -1.37 C L$9Y -1.16 C 1.50 -1.16 1.39 -0.77 1.39 -0.39 1.39 .0.77 1.34 -1.20 1.60 -1,16 1.09 -IsO 1.13 .1.11 0.43 -0.21 0.14 -0.70 0.13 -0.20 -072 0.29 53 0.54 .2.06 1.23 -2.63 1.23 -2.63 1.04 .234 1,41 -2A2 1.3 -27 1.20 -2.78 1.09 0.95 1.39 1,15 1.13 2.12 1.15 4.19 F 1.30 4.35 F L90 4-31 I 0.90 4.51 F 0.90 .612 F 0.90 2.91 F 0.90 2.15 F 0.90 1.66 P 0.45 0.51 F -0.15 0.32 0.30 0.32 0.30 0.78 F 0.90 1.06 F 0.90 1.37 F 0.90 4.44 F 0.90 5.33 F 0.9 5.33 F 0.90 2.M F 0.00 2.24 P 0.90 2.24 F 0.75 0.69 F 0.45 0-52 0.60 0.35 0.30 0-30 0.30 0.13 -0.30 0.11 030 0.40 0.45 1.08 F 1.10 1.39 F 1-50 2.66 F 1.34 4.98 F 1.98 4.32 F 2.52 1.64 F 2.86 1-60 F 3.40 1.24 F 2-36 3.24 F 2.46 1.60 F 2-46 2.00 P 3.06 2.67 F 3.06 2-72 F 3.40 1.67 F 2.66 1.03 F 1.77 os2 0.91 0.31 0.04 0.46 -0.60 0.19 -0.60 0.1* -0.60 0.19 -0.60 0.19 C IAIIli -0.60 0.09 -0.60 0.14 -0.60 0.09 -0.20 0.6

T

T

1' T

T

T T T T T T

T

A

A A A A A A A A A A A A A A

A

A A A A A A

A

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062637999 NO.640 953 00 4 00 Table I (continued) Res Ponon I II 1I IV V VI VIl Vill IX X XI Xl XIII Xlv Secr 58 A T 2.28 1.09 -0,20 0.05 Cys 59 A T -2.32 1.07 -0.20 0.09 Cys 60 A T -2.59 1.03 -0.20 m0.0 LU 61 B 5 -2.08 0.99 -0.60 0.04 Thr 62 a B -1.97 099 -0.60 0.11 Val 63 B B -1.91 1.20 .0.60 0.17 Val 64 B B -124 1.39 -0.60 0.33 See 65 B B -143 1.10 -0.60 0.40 Phe 66 A a -1-21 1.26 -0.60 0.40 Tyr 67 A B -149 1.11 -0.60 0.54 laP 63 A B -1.44 0M97 -0.60 041 Val 69 A B -0.59 1.27 .0.60 0.3 SAla 70 A B -0.63 89 60 043 Ala 7 A B 0.07 056 -O.60 0.25 00 Lau 72 A T -00 0.16 0.10 0.55 Gin 73 A T -1.09 0.20 F 0.25 0.45 Gly 74 A T -0.53 0.20 F 025 OtS.

C Asp 75 A T -076 0.09 F 0.25 Le 76 A A .0.6 0.09 F -0.13 O.a, Ala 77 A A 0.17 -0.31 0.30 069 Scr 78 A A 0.17 -0.24 0.30 0.43 Len 79 A A -030 -0.24 0.30 O8 Arg 80 A A -0.30 -024 0.30 0.7' Ala 81 A A 017 -0.34 0.30 0.93 Gli 82 A A 0.72 -0.30 0.45 1.11 Lou S3 A A 0.99 -0.49 0.30 0.77 Gin 84 A A 1.21 0.01 -0.15 1.04 Gly 835 A A 1.10 l0.01 -030 0.61 His 86 A A 1.73 0.01 -0.15 1.27 IIts 7 A A 0.92 -0.67 0.75 1.47 Ala SB A A 1.52 -039 0.45 1.22 Glu 89 A A 0.93 -0.30 0.45 1 Lys 90 A A 0.93 -0.39 F 0.60 1.03 Lau 91 A T 0.38 -0.46 0.85 1.01 Pro 92 A T 0.07 -0.46 0.70 0.59 Ala 93 A T 0.07 -0.03 0-70 0.29 Gly 94 A T -0.14 0.47 .0.20 0.36 Ala 95 A -0.14 0.21 -0.10 0.36 Gly 96 A 0.08 -0.21 F 0.65 0.73 Ala 97 A -0.06 -0.21 F 0.65 07 Pro 98 A -0.21 -0.21 F 0.65 Lys 99 A A 0.07 -003 F 0.45 Ala 100 A A 0.66 -o.4A6 F 0.60 1.01 Gly 101 A A 0.41 -0.96 F 0.90 1,13 Leu 102 A A 0.79 -089 F 0.75 0.57 Glu 103 A A 0.41 .0.46 F 0.45 0.88 Gl 104 A A -0.49 -046 F 0.45 0.89 Ala 105 A A -021 -0.24 0.30 0.81 Pro 106 A A -A0.46 -044 0.30 0,67 Ala 107 A A 0.01 0.06 -030 0.39 Val 108 A A -0.80 0.49 4.60 0.38 Thr 109 A A -0.76 0.67 -0.60 0.20 Ala 110 .06 U.L4 -U.JU U.411 Gly Ill A A 1-54 0.43 -0460 0.38 Len I112 A A -0.96 0.57 -0.60 0.23 Lys 113 A B -0.31 0.09 -0.30 0.39 lic 114 A B -0.21 0.01 -0.30 0.63 COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/3/200e 16:05 61 2 92586999 4 062837999 N.640 PD54 Table I (continued) VI VII Vill Ix x xi Res PoSIuoM" I II Iv v XII Xiii xiv 00

C

Pro Pro Ala Pro Cry Clu Gly Am IS Scr

GIN

Am 5cr Ar Asn LyS Arg Ala val Gin Gly

PM

Glu Glu Thr Val Thr Gin Asp Cys Ltu Gin Len lk Ala Asp St Glu Thr pro Thr Do0 Gin Lys Gly Tyr 11w Phe

VI

Pro Trp Leu LCn

T

T

T T T T

T

T

T

T

A B

A

A

A

A

A

A

A

A

A

A A A A A A A A

A

T

T

T T

T

T

T

T

T

T T

T

T

T

-0.21 0.01 C -00M -0.17 C 0.39 0.26 C O.-M aM0 C 0.89 -0.79 C 1.59 -0.36 3.29 -0.39 1,20 0.43 C 1.4 -0.54 C .00 -0.57 C 1.91 -0.60 C 21.37 21 C 2.37 .0,64 C 2.76 -0.64 C 2.87 -1.03 2.58 -1.41 102 -1.31 2.02 -1.07 1.69 -1-06 C 1.77 -0.63 C 1.66 '-60 C 1.66 -0,60 C 1.30 -0-60 C 0.33 -16 0.61 -0-61 1.47 -0.53 1.47 -0.56 1.14 .199 0.54 -0.41 054 0.27 -0.27 0.19 -0.84 0.23 -0.511 1.43 -0.27 0.53 -0.57 0.53 -0.57 0.34 C -021 -0.34 0.39 -0.26 C 0.08 4-51 C -0.0O 471 C 0.99 -0-53 C 1.52 -0.13 1.18 .0.51 1.18 -0.09 093 -119 093 0.14 0.44 0.14 010 0.24 0.58 0.49 0.2N 0.91 457 1.40 1.D, 1,70 -1.03 1.63 -lA9 1.53 -1.13 1.53 -0.32 0,89 0-.1 F 1.25 F 1.10 F 2.20 F 3.00 F 2.25 F 2 I S2-00 S1,60 F iSw F 1.50 F F 5.54 P 2.18 F 32 F 86 F 3.40 F 3.06 F 2.7' F 2.18 V- 1.64 F 1.49 F 1.83 F .52 P 2.86 F 3.40 F 2.66 F 2.12 F 1.78 F: 1.19 F lye5 F 0.25 r 0.25 0.1 -0.60 -0.30 00 F 2.45 F 3.00 F 2.70 F 240 F 1.56 F 1.9i F 1.03 F 2.04 F 1.60 F 1,44 F 1.28 0.12 *0.44 -0.60 -0.60 -0.60 -0.60 -0.60 -0.30 14 B 13 B B B B B B

B

B

B

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03200 16:05 62 2 92596999 4 062937999 NO.640 055 Res Position I Table I (continued) If IN IV V VI Vi VIll ix x XI xii xyil xiv Ter Phe Lys Art Oly Sor Ala Lao Clu Clu IS Lys Glu Asn Lys lie Jau Val Lys alt' Thr dy1 Tyr Pta Phe lIt Tyr Gly Gin

VI

Leu Tyr Th Asp Lys Thr Tyr Ala Met Gly His Ltu Ile Gin Arg Lys Lys Vat Iis Val Phe Gly Asp Let Sec Leu

VI

171 17' 173 174 '75 176 177 378 179

ISO

382 183 1M4 185 186 187 UsR 389 190 191 192 193 194 '95 196 '97 198 199 201 202 203 201 205 206 207 20S 209 210 211 212 213 214 215 216 217 21 219 220 221 22Z 224 225 226 227

A

T

T I

T

T

7

A

A A A A A A A A A A A A A A A A A A A A A A A A

A

T

T T T T T 7 B B B B T B B B T T T T

T

0.19 0.46 0.10 -0-03 -020 -0.33 C -420 -0.5 C 0.61 -021 09 -1.00 1.66 .1.00 1.61 -1.00 1-50 -I.A3 1.9 1.30 -1.91 1.09 .91g 1.03 -1.23 1.0 -0.59 1.06 -0.59 0.72 -0.59 0.32 -0.50 0.13 -0.07 -0.61 0.00 -0.42 0.10 -030 0.24 0.II 0.93 -028 1.69 -0.29 1.63 -0,82 1.60 -1.29 149 .1.2 1.39 *0.90 136 C -0.20 1.16 C 0-3 040 0.67 -103 0.77 0.06 0.18 0.17 0.43 -0.01 0.90 -116 1.11 -0.21 0.6j 0.29 -0.28 0.97 -0.32 1.17 0.10 1.81 0.39 0.31 1.02 -0.30 0.77 -0.73 1.08 -0.59 0.26 -077 0.37 -81 0.91 -0.43 0.91 4100 0.80 -0.00 -0.06 -0.00 -0.40 0.04 -0.36 -0.07 -I.i v0 6] -0.17 *0.74 -0.11 -1.10 0.57 -0.9 1-36 0.20 180 F 2.6 F 3.00 F 2.25 P 2-w F 135 F 1.20 F 0.90 F 0.90 Ir 0.90 F 090 F 0.90 F 0.75 0.60 ,60 0.30 F 0-45 P 0.40 F 0.3o F 0.65, 0.20 -060 -0.60 -060 -0.20 -0.20 -0140 -0.10 1.25 F 0.80 F 0.10) F 1.00 TF 0.60 0.45 -0.30 -o"n -0.60 -0.60 -0.30 0.45 0.75 P 0.90 F 090 F 0.90 0.30 0.30 D.39 0.30 -0.30 D-50 U.S0 0.30 0.30 -0-60D -0.60 0.73 0.85 1.32 1.4 0.99 &76 1-54 1.93 3.16 7.66 3.10 1.48 0.55 a63 0.68 1.54 0.67 0.27 0.29 0.29 0.32 0.23 0.26 0.59 0.54 0.92 1.06 2.06 3.91 lii 1.73 1.03 0.70 0.40 0.13 1.80 1.62 3.14 1.35 0.611 6.29 0.25 0.57 0.31 0.63 u.6&, 0.45 0.39 0.18 0.11 COMS ID No: ARCS-i83608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/o3/2ooe 16:05 61 2 92596999 4 062837999 ND.G40 9D56 Table I (continued) Res Position I H III IV V VI VII Vill IX X XI XII XIII XIV 00 ci STmr Ltu Phe Atg CyS

II

Glin As.

Me Pra GIu Tbr LAu Pro

AM

Ast Scr Cvs Tyr Ala dly lie Ala

LYS

Lou ClIO Gilu Gly Asp Glu Lau Gin Lcu Ala Ie.

Pro A r Giu Asn Ala OlN lie L&u Asp Gly Asp Val Thr Phe Ftc Ca..

Ala Lys Lou ICu

B

B

B

B

1T B '1 B T

B

B

B

-1.66 -1,73 -I.43 -0.62 -0-17 -0.21 0.54 C 0.93 C 0.01 C 0.47 1.36 C 1.36 C 1.06 0.99 0.96 0.66 0.3R 0.84 0.17 -042 -0.39 -Q89 -4.22 0.02 -402 0.57 0-L91 0.99 1.58 0.72 0.94 0.06 -0-16 0.30 0.10 0.30 0.2 0.52 0.44 0.73 0.81

UP-

02 0.57 C 0.57 C -0.29 -0.01 -0.71 -0-52 -0,57 *0.34 1.16 -0.77 1.53 1,53 -1.61 -1.30 -0.99 1.03 0.6 0,86 0.86 0.3 0.46 0,10 0.10 0.1 0.01 aoa -0.17 -0.21 036 063 O140 0L43 071 0.23 0.26 0.19 -0.20 -0.70 -0.70 -1.39 -1.20 149 -0980 -0.30 -0.04 0.39 -0.30 -0.30 -0.49 -0.59 -0.59 -0.63 0.06 0.06 0.09 -OA1 -0.17 -0.37 0.03 0.13 0.56 0.63 1.31 U.6-1 0.87 0.77 IL41 0.41 0."W -460 -0.60 -6.60 -0.60 -0.20 alti 0.05 F 020 F 0.44 F 1.06 P 1.12 F 1.96 F 240 F L41i r 1.22 F 1.13 0.74 0.20 -0.411 -0.60 -0-30 -0.30 0.30 F 0-1)0 F 0.90 F 0.90 F 0.9)0 F 0.90 P 0.75 030 0.30 -0.30 -0.30 0.30 F 1.00 F 1.00 F 1.34) F 1..V 0.95 to 0.13 060 F 1.60 F 2.25 F F 1.b.

F 1.00 -0.10 -0,.35 -u0u -060

-OW

.0.60 -0.60 -0.30 T 7 T T T T

T

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 062837999 NO.640 P57 00

O

Additional preferred nucleic actd fragments of the present invention include Snucleic acid molecules comprising, or alternatively consisting of a sequence encoding one or more eptope-beanng portions of Neutrokine-alpha. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules comprising, or alternauvely consisting of, a sequence encoding a polypepude selected from: from about Phe-115 to about Leu-147 from about Ile-150 to about Tyr-163, from about SSer-171 to about Phe-194, from about Glu-223 to about Tyr-246, and from about 0 Ser-271 to about Phe-278. of the amino acid sequence of SEQ ID NO:2. In this 00 l0 context, about" means the paricularly recited ranges and ranges larger or smaller by 0 several, a few 5. 4. 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini. Polypeptides encoded by these nucleic acid molecules are also encompassed by the inventon. Polypeptde fragments which bear antgenc epitopes of the Neutrokme-alpha may be easily determined by one of skill in the art using the Is above-described analysis of the Jameson-Wolfantigenie index, as shown in Figure 3 Methods for determining other such epitope-beanng portions of Neutrokme-alpha are described in detail below Additional preferred nucleic acid fragments of the present invention include nucleic acid molecules comprising, or alternatively consisting of a sequene encoding one or more epitope-bearng portions of Neutroknc-alphaSV In paricular such nucleic acid fragments of the present invention include nucleic acid molecules comprsing, or alternatively consisting of a sequence encoding a polypepude selected from about Pro-32 to about Leu-47 from about Glu-I 16 to about Ser 143, from about Phe 153 to about Tyr-173, from about Pro-218 to about Tyr 227 from about Ser-252 to about Thr-258, from about Ala-232 to about Gin-241 from about Ie-244 to about Ala-249- and from about Ser-252 to about Val-257 of the amino acid sequence of SEQ ID NO 19 In this context, about" means the particularly recited ranges and ranges larger or smaller by several, a few 5. 4. 3, 2 or amino acid residues at either or both the aminn- and r rboy..-ermni. D-P-pepdun.t. cod by te nuc.ic aciL i ic are also encompassed by the invention, Polypputde fragments which bear antigenmc epitopes of the Neutrokine-alpha may be easily determined by one of skill in the ar using the above-described analysis of the lameson-Wolf antigenic ndex. Methods for COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2888 16:05 61 2 92586999 4 062837999 NO.640 D58 00 o 54 c- determining other such epitope-beanrg portions of Neutrokine-alphaSV are described in detail below SIn specific embodiments, the polynucleondes of the invention are less than 1O00,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb. 300 kb. 250 kb.

s 200 kb, 175 kb, 150 kb, 125 kb, 100 kb. 75 kb. 50 kb, 40 kb. 30 kb, 25 kb, 20 kb, kb, 10 kb, 7.5 kb, or 5 kb m length.

In further embodiments, polynucleotides of the mventon comprise at least at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000

O

Cg contiguous nucleotades of Neutrokme-alpha coding sequence, but consist of less than or 00 equal to 1000 kb, 500 kb, 250 kb, 200 kb. 150 kb, 100 kb. 75 kb, 50 kb, 30 kb. 25 kb, kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1A and IB (SEQ ID NO-1) or Figures 5A and 5B (SEQ ID NO'18).

In further embodiments, polynucleotides of the invention comprise at least 15. at least at least 50, at least 100, or at least 250, at least 500, or at least 1000 conuguous nucleotdes of Neutrokme-alpha coding sequence, but do not comprise all or a portion of any Neutrokme-alpha intron. In another embodiment, the nucleic acid comprising Neutrokine-alpha coding sequence does not contain coding sequences of a genomic flanking gene 5' or 3' to the Neutrokme-alpha gene in the genome). In other embodiments, the polynucleondes of the mvention do not contain the coding sequence of more than 1000, 500,250, 100. 50,25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

In another embodiment, the mvention provides an isolated nucleic acid C molecule comprising a polynucleoade which hybridizes under stnngent hybridization conditions to a portion of the polynucleotde in a nucleic acid molecule of the inventon described above, for instance, the sequence complementary to the coding and/or noncoding sequence depicted in Figures IA and IB (SEQ ID NO-1), the sequence of the cDNA clone contained m the deposit having ATCC accession no. 97768. the sequence complementary to the coding sequence and/or noncoding sequence depicted in Figures 5A and 5B (SEQ !D NO: -hc seqence cDN clone conliainu the deposit having ATCC accession no. 203518, the sequence complementary to the coding sequence and/or noncoding sequence transcribed, untranslated) depicted in SEQ ID NO:2 the sequence complementary to the coding sequence and/or noncoding sequence depicted in SEQ ID NO:22, the sequence complementary to the coding COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO.640 959 0 Ssequence and/or noncoding sequence depicted in SEQ ID NO:27 the sequence complementary to the coding sequence and/or noncoding sequence depicted in SEQ ID C NO:29 or fragments (such as, for example, the open reading frame or a fragment thereof) of these sequences, as described herein. By stringent hybndization s conditions" is intended overnight incubation at 42C in a solution compnsing: formamide, 5x SSC (750 mM NaCI, 75 mM tnsodium ctrate), 50 mM sodium phosphate (pH 5x Denhardt's solution, 10% dextran sulfate, and 20 pg/mn Sdenatured, sheared salmon sperm DNA, followed by washing the filters m 0. x SSC at Sabout 65 0

C.

0 0 to By a polynucleotide which hybridizes to a portion of a polynucleotide is O intended a polynucleotde (either DNA or RNA) hybndizing to at least about 13 nucleoudes and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 40, 50, or 60) nucleodes, and even more preferably about any integer i the range of 30-70 or 80-150 nuclcotides, or the entire length of the reference polynucleotde. These have uses, which include, but are not limited to, diagnostic probes and primers as discussed above and an more detail below By a portion of a polynucleotde of at least about 20 nt an length, for example, is intended to include the particularly recited ranges, larger or smaller by several 5, 4. 3, 2. 1 or 0) anuno acids, at either extreme or at both extremes of the nucleotide sequence of the reference polynucleotide the sequence of one or both of the deposited cDNAs. the complementary strand of the nucleotide sequence shown an Figures IA and IB (SEQ ID NO the complementary strand of the nucleotide sequence shown in Figures SA and 5B (SEQ ID NO 18), the complementary strand of the nucleotide sequence shown in SEQ ID NO:21. the complementary strand of the nucleotde sequence shown tn SEQ ID NO:22, the complementary strand of the nucleotide sequence shown in SEQ ID NO:27 and/or the complementary strand of the nucleotde sequence shown in SEQ ID NO:29). Of course, a polynucleotde which hybridizes only to a poly A sequence (such as the 3' terminal poly tract of the Neutrokine-alpha cDN.A shown n igyres Ad B (SEQ ID NO-j. the 3' ier-nia poly(A) tract of the Neutrokme-alphaSV eDNA shown in Figures 5A and 5B (SEQ ID NO: 18) or the 3' terminal poly(A) tract of the Neutrokne-alphaSV cDNA shown in SEQ ID NO:22), or to a complementary stretch of T (or U) residues, would not be included n a polynucleotide of the Invenuon used to hybridize to a pormon of a nucleic COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 960 0 cI acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly stretch or the complement thereof practically any double-stranded cDNA clone generated using oligo dT as a prmer).

SAs indicated, nucleic acid molecules of the present invention which encode a s Neutrokine-alpha polypepude or a Neutrokme-alphaSV polypeptide may include, bui are not limited to, polynucleotdes encoding the armno acid sequence of the respective extracellular domains of the polypeptides, by themselves; and the coding sequence for the extracellular domains of the respective polypepudes and additional sequences, such Ci as those encoding the intracellular and transmembrane domain sequences, or a pre- or 0O o 10 pro- or prepro- protein sequence; the coding sequence of the respective extracellular o domains of the polypeptides. with or without the aforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above protein sequences together with additional, non-coding sequences, including for example, but not limited to, introns and non-coding 5' and 3' sequences, such as the transcribed. non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example, ribosome binding and stability of mRNA, an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this embodiment of the Cminvention, the marker amino acid sequence is a hexa-htstidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue. Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentr et al., Proc. Nal. Acad. Sca. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA tag is another peptide useful for puification which corresponds to an epitope derived from the influenza phcmagglatiin protemn. which ht bcun uc.i'b.cu by WilWs ci us., Cl 37 767 98 4 3o As discussed below other such fusion proteins include the Neutrokine-alpha or the Neutrokine-alphaSV polypepudes fused to Fe at the N- or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions. analogs or denvatives of the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO. 640 961 0 Neutrokne-alpha or Ncutrokne-alphaSV polypepudes of SEQ ID NO:2. Variants may Soccur naturally such as a natural allelic variant. By an allelic vanant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewm, ed., John Wiley Sons, New York (1985).

s Non-naturally occurrng variants may be produced using art-known mutagenesis techniques, which include, but are not lirmted to oligonucleoude mediated mutagenesis, 4alanme scanning, PCR mutagenests, site directed mutagenesis (see Carter et al., SNucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), 0 cassette mutagenesis (see Wells er Gene 34:315 (1985)), restriction selection 0 10 mutagenesis (see e.g. Wells er al., Philos. Trans. R. Soc. London SerA 317-415 0 (1986)).

Such variants include those produced by nucleoude substitutions, deletons or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered m coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amno acid substituttons, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Neutrokmne-alpha and/or Neutrokine-alphaSV polypepudes or portions thereof. Also especially preferred m this regard are conservative substitutions.

Additional embodiments of the invention are directed to isolated nucleic acid molecules comprising a polynucleotde which encodes the anuno acid sequence of a Neutrokne-alpha and/or Netrokne-alphaSV polypeptide a Neutrokme-alpha and/or Neutrokine-alphaSV polypeptide fragment described herein) having an amino acid sequence which contmns at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutons, even more preferably not more than 40 conservative anuno acid substtutions, still more preferably not more than conservative amino acid substitutions, and still even more preferably not more than conservative amino acid substitutions, 10-20 conservative amino acid substitutions, tUn onser-.ae am no acd substatutions, 1-5 ConiSCrvaiv aiilln ilu Nu 3-5 conservative amino acid substtutions, or 1-3 conservative amino acid substitutions.

Of course, in order of ever-ncreasing preference, i is highly preferable for a polynucleotide which encodes the amno acid sequence of a Neutrokine-alpha and/or COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16*05 61 2 925e6999 4 062e37999 N0.640 D62 o 00 c, Neutrokine-alphaSV polypeptide to have an anuno acid sequence which contains not more than 10, 9 8,7 6,5, 4, 3, 2 or I conservative amino acid substitutions.

SFurther embodiments include an isolated nucleic acid molecule comprising or 0\ alternatively consisting of, a polynucleotide having a nucleoude sequence at least 85%, or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide selected from the group consisting of: a nucleotlde sequence encoding the Neutrokne-alpha polypeptide having the complete annno acid sequence in Figures IA and 1B positions I to 285 of SEQ ID NO:2); a Snucleoude sequence encoding the Neutrokine-alpha polypeptide having the complete 00 o 10 amino acid sequence in SEQ ID NO:2 excepting the N-terminal methiomne c postuons 2 to 285 of SEQ ID NO:2); a fragment of the polypepude of having Neutrokine-alpha functional activity anugenmc or biological activity): a nucleoude sequence encoding the predicted extracellular domain of the Neutrokine-alpha polypeptide having the amino acid sequence at positions 73-285 in 1S Figures IA and I B (SEQ ID NO:2); a nucleotide sequence encoding the Neutroklne-alpha polypepttde having the amino acid sequence at positions 134-285 in Figures I A and IB (SEQ ID NO:2); a nucleotide sequence encoding the Neutrokme-alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone contained i the deposit having ATCC accession number 97768. a nucleotide sequence encoding the extracellular domain of the Neutrokmne-alpha polypeptide having the amino acid sequence encoded by the cDNA contained in the deposit having ATCC accession number 97768; and a nucleotde sequence Scomplementary to any of the nucleotide sequences in or (h) above.The present invention also encompasses the above polynucleoude sequences fused to a heterologous polynucleotide sequence. Polypepudes encoded by these polynucleotdes and nucleic acid molecules are also encompassed by the invention.

Highly preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatively consisting of a polynucleotide having a nuclentide sequence at east 80O% 85%, M ;iinca anu iuic pilrcraoly at lcas 95%. 96%. 97%, 98%, 99% or 100% identical to a polynucleotde sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at posiions 134-285 in Figures IA and 1B (SEQ ID NO:2). Preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatively consisting of a COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 925eG999 4 062837999 N0.640 D63 00 o polynucleoude having a nucleotde sequence at least 90% identical to a polynucleotide sequence encoding the Neutrokme-alpha polypeptide having the amino acid sequence at posmons 134-285 m Figures IA and IB (SEQ ID NO:2). More preferred embodiments of the invention are directed to nucleic acid molecules comprnsng, or s alternatively conssting of a polynucleotde having a nucleotde sequence at least Identical to a polynucleotide sequence encoding the Neutrokme-alpha polypeptide having the anuno acid sequence at positions 134-285 m Figures IA and IB (SEQ ID SNO:2). More preferred embodiments of the mvention are directed to nucleic acid o molecules comprising, or alternatively consistng of a polynucleotide having a 00 to nucleotide sequence at least 96% identical to a polynucleotide sequence encoding the 0 Neutrokine-alpha polypeptide having the ammo acid sequence at positions 134-285 in Figures IA and 1B (SEQ ID NO:2).

Additionally more preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatively consisting of a polynucleotide having a 1a nuclcoude sequence at least 97% to a polynucleoude sequence encoding the Neutrokine-alpha polypeptde having the amino acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2). Additionally more preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternauvely consisting of a polynucleotide having a nucleotde sequence at least 98% to a polynucleotde sequence encoding the Neutrokane-alpha polypeptide having the amino acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2). Additionally more preferred embodiments of the invenuon are directed to nucleic acid molecules comprising, or alternatvely consistng of a polynucleoude having a nucleotide sequence at least 99% identical to a polynucleoutde sequence encoding the Neutrokne alpha polypeptude having the amino acid sequence at positions 134-285 in Figures 1A and IB (SEQ ID NO:2).

A further embodiment of the mventon relates to an isolated nucleic acid molecule compnsing a polynucleotide which encodes the amino acid sequence of a Neutrkine-alnhaSV polpeptde Neu:rok:ne-alphaS" polypeptuuc fagm nt described herein) having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably not more than 40 conservative amuno acid substitutions, still more preferably not more than 30 conservative amino acid COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/20088 16:05 61 2 92586999 4 062837999 NO.640 064 00 o O substitutions, and still even more preferably not more than 20 conservative anuno acid substtutions. Of course, in order of ever-increasing preference, it is highly preferable f. for a polynucleotide which encodes the ammo acid sequence of a Neutrokne-alpha polypeptide to have an amuno acid sequence which contains not more than 7-10, 5-10, s 3-7 3-5.2 1 5,1 3, 10,9 8.7 6, 5, 4, 3, 2 or I conservative ammino acid substitutions.

Further embodiments include an isolated nucleic acid molecule comprsing, or alternatively consisting of a polynucleoude having a nucleotide sequence at least o 85% or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% 00 io identical to a polynucleoude selected from the group consisting of: a nucleotde O r" sequence encoding the Neurokmne-alphaSV polypepude having the complete amino acid sequence in Figures 5A and 58 positions I to 266 of SEQ ID NO:19): a nucleoude sequence encoding the Neutrokne-alphaSV polypeptde having the complete amino acid sequence in SEQ ID NO:19 excepting the N-terminal methionnc is positions 2 to 266 of SEQ ID NO:2); a nucleotide sequence encoding the predicted extracellular domain of the Neutrokine-alphaSV polypeptde having the amino acid sequence at positions 73-285 in Figures 5A and 5B (SEQ ID NO'19); a nuclcotide sequence encoding the Neurrokme-alphaSV polypeptide having the complete ammo acid sequence encoded by the eDNA clone contained in the deposit having ATCC accession number 203518, a nucleotide sequence encoding the extracellular domain of the Neutrokme-alphaSV polypeptude having the amino acid sequence encoded by the cDNA clone contained m the deposit having ATCC accession C number 203518; and a nucleotide sequence complementary to any of the nucleoutde sequences m or above.

Further, the invention includes a polynucleotide comprising, or alternatively consislng of, a sequence at least 90%, or at least 95%, identical to any portion of at least about 10 contiguous nucleotides, about 20 contiguous nucleotides, about contiguous nucleotides, or about 30 contiguous nucleotides, preferably at least about rnlle.-irtl r a t least about f nm-lItIdetr Of' the Uean.e. ft I to nucleoude 1082 in Figures IA and IB (SEQ ID NO; preferably excluding the nucleotide sequences determined from the above-listed 4 cDNA clones and the nucleotide sequences from nucleotide 797 to 1082. 810 to 1082, and 346 to 542. The invention also includes a polynucleotide compnsing, or alternatively consisting of, a COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 NO. 640 00 '1 0 sequence at least 90%, or at least 95%, identical to any portion of at least about Scontiguous nucleotides, about 20 contiguous nucleotides, about 25 contiguous t nucleoudes, or about 30 contiguous nucleoudes, preferably at least about nucleotides, or at least about 50 nucleotides, of the sequence in Figures 5A and (SEQ ID NO: 18). preferably excluding the nucleotide sequences determined from the above-listed 4 cDNA clones. The invention also includes a polynucleotide comprising, or altematively consisting of a sequence at least 90%, or at least 95%, identical to any portion of at least about 10 contiguous nucleotdes, about 20 contIguous nucleotides, about 25 contiguous nucleotides, or about 30 contguous nucleotdes, preferably at least 00 tO about 40 nucleoudes, or at least about 50 nucleoudes, of the sequence in SEQ ID NO:21, preferably excluding the nucleotide sequences determined from the above- Ci listed 4 cDNA clones. The invention also includes a polynucleotide comprising a sequence at least 90%. or at least 95%, identical to any portion of at least about contiguous nucleotdes, about 20 contiguous nucleotides, about 25 contiguous nucleotides, or about 30 contiguous nucleotldes, preferably at least about nucleotades, or at least about 50 nucleotudes, of the sequence in SEQ ID NO:22, preferably excluding the nucleotide sequences determined from the above-listed 4 cDNA clones. The invention also includes a polynuclcotide comprising a sequence at least 90%, or at least 95%, identical to any portion of at least about 10 contiguous nucleotudes, about 20 contiguous nucleotdes, about 25 contiguous nucleotides, or about 30 contiguous nucleotides, preferably at least about 40 nucleotides. or at least about 50 nucleoudes. of the sequence m SEQ ID NO:27 preferably excluding the nucleoude sequences determined from the above-listed 4 cDNA clones. The invention also mcludes a polynucleotide comprising a sequence at least 90%. or at least identical to any portion of at least about 10 contguous nucleondes, about contiguous nucleotldes, about 25 contiguous nucleotdes, or about 30 contiguous nucleotides, preferably at least about 40 nucleotides, or at least about 50 nucleotides. of the sequence m SEQ ID NO:29 preferably excluding the nucleotide sequences determined frnm the abve.-lisrd 4 rDNA Clones. In this context about" includes the particularly recited ranges, larger or smaller by several 5, 4,3,2 or 1) ammo acids.

at either extreme or at both extremes.

By a polynucleotde havng a nucleoude sequence at least, for example, "identical" to a reference nucleotide sequence encoding a Neutrokine-alpha and/or COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 966 00 0, Neutroknc-alphaSV polypeptide is intended that the nucleotide sequence of the k polynucleotde is identical to the reference sequence except that the polynucleonde sequence may include up to five mismatches per each 100 nucleotides of the reference Snucleotide sequence encoding the Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides n the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may C be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or O anywhere between those ternmnal posmions, interspersed either individually among nuelcotides m the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire nucleonde sequence encoding Neutrokme-alpha or Nentrokine-alphaSV as shown an Figures I A is and IB (SEQ ID NO-1) and Figures 5A and 5B (SEQ ID NO:18), respectively or any Neutrokne-alpha such as. for example, the Neutrokme-alpha polynucleoudes shown as SEQ ID NOs:21 22,27 or 28, or any Neutrokme-alpha or Neutrokme-alphaSV polynucleotide fragment as described herein.

As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 95%. 96%. 97%, 98% or 99% identical to. for instance, the nucleotide sequences shown in Figures IA and IB, or the nucleotide sequences shown i Figures and 5B, or to the nucleodes sequence of the deposited cDNA clones, or to any Neutrokine-alpha polynucleotude such as, for example, the Neutrokine-alpha polynucleotdes shown as SEQ ID NOs:21, 22, 27 or 28, or fragments thereof, can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix. Genetics Computer Group. University Research Park, 575 Science Drive, Madison, WI 53711).

Bestfit uses the local homology algorithm of Smith and Waterman to find the best segment of hnnmongy between twn sequhences .tAd Applied hemati. c 2:482-489 (1981)). When using Besfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present nvention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleoude COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 9258999qq 4 O?Qa>7a NO.640 967 00 63 Ssequence and that gaps in homology of up to 5% of the total number of nucleondes in the reference sequence are allowed.

SIn a specific embodiment, the identity between a reference (query) sequence (a sequence of the present mvention) and a subject sequence, also referred to as a global sequence alignment, is deternmed using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In a Ssequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global 0 sequence alignment is in percent identity Preferred parameters used in a FASTDB 00 10 alignment of DNA sequences to calculate percent identity are: Marnx=Unitary Sk-tuple=4, Mismatch Penalhy=l Jotung Penaly=30, Randomization Group Length=0, Cutoff Score=, Gap Penalty=5. Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleonde sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of intemal deletions, a manual correction is made to the results to take into consderation the fact that the FASTDB program does not account for and 3' truncauons of the subject sequence when calculating percent identity For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the above FASTDB program using the specified parameters, to arrve at a final percent identity score. This corrected score is what s used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence. as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is alignea to a 00 nase query sequence wt ucntiiinc percen t identity The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases m the query sequence) so 10% s subtracted from the COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/208 16:05 61 2 92586999 4 062837999 N0.640 D68 00 C percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a C 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject S sequence which are not matched/aligned with the query In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of 0 this embodiment.

o 10 The present application Is directed to nucleic acid molecules at least 80%, o 90%. 92%, 95%. 96%. 97%, 98% or 99% identical to the nucleic acid sequences polynucleoudes) disclosed herein those disclosed m Figures IA and IB (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNAs), irrespective of whether they encode a polypeputde having Neutrokne-alpha and/or Neutrokine-alphaSV is functional activity biological activity). In addition, the present application is also directed to nucleic acid molecules at least 80%, 85%, 90%, 92%. 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 5A and 5B (SEQ ID NO 18) or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeplide having Neutrokine-alphaSV activiy Moreover the present application is also directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 96%, 97%, 98%, 99% identical to the nucleic acid sequence shown in SEQ ID NOs:21 22, 27 or 28, irrespective of whether they encode a polypeptide having (Neutrokine-alpha activity This is because even where a particular nucleic acid molecule does not encode a polypeptide having Neutrokine-alpha and/or Neutrokme-alphaSV activity one of skill in the an would still know how to use the nucleic acid molecule, for instance, as a hybndization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having Neutrokne-alpha and/or Neutrokne-alphaSV activity includc. i.ter alia, isolani thc Ncut.okinse alpna anror Neutrokine-alphaSV gene or allelic variants thereof in a eDNA library, in sun hybridization "FISH") to meraphase chromosomal spreads to provide precise chromosomal location of the Neutrokine-alpha and/or Neutrokine-alphaSV gene, as described in Verma et al.. Human Chromosomes: A Manual of Basic Techniques, COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 P69 00 O Pergamon Press, New York (1988); and Northern Blot analysis for detecting Neutrokine-alpha and/or Neutrokine-alphaSV mRNA expression in specific tissues.

SPreferred, however, are nucleic acid molecules having sequences at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein the nucleotide sequence shown in Figures IA and IB (SEQ ID NO: 1) and the nucleic acid sequence of the deposited cDNAs, or fragments thereof), which do, in fact, encode a polypeptide having Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide functional activity biological activity). Also O preferred are nucleic acid molecules having sequences at least 80%, 85%, 90%. 92%, 00 o0 95%, 96%, 97%. 98% or 99% identical to the nucleic acid sequence shown in Figures and 5B (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNA which do. in fact, encode a polypeptide having Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide functional activity biological activity). Also preferred are nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown SEQ ID NOs:21, 22, 27 or 28 which do, in fact, encode a polypeptide having Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide functional activity biological activity).

By "a polypeptide having Neutrokine-alpha polypeptide functional activity" biological activity) and "a polypeptide having Neutrokine-alphaSV polypeptide functional activity" biological activity) are intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the extracellular domain or the full-length Neutrokine-alpha or Neutrokine-alphaSV polypeptides of the invention, as measured in a particular functional assay immunological or biological assay). For example, Neurokine-alpha and/or Neutrokine-alphaSV polypeptide functional activity can be measured by the ability of a polypeptide sequence described herein to form multimers homodimers and homotrimers) with the complete Neutrokine-alpha and/or Neutrokine-alphaSV or extracellular domain of Neutrokine-alpha and/or Neutrokine-alphaSV, and to bind a Neutrokine-alpha and/or Neutreoine-alphaSV ligand Neurckine-alpha and/or Neutrokinc-alphaSV polypeptide functional activity can be also be measured by determining the ability of a polypeptide of the invention to induce lymphocyte B cell) proliferation, differentiation or activation and/or to extend B cell survival. These functional assays can be routinely performed using techniques described herein see Example 6) and otherwise COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/2008 16:05 61 2 92586999 4 062837999 N0.640 070 0 0 o known in the an. Additionally Neutrokinc-alpba or Neutrokne-alphaSV polypepudes of the present invention modulate cell proliferation, cytotoxlcity cell survival and cell Sdeath. An m vitro cell proliferaton, cytooxicity cell survival, and cell death assay for measunng the effect of a protein on certain cells can be performed by using reagents well known and commonly available in the art for detecting cell replicaton and/or death. For instance, numerous such assays for TNP-related protein activities are described m the various references in this disclosure. Bnefly an example of such an assay involves collectmg human or animal mouse) cells and mxmng with (1) o transfected host cell-supernatant contamning Neutrokine-alpha protein (or a candidate C0 10 polypeptide) or nontransfected host cell-supematant control, and measuring the 0 effect on cell numbers or viability after incubation of certain period of time. Such cell proliferation and/or survival modulation activities as can be measure in this type of assay are useful for treating tumor, tumor metastasis, infections. autoimmune diseases, inflammation and other immune-related diseases.

Neutrokine-alpha modulates cell proliferation and differentiation in a dose-dependent manner in the above-described assay Accordingly it is preferred that a polypeptide having Neutrokane-alpha polypeptde functional activnty" biological activity) includes polypeptides that also exhibit any of the same cell modulatory (particularly immunomodulatory) activities in the above-described assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be identical to that of the Neutrokme-alpha polypeptdes, preferably a polypeptide having Neutrokne-alpha polypeptde functional activty" will exhibit substanually similar dose-dependence in a given activity as compared to the Neutrokine-alpha polypeptides the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably not more than about tenfold less activity relative to the reference Neutrokine-alpha polypeptides).

In certain preferred embodiments, a polypepude having Neutrokine-alpha polypeptide functional activity" biological activity) and a polypeptide having Neutrokme-alphaSV polypeptlde functional activtv" tc.g hiologra! lr v actv\ incude polypeptides that also exhibit any of the same B cell (or other cell type) modulatory (particularly immunomodulatory) activities described in Figures SA, SB, 9A, 9B. I I 12A, and 12B, and in Example 6.

COMS ID No: ARCS-183608 Received by IP Australia: Time 16:30 Date 2008-03-19 19/03/200 16:09 BLAKE DAWISON WALDRON LAWYERS 4 062837999 NO.197 D01 FAX TRANSMISSION No of pages (includlng this sheet) Level 36, Grosvenor Place 225 George Street Sydney NSW 2000 Australia Blake Dawson PATENT ATTORNEYS To The Commissioner of Patents IP Australia F 02 6283 7999 Human Genome Sciences, Inc New Australian divisional patent application Title: Neutrokine-alpha and neutroklne-alpha splice variant PART 2 OF 6 T 61 2 9258 6000 F 61 2 9258 6999 DX 355 Sydney Locked Bag No 6 Grosvenor Place Sydney NSW 2000 Australia www.blakedawson.co 19 March 2008 Our referenee DGC SJ 02 1430 4452 Partner David Clark T 61 2 9258 6839 clavid,lark @blakedawson.com Please check that you have received this document In lull, II not, please telephone the sender or call 61 2 9258 6000.

Conndentlallty This document is confidential and may contain legally privileged information. If you are not a named or authorised recipient you must not read, copy, distribute or act in reliance on it. If you have received this document In error, please telephone our operator immediately on 61 2 0258 6000 and return the document by mail.

Mofbourne Brsbane Perth Canberm 203937378_1 COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D02 00 o Like other members of TNF family Neutrokine-alpha exhibits activity on leukocytes including, for example, monocytes, lymphocytes B cells) and Sneutrophils. For this reason Neutrolune-alpha is active in directing the proliferation.

differentiation and migration of these cell types. Such activity is useful for immune S enhancement or suppression, myeloprotection, stem cell mobilization, acute and chronic inflammatory control and treatment of leukemia. Assays for measunng such activity are known in the art. For example, see Peters et al., Immun. Today 17:273 (1996); Young et al., J. Exp. Med. 182:1111 (1995); Caux et al.. Nature 390:258 o (1992); and Santago-Schwarz et al., Adv Exp. Med. Biol. 378:7 (1995).

00 10 Of course, due to the degeneracy of the genetic code, one of ordinary skill in the O art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%. 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence contained in cDNA clone deposited in ATCC accession no.

97768, or the nucleic acid sequence shown in Figures IA and IB (SEQ ID NO'1), or fragments thereof, will encode a polypeptide "having Neutrokine-alpha polypepude functional activity" biological activity). One of ordinary skill in the art will also immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence contained in cDNA clone deposited in ATCC accession no.

203518 or the nucleic acid sequence shown in Figures 5A and 5B (SEQ ID NO: 18) will encode a polypeptide "having Neutrokme-alphaSV polypeptide functional activity" biological activity). In fact, since degenerate variants of these nucleoude sequences all encode the same polypepude, this will be clear to the skilled artisan even i without performing the above described comparison assay It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having Neutrokne-alpha and/or Neutrokine-alphaSV activity This is because the skilled artisan is fully aware of arruno acid substitutions that are either less likely or not likely to significantly effect worene function ranlacrng one aliphatiu ammo acia w:th a secona liphatc amiino acid), as further described below Similarly polynucleotides encoding polypeptides which contain all or some ponion of the region V 142 through K 160 of SEQ ID NO:2 are likely to be valuable diagnostic and therapeutic polynucleotides with regard to detecting and/or altering COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 I9/032U08 1:09 BLPKE DAWSON WALDRON LAWJYERS 092937999NO17 Q3 NO. 197 903 Cl expression of either Neutrokine-aipha or Neurrokmne-alphaSV polynucleotsdes. In Ct addition, polyniicleotides which span the junction of amino acid residues T- 141 and 0- 142 of the Ncuu-rokine-alphaSV polypepuide shown in SEQ ID NO-019 (in between ON which the V 142 through K 160 ammno acid sequence of Neutrokine-aipha is apparently inserted), ar also likely to be useful both diagnostically and therapeutically Such T 141U& 142 spanning polyntucleoudes will exhibit a much higher likelihood of hybndization with Neutrokine-alphaSV polynueleotides than with Neurrokine-alpha polynuclcotades. A pmrtial, non-limiting, non-exclusive list of such Neutrokine-aiphaSY polypeptades which are encoded by polynucleotides of the 00 o 1 invention includes polypepuides comprng, or ahterauvely consisting of, an amino oacid sequence selected from the following: 0- 121 through S-I 163 E- 122 throu gh E-163; 0-123 through E-163. N-124 through E-163 5-125 through E 163,S5-126 through E-163; Q-1'27 through E 163, N-128 through E-163. S-129 through E 163 R 130 through H-163, N-131 through E5163; K 132 through E-163, R 133 through is E 163; A-134 through E 163,,V 135 tough E 163, Q-I136 through E- 163, G- 137 through F_-163: P 138 through E- 163 E-l139through E 163, E- 140 through E 163 T 341 through E- 163; G- 142 through E- 163; S- 143 through E- 163, Y 144 through E- 163 145 through E- 163. P-.146 through E 163; V-l147 throughl.- 163; P 148 Lhrough E 163. W 149 through E 163; L 150 through E t63, L 151 through E 163 5-152 through E- 163, F- 153 through E- 163; K 154 through E- 163 R-l155 through E- 163; G- 156 through E- 163. S- 157 through E- 163 A-I158 through E- 163, L 159 through E 163; E-160through E- 163, E- 161 through E 163. K 162zthrough E- 163 (J G-121 through K 162; G-121 through E-161 G-121 through E 160; G-121 through L 1590 -121 throughbA-158; G-121 through S-157-'G-121 through6G-156;G0-121 ?s through R 155; G-121 through K 154; G-121 throughPF-153.,G-121 through5S-152; 0- 121 through L15 G-121through L 150: 0-121 thrugh W 149G -121 through P 148; G- 121 through V 147 121 through F- 146 G3-121 through T 145- G- 121 through Y 344; 0- 121 through S- 143 0-1 21 through 6- 142; G- 121 through T 141 0- 121 through F ]An- G-12 rhrougn E-13 G-12. niArough P i 38. 0-iCa ihrougn 43-137-G-121 through Q-136;G-L21 through V 135- G-121 through A-134 G-121 through R 133,6- 121 through K 132; G-121 through N- 131 G_-121 through R 130: G-121 through S- 129- 0-121 through N-18; 0-123 through Q- 127- 0-121 through S-126; G-121 through S-125;0-121 ihrouhN-124:G-121 throuehG-123, and 0-121 COMS ID No: ARCS-183615 Received by IP Australia: Time (1-Pm) 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 ND.197 904 00 0 0 through E-122 of SEQ ID NO 19 Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Vectors and Host Cells The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, or which are otherwise engineered to produce the polypeptides of the invenon, and the production of Neutrokne-alpha and/or Neutrokmne-alphaSV O polypepudes, or fragments thereof, by recombinant or synthetic techniques.

00 In one embodiment, the polynucleotides of the invention are joined to a vector S0to a cloning or expression vector). The vector may be, for example, a phage, plasmd. viral or retroviral vector Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. The polynucleotdes may be joined to a vector containing a selectable marker for propagation in a host. Introducton of the vector construct into the host cell can be effected by techniques known in the an which include, but are not linuted to, calcium phosphate transfecton, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporatton, transduction, infection or other methods. Such methods are described n many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

Generally recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell. the ampicillin resistance gene of E. coli and S. cerewsme TRPI gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.

Such promoters can he derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate knase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled i appropriate phase with translaton initaton and termmaon sequences, and preferably a leader sequence capable of directing secren on of ranslated pro -s tne per.p4smic space or extracellular medium. Optionally the heterologous sequence can encode a fusion protein including an N-terminal identification pepide imparting desired characteristics for example, stabilization or simplified purfication of expressed recombinant product.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 00 0 C In one embodiment, the DNA of the invention Is operatively associated with an t appropriate heterologous regulatory element promoter or enhancer), such as, the Sphage lambda PL promoter, the E. coli lac, trp, phoA. and tac promoters, the 0 early and late promoters and promoters of retroviral LTRs, to name a few Other suitable promoters will be known to the skilled artisan.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycm resistance for eukaryotc cell culture and tetracycline, kanamycin or ampacillin C- resistance genes for cultunng in E. coli and other bactena. Representative examples of appropnate hosts include, but are not limited to, bacterial cells, such as E. coli, o Strepinmyces and Salmonella typhzmurrtm cells; fungal cells, such as yeast cells Saccharomyces cerewssae or Pichia pastorts (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Appropnate culture mediums and is conditions for the above-described host cells are known in the art.

The host cell can be a higher eukaryotic cell, such as a mammalian cell a human derived cell), or a lower eukaryouc cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion destred Expression from certain promoters can be elevated in the presence of certain mducers; thus expression of the genetically engineered polypepude may be controlled. Furthermore, different host cells have charactenstcs and specific mechansms for the translational and post-translational processing and modification phosphorylauon, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed. Selection of appropnate vectors and promoters for expression in a host cell is a well-known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in me nost are routine stKils in me art.

Useful expression vectors for bactenal use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation nitiation and termination signals in operable reading phase with a functional promoter.

The vector will comprise one or more phenotypic selectable markers and an origin of COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 G06 00 71 0 0 replication to ensure maintenance of the vector and to, if desrable, provide amplification within the host. Suitable prokaryonc hosts for transformation include E.

Scoli, Bacillus subtilis, Salmonella typhimurtum, and various species within the genera SPseudnoonas, Streptomyces, and Staphylococcus, although others may also be s employed as a matter of choice. As a representative, but nonlimitmg example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasnmds comprsing genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial O vectors include, for example, pKK2 23 3 (Phannacia Fine Chemicals, Uppsala, 00 0to Sweden) and GEMI (Promega Btotec, Madison, WI. USA). These pBR322 S"backbone sections are combined with an approprnate promoter and the structural sequence to be expressed. Among vectors preferred for use in bacteria include pHE4-5 i.

(ATCC Accession No. 209311 and vanations thereof), pQE70, pQE60 and pQE-9 available from QIAGEN, Inc., supra; pBS vectors, Phagescnpt vectors, Bluescript is vectors. pNH8A, pNH 16a, pNHISA, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred expression vectors for use m yeast systems include, but are not limited to, pYES2, pYDI. pTEFlZeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9 pPIC3.5, pHIL D2, pHIL SI pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlsbad, CA). Among preferred eukaryotic vectors are pWLNEO. pSV2CAT p0G44, pXTI and pSG available from Stratagene; and pSVK3, pBPV pMSG and pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to the skilled artisan. Followmg transformation of a suitable host strain and growth of the host strain to an appropriate cell density the selected promoter is induced by appropriate means temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centifugauon, disrupted by physical or chemical means, and the resulting crude extract retained for further punfication.

Microti3a cells cmploy i;n expression u pri'ocins u;u veu duupicu by any convenient method, including freeze-thaw cycling, sonicaton, mechanical disruption, or use of cell lysmg agents, such methods are well know to those skilled in the art.

In one embodiment, the yeast Pichra pastoris-is used to express Neurrokinealpha protein in a eukaryouc system. Pichm pastons is a methylotrophic yeast which COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P07 00 C can metabolize methanol as its sole carbon source. A main step in the methanol cmetabolization pathway is the oxidation of methanol to formaldehyde using This reaction as catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastons must generate high levels of alcohol oxidase due, in pan, to the relatively low affinity of alcohol oxidase for Consequently in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of o melhanol, alcohol oxidase produced from the AOXI gene comprises up to 0 approximately 30% of the total soluble protein in Pichia pastorns. See, Ellis, et o al., Mol. Cell. Brl. 5:1111 21 (1985); Koutz, PJ, et al.. Yeast 5:167 77 (1989); c- Tschopp, et aL. Nucl. Acids Res. 13:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a Neutrokine-alpha or Neutrokine-alphaSV polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOXI regulatory sequence is expressed at excepuonally high levels in Pichta yeast grown in the presence of methanol.

In one example, the plasnnd vector pPIC9K is used to express DNA encoding a Neutrokine-alpha or Neutrokine-alphaSV polypepude of the invention, as set forth herein, in a Pichea yeast system essentially as described in Pacha Protocols: Methods in Molecular Biology D.R. Higgms and J Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a Neutrokinealpha or Neutrokme-alphaSV protein of the invention by virtue of the strong AOXJ promoter linked to the Pichta pastons alkaline phosphatase (PHO) secretory signal peptide leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used i place of pPIC9K, such as, pYES2, pYDI, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9 pPIC3.5, pHIL D2. pHIL-S pPIC3.5K, and PA0815, as one skilled in the an would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcrnpion, translaton, secreton (if desired), and the like, including an inframe AUG as required.

In one embodiment, high-level expression of a helerologous coding sequence.

such as, for example, a Neutrokie-alpha or Neutrokine-alphaSV polynucleoude of the COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 D98 00 0 present inventon, may be achieved by cloning the heterologous polynucleotide of the nvention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and Sgrowng the yeast culture in the absence of methanol.

Transcnption of the DNA encoding the polypepudes of the present invention by higher eukaryotes is increased by mseing an enhancer sequence into the vector.

Enhancers are cis-acung elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase is transcnpton. Examples including the SV40 enhancer on the late side of the replication ongmt bp 100 to 270, a cytomegalovirus early promoter o enhancer, the polyoma enhancer on the late side of the replication origin, and 00 to adenovirus enhancers.

0 Vanous mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman (Cell 23.175 (1981)), and other cell lines capable of expressing a compatible vector, for example, the C127 3T3, Ss CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an ongin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site. splice donor and acceptor sites, transcnptonal termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

In a specific embodiment, constructs designed to express a poruon of the extracellular domain of the Neutrokine-alpha amno acid residues Ala-134 through Leu-285) are preferred. One of skill m the an would be able to use the polynucleoude and polypeptide sequences provided as SEQ ID NO: 1 and SEQ ID NO:2, respectively or SEQ ID NO: 18 and SEQ ID NO 19 respectively to design polynucleotide primers to generate such an expression construct.

In another embodiment, constructs designed to express the entire predicted extracellular domain of the Neutrokine-alpha anuno acid residues Gln-73 through _Au-285' amt preerred. One o: sd i tinc art would be able io use the pulynucleotide and polypeptide sequences provided as SEQ ID NO- I and SEQ ID NO:2. respectively or SEQ ID NO: 18 and SEQ ID NO: 19 respectively to design polynueleotide primers to generate such an expression construct.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D09 00 71 0 0 In additon to encompassig host cells containing the vector constructs discussed herein, the invention also encompasses prmary secondary and immortalized Shost cells of vertebrate origin, particularly mammalian ongin, that have been 0\engineered to delete or replace endogenous geneuc material Neutroklne-alpha coding sequence), and/or to include genetic material heterologous polynucleotide sequences) that is operably associated with Neutrokme-alpha polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous Neutrokne-alpha polynucleotdes. For example, techniques known in the an may be used to operably- 0 associate heterologous control regions promoter and/or enhancer) and 0 10 endogenous Neutrokmc-alpha polynucleotde sequences via homologous o recombination (see, U.S. Patent No. 5.641.670, issued June 24, 1997- 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. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435- 438 (1989), the disclosures of each of which are incorporated by reference in their entreties).

The host cells described nfra can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively cell-free translation systems can also be employed to produce the polypepudes of the invention using RNAs denved from the DNA constructs of the present invention.

The polypeptide of the invention may be expressed or synthesized in a modified form, such as a fusion protein (compnsng the polypeptide joined via a pepude bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals, but also additional heterologous functional regions. Such a fusion protein can be made by ligatmg polynucleotides of the invention and the desired nucleic acid sequence encoding the desired ammo acid sequence to each other, by methods known m the art, in the proper reading frame, and expressing the fusion protein product by methods known in the art Alternatvely such a fusion protein can be made by nmnicn yvnthetic techunues-t, g. y use oA a pcptde synthcsize. Thus, for instance, a region of additional ammo acids, particularly charged amino acids, may be added to the N-termmus of the polypeptde to improve stability and persistence m the host cell, dunng purification, or durng subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purifiation. Such COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO.197 910 00 0 regions may be removed pror to final preparation of the polypepude. The addition of Speptide moieties to polypepudes to engender secretion or excretion, to improve stability St and to facilitate purification, among others, are familiar and routine techniques n the art.

In one embodiment, polynucleotides encoding Neutrokme-alpha and/or Neutrokane.alphaSV polypeptudes of the invention may be fused to the pelB pectate 4 lyase signal sequence to increase the efficiency to expression and punfication of such polypeptides m Gram-negative bactena. See, U.S. Patent Nos. 5.576,195 and o 5,846,818, the contents of which are herein incorporated by reference m their entiretcs.

00 10 A preferred fusion protein comprises a heterologous region from 0 0 immunoglobulin that is useful to stabilize and punfy proteins. For example. EP-AO- (N 464 533 (Canadian counterpart 2045869) discloses fusion proteins compnsing various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fe part in a fusion protemn is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacoklneuc properties (EP-A 0232 262), On the other hand, for some uses it would be desirable to be able to delete the Fe pan after the fusion protein has been expressed, detected and punfied m the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery for example, human proteins, such as hIL 5 has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL See, D Bennett et al., J Molecular Recognimon 8:52-58 (1995) and K. Johanson ei a. J. Bwl. Chem. 270:9459-9471 (1995).

Polypeptides of the present invention include naturally punfied products, products of chemical synthetic procedures, and products produced by recombmant techmnques from a prokaryotic or eukaryouc host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed m recoMwean'a prounctoa procc..ur, the puypeplau Ui e pi;r IclSlesct iilvclaoiii may D glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionme residue, in some cases as a result of host-mediated processes.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P11 00 7'L 0 Polypepudes of the minvention can be chemically synthesized usming techniques known min the art see Creighton, 1983. Proteins: Structures and Molecular Prnciples, W.H. Freeman Co., and Hunkapiller, et al., 1984. Nature 310:105-111i). For example, a pepude corresponding to a fragment of the complete Neutrokine-alpha or Neutrokme-alphaSV polypeptides of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical ammo acid analogs can be Introduced as a substitution or addition into the Neutrokine-alpha or Neutrokme-alphaSV polynucleoude sequence.

o Non-classical anmo acids include but are not limted to, to the D-isomers of the 00 10 common amino acids, 2,4-diarnnobutyne acid, a-amino isobutyrnec acid, 4- 0 amminobutyric acid, Abu. 2-amino butynrc acid, g-Abu, e-Ahx, 6-amino hexanoe acid.

Aib, 2-ammino isobutyrc acid, 3-amino propiomc acid, ornahme, norleucine. norvaline, hydroxyproline, sarcosine. citrulline, homocitrulline, cysteic acid, t-butylglycine, tbutylalanine, phenylglycine. cyclohexylalaune, b-alarune, fluoro-amino acids, designer armno acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amano acids, and amino acid analogs in general. Furthermore. the amino acid can be D (dextrorotary) or L (levoratary).

The invention encompasses Nenurrokmne-alpha or Neutrokine-alphaSV polypeptides which are differentially modified dunring or after translanon, by glycosylation, acetylation, phosphorylation, amidation, denrivatization by known protectmg/blocking groups. proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modificantions may be cared out C by known techniques meincluding but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain. V8 protease, NaBH,, acetylaion, formylation, oxidation, reducton, metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-termnal or C-terminal cnls'%. anachnient of Cne.mica m.ic to the amninu aw backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or delceton of an N-terminal mcrhionine residue as a result of procaryotic host cell expression. The polypcptides may also be modified with a detectable label. such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolaton COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D12 00 -rr 0 O of the protein. In addition, polypeptides of the invention may be modified by aodination.

SIn one embodiment, Neutrokne-alpha and/or Neutrokme-alphaSV polypeptdes of the invention may also be labeled with bioun. In other related embodiments, s botnnylated Neutrokine-alpha and/or Neutru-one-alphaSV polypeptldes of the invention may be used, for example, as an imaging agent or as a means of identifying one or more Neutrokine-alpha and/or Neutrokine-alphaSV receptor(s) or other coreceptor or coligand molecules.

O Also provided by the invention are chemically modified derivatives of 000 0 Neutrokme-alpha or Neutrokrne-alphaSV which may provide additional advantages O such as increased solubility stability and in vivo or in vitro circulating time of the N polypeptude, or decreased immunogemncty (see U S. Patent No. 4,179,337). The chemical moieties for denvitzation may be selected from water soluble polymers such as polyethylene glycol. ethylene glycol/propylene glycol copolymers, is carboxymethylcellulose, dextran, polyviyl alcohol and the like. The polypepudes may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about I kDa and about 100 kDa (the term about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease m handling and manufactunng. Other sizes may be used, depending on the destred therapeutic profile the duration of sustained release desired, the effects, if any on biological activity the ease in handling, the degree or lack of antigencity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000. 1500, 2000, 2500, 3000. 3500, 4000, 4500, 5500, OCZ3. 6500, r00,. '500, ,SOU, t500, 9000, 9500. 10,000, 0.500. a 1,.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, 55,000, 60.000,65,000,70,000, 75.000, 80,000, 85.000.

90,000. 95,000, or 100,000 kDa.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 013 00 0 As noted above, the polyethylene glycol may have a branched structure.

Branched polyethylene glycols are described, for example, in U.S. Patent No.

S5,643,575; Morpurgo et aL, AppL Biochen. Brotecno. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleondes 18:2745-2750 (1999); and Calicen et aL, Buoconjug.

Chem. 10.638-646 (1999), the disclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moietecs) should be attached to the protein with consideration of effects on funcuonal or antigenic domains O of the protein. There are a number of attachment methods available to those skilled m 00 to the art, EP O 401 384, herein incorporated by reference (coupling PEG to G-CSF), o see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through armno acid residues via a reactive group, such as, a free ammo or carboxyl group. Reactive groups are those to which an activated polyethylene glycol 13 molecule may be bound. The amino acid residues havmg a free ammno group may include, for example, lysme residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutarmc acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysme group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of armno acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, bistdine, asparuc acid, glutanuc acid, or cystene residues. One or more reaction chemistines may be employed to attach polyethylene glycol to specific amino acid residues lysine.

histidine, aspartc acid, glutamec acid, or cystene) of the protein or to more than one type of amino acid residue lysme, histidine, aspartc acid, glutamic acid. cystcme and combinat;ons thereft nif the protEm.

One may specifically desire proteins chemically modified at the N-terrmnus.

Using polyethylene glycol as an illustration, one may select from a varety of polyethylene glycol molecules (by molecular weight. branching, etc.), the proportion of polyethylene glycol molecules to protein (or pepude) molecules in the reaction mix, the COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 314 00 73 0 Stype of pegylanon reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparaton separating this moiety from other monopegylated moieties if necessary) may be by punfication of the N-terminally pegylated maternal from a s population of pegylated protein molecules. Selectve proteins chermcally modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amno groups (lysne versus the N-tenrmnal) available for dervauzatton n a parucular protein. Under the appropnate reaction conditions, substantially selective derivatzation of the protein at 0 10o the N-termnus with a carbonyl group containing polymer is achieved.

SAs indicated above, pegylauon of the proteins of the invention may be C< accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker Linkerless systems for attaching polyethylene glycol to proteins are described m Delgado er at., Crr. Rev.

Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et Intern. J. ofHematol. 68:1 18 (1998): U.S. Patent No. 4,002,531 U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchlonde

(CISO

2

CH

2 CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the Invention includes protein-polyehylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2 2 .2-tnfluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, the entire disclosure of which is incorporated herem by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polvethvlene givcol is arracherd i the pirnin by a liner can also be proauccd by reaction of proteins with compounds such as MPEG-succinimidylsuccinate,

MPEG

activated with 1,1 -carbonyldiimndazole. MPEG-2.4.3-mchloropenylcarbonate, MPEG-p-ntrophenolcarbonaie, and various MPEG-succinatc derivatives. A number addiional polyethylene glycol derivatives and reaction chemistries for attaching COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 062837999 NO.197 00 o polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which Is incorporated herein by reference. Pegylated protein products produced using Sthe reaction chemistres set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each protein of the mvention the degree of substitution) may also vary For example, the pegylated proteins of the invention may be linked, on average, to 1 2,3, 45, 6,7 8,9 10. 12, 17 20, or more polyethylene glycol molecules. Similarly the average degree of substitution within ranges such as 1 3, 2-4, 3-5, 4-6, 5-7 6-8. 7-9 8-10, 9-11, 10-12, S11 13, 12 14, 13-15, 14-16, 15-17 16-18, 17-19 or 18-20 polyethylene glycol moieties 00 10 per protein molecule. Methods for determining the degree of subsututon are S discussed, for example, in Delgado ea at, Cr:t. Rev. Thero. Drug Carrier Sys. 9:249- CI 304(1992).

The Neutrokine-alpha and/or Neutrokine-alphaSV polvpeptdes can be recovered and purified by known methods which include, but are not lirmted to, ammonium sulfate or ethanol precipitation, acid extracton, anion or caton exchange chromatography phosphocellulose chromatography hydrophobic interaction chromatography affinity chromatography hydroxylapaute chromatography and lectin chromatography Most preferably high performance liquid chromatography ("HPLC") is employed for purification.

Neutrokine-alpha Polypeptides The Neutrokine-alpha and/or Neutrokme-alphaSV polypepltdes of the nvention 3 may be in monomers or multimers dimers, tmers, teiramers and higher mulumers). Accordingly the present mventon relates to monomers and multimers of the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the invention, their preparation, and compositions (preferably pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, tnmers or letramers. In additional embodiments, the multimers of the nvention are at least dimers. at least tnmers. nr at least tctrameitrs.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer. refers to a multimer containing only Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the invention (including Neutrokine-alpha and/or Neutrokine-alphaSV fragments, vanants. and fusion proteins, as described COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 P16 00 Si 0 o heremin). These homomers may contain Neutrokine-alpha and/or Neutrolunc-alphaSV polypepudes havming idenucal or different armno asd sequences. In a specific Sembodiment, a homomer of the invention is a mulmer containing only Neutrolune-alpha and/or Neutrokmne-alphaSV polypoptides havmg an identcal amino acid sequence. In another spectfic embodiment, a homomer of the invention ts a multimer contammg Neutrokmt-alpha and/or Neutrokine-alphaSV polypeptides having different ammo acid sequences. In speeific embodiments, the muluimer of the invention is a homodimer contamining Neutrokmne-alpha and/or o Neutrokunc-alphaSV polypepudes havming identical or different ammo acid sequences) 00 to or a homotrimer containing Neutrokune-alpha andfor Neutrobnc-alphaSV polypeptides having identical or different amino acid sequences). In a preferred embodiment. the mnultimer of the invention is a homorimer In additional embodiments, the homomerie multimer of the invention as at least a honodimer, at least a homotrimer. or at least a homotetramrner.

As used herein, the term heteromer refers to a multimer containing heterologous polypeputides polypeptides of a different protein) min addition to the Neutrokmne-alpha and/or Neutrokine-alphaSV polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotnmer, or a heterotcramer. In additional embodiments, the heceromeric multimer of the invention is at least a beterodimer, at least a heterotrimer, or at least a beterotetramer In a further nonexclusive embodiment, the heteromers of the invention contain ligand polypeptide sequence(s), or biologically active fragment(s) or varlant(s) thereof.

Multimers of the invention may be the result of hydrophobic, hydrophilic, iomenic and/or covalent associations and/or may be Indirectly linked, by for example, liposorne founaaon. Thus, an one embodiment, mulumers of the invention, such as, for example, homodimers or homommers, are foranned when polypepudes of the invention contact one another in solution. In another embodiment, heteromulumers of the invention.

such as, for example, heterotnmers or heterotetramers, are formed when polypeptides or the :nventor contact arubodics to the polyptpiuecs Oi the invenution (including antibodies to the heterologous polypeptde sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the Neutroline-alpha and/or Neutrokine-alphaSV polypeptides of the invention. Such covalent associations may COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 i17 00 O0 O involve one or more ammno acid residues contained in the polypepude sequence Sthat recited in SEQ ID NO:2 or SEQ ID NO-19 or contained in the polypeptide tencoded by the clones deposited t connecuon with this application). In one instance, the covalent associations are cross-linking between cystene residues located within the polypeptide sequences which interact in the native naturally occurng) polypepude. In another mstance, the covalent associations are the consequence of Schemical or recombinant mampulation. Alternatively such covalent associations may involve one or more amuno acid residues contained in the hetcrologous polypepude 0 sequence in a Neutrokine-alpha and/or Neutrokine-alphaSV fusion protein. In one 00 to example, covalent associations are between the heterologous sequence contained in a O C fusion protein of the invention (see, US Patent Number 5,478.925). In a specific example, the covalent associations are between the heterologous sequence contained in a Neutrokine-alpha-Fc and/or Neutrokne-alphaSV -Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypepude sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegern (see, International Publication No. WO 98/49305 the contents of which are herein incorporated by reference in its entirety). In another specific example, covalent associations of fusion proteins of the Inventon are between heterologous polypcptide sequence from CD40L. or a soluble fragment thereof. In another embodiment, two or more Neutrokine-alpha and/or Neutrokine-alpha polypeptides of the invention are joined through synthetic linkers C_ peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described m U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple Neutrokne-alpha and/or Neutrokine-alphaSV polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology Another method for prepanng multimer Neutrokine-alpha and/or Neutrokme- !lphasT p:ypcF:,,as IneS msantiion imvolves use of Neutroxine-aipna anaor Neutrokine-alphaSV polypeptides fused to a leucine zipper or isoleucne zipper polypeptide sequence. Leucne zipper or isoleucine zipper domains are polvpepttdes that promote multlmenzation of the proteins in which they arc found. Leucne zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D18 00 o 240:1759 (1988)), and have since been found in a variety of different proteins. Among the known leucne zippers or soleucmne zippers are naturally occurring peptidts and Sderivatives thereof that dimenze or tnmenze. Examples of leucine zipper domains suitable for producing soluble multimenc Neutrokine-alpha and/or NeutrolknealphaSV proteins are those described in PCT application WO 94/10308, hereby icorporated by reference. Recombinant fusion proteins comprising a soluble Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide fused to a peptide that dimenzes or inmenzes i solution are expressed in suitable host cells, and the resulting o soluble multimene Neutrokme.alpha and/or Neutrokne-alphaSV is recovered from the 00 10 culture supernatant using techniques known m the art.

Q Certain members of the TNF family of proteins are believed to exist m nine nc form (Beutler and Huffel. Science 264:667 1994, Banner ct al., Cell 73:431 1993).

Thus, trmen Neutroktne-alpha and/or Neutrokine-alphaSV may offer the advantage of enhanced biological activity Preferred leucne zipper moieties are those that is preferentially form tnmers. One example is a leucne zipper derived from lung surfactant protein D (SPD), as described in Hoppe et at. (FEBS Letters 344-191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring tnmeric proteins may be employed in preparing trimenc Neutrokine-alpha and/or Neutrokne-alphaSV In another example, proteins of the mvention are associated by interactions between the Flag@ polypeptide sequence contained in Flag®-Neutrokmne alpha or Flag®-Neutrokine-alphaSV fusion proteins of the invention. In a further embodiment, proteins of the invention are associated by nteractions between the heterologous polypeptide sequence contained in Flag®-Neutrokine-alpha or Flag®-NeutrokinealphaSV fusion proteins of the invention and ant-Flag® antibody The mulumers of the invention may be generated using chemical techniques known in the an. For example, polypeptudes desired to be contained in the mulumers of the invention may be chemically cross-linked using linker molecules and linker molecute engin opninm:z :at tecnntques Known m the art (sec. JS atc,.t umAer 5,478,925, which is herein incorporated by reference in is entirety). Additionally mulumers of the invention may be generated using techniques known m the arc to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multinmr (see, US COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 919 00 a 94 0 Patent Number 5,478,925. which is herein incorporated by reference m its entirely).

Further, polypeptides of the invention may be routinely modified by the addition of Scystemie or bioun to the C terminus or N-terminus of the polypeptide and techniques 0known in the an may be applied to generate multimers containing one or more of these modified polypepudes (see, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally techniques known m the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, US Patent Number o 5,478.925, which is herein incorporated by reference in its enurety).

0 0 10 Alternatively mulumers of the invention may be generated using genetic o engineering techniques known in the art. In one embodiment, polypeptides contamed in multimers of the inventon are produced recombinantly using fusion protein technology described herein or otherwise known m the an (see, US Patent Number 5,478,92>, which Is herein incorporated by reference m its entrety). In a specific embodiment, polynucleoudes coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleoudc encoding the translated product of the polypeptide in the reverse orientation from the orginal C-terminus to the N-terminus (lacking the leader sequence) (see, US Patent Number 5,478,925, which is herein incorporated by reference in us entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptdes of the mvention which contain Sa transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, US Patent Number 5,478.925. which is herein incorporated by reference n its entirety).

In one embodiment, the invention provides an isolated Neutrokune-alpha polypeptide having the anuno acid sequence encoded by the cDNA clone contained an ATCC No. 97768, or the amino acid sequence in Figures IA and IB (SEQ ID NO:2).

or a nolvocntde cnmpristng a. purtnn a ragment) or ic above polypeptuc., iI another embodiment, the invention provides an isolated Neutrokme-alphaSV polypepude having the amino acid encoded by the cDNA clone contained in ATCC No. 203518, or the amino acid sequence an Figures 5A and 5B (SEQ ID NO-19), or a polypeptide comprising a ponmon fragment) of the above polypeptides.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 00 o O Polypeptide fragments of the present invention include polypeptides comprising or altematively consisting of, an amino acid sequence contained in SEQ ID N0;2, encoded by the cDNA contained in the plasmd having ATCC accession number 97768, or encoded by nucleic acids which hybridize under stringent hybridization s conditions) to the nucleoude sequence contained in the deposited clone, or the complementary strand of the nucleode sequence shown in Figures IA-B (SEQ ID

NO-I.

Additionally polypepude fragments of the present invention include o polypeptides comprsing or alternatively consisting of, an amino acid sequence contained n SEQ ID NO:19 encoded by the cDNA contained in the plasmid having ATCC accession number 203518. or encoded by nucleic acids which hybridize C under stringent hybridization conditions) to the nucleoude sequence contained in the deposited clone, or the complementary strand of the nucleotde sequence shown in Figures SA-B (SEQ ID NO'18).

IS Additionally polypeptude fragments of the present invention include polypeptides compinsng or alternatively consisting of, an amino acid sequence encoded by nucleic acids which hybridize under hybridization conditions described herein) to the complementary strand of the nucleotide sequence shown in SEQ ID NO:21.

Polypeptide fragments of the present invention also include polypepudes comprising or alternatively consstimg of, an armno acid sequence contained in SEQ ID NO:23, or encoded by nucleic acids which hybridize under hybndization conditions described herein) to the complementary strand of the nucleoude sequence shown in SEQ ID NO:22.

In additon, polypcptide fragments of the present invention include polypeptdes comprising or alternavely consisting of, an armno acid sequence contained in SEQ ID NO:28, or encoded by nucleic acids which hybridize under hybndizaton conditions described herein) to the complementary strand of the nucleotide sequence shown mn SEQ C NO0::- Additionally polypeptde fragments of the present invention include polypepudes comprising or alternatively consisting of, an anuno acid sequence contained m SEQ ID NO:30, or encoded by nucleic acids which hybndite under COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D21 00 0 c hybndization conditions described herein) to the complementary strand of the k nucleotde sequence shown m SEQ ID NO:29 Polypeptide fragments of the present invention include polypeptides comprising or alternatively consisting of, an amino acid sequence contamed in SEQ ID NO:2, s encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize under stnngent hybrdization conditions) to the nucleoude sequence contained in the deposited clone, or shown in Figures IA and IB (SEQ ID SNO 1) or the complementary strand thereto. Protein fragments may be "free-standing, 0 or comprsed within a larger polypeptide of which the fragment forms a part or region, 00 io most preferably as a single continuous region. Representative examples of polypeptide O fragments of the invention, include, for example. fragments that compnse or alternatively consist of from about amuno acid residues: 1 to 50, 51 to 100. 101 to 150, 151 to 200,201 to 250, and/or 251 to 285 of SEQ ID NO:2. Moreover, polypeptde fragments can be at least 10, 20. 30, 40,50, 60. 70, 80, 90, 100, 110, 120, 130. 140, 150, 175 or 200 amino acids in length.

In specific embodiments, polypeptide fragments of the invention comprise, or alternatively consist of, amino acid residues: 1-46, 31-44, 47-72, 73-285. 73-83, 94-102, 148-152, 166-181, 185-209 210-221,226-237 244-249 253-265, and/or 277-284, as depicted m Figures IA and IB (SEQ ID NO:2). Polynucleotides encoding these polypeptides are also encompassed by the nvention.

It will be recognized by one of ordinary skill in the an that mutations targeted to regions of a Neutrokine-alpha polypepude of the invention which encompass the nineteen ammo acid residue insertion which is not found in the Neutrokme-alphaSV polypeptide sequence ammo acid residues Val-142 through Lys-160 of the sequence presented in Figures IA and IB and m SEQ ID NO:2) may affect the observed biological activities of the Neutrokme-alpha polypepude. More specifically a partial, non-limitmg and non-exclusive list of such residues of the Neurrokme-alpha polypeptide sequence which may be targeted for mutation includes the following amino acid residues of tne NeutrcKine-.apia polypcpudc scquence as shwi iii SEQ ID r'0:.

V 142;T 143 Q-144;D-145;C 146; L 147 Q-148; L 149- 1-150; A-131 D-152: S-153: E 154: T 155: P 156; T 157" 1-158; Q-159- and K 160. Polynucleoudes encoding Neutrokine-alpha polypeptdes which have one or more mutatons in the COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D22 0 0 region from V 142 through K 160 of SEQ ID NO:2 are contemplated. Polypeptsdes encoded by these polynucleotides are also encompassed by the mvention.

Polypeptide fragments may be "free-standing, or comprised within a larger polypeptlde of which the fragment forms a part or region, most preferably as a single Scontinuous region. Representative examples of polypeptde fragments of the mvention, include, for example, fragments that comprse or alternatively consist of from about amino acid residues: 1 to 15. 16-30, 31-46,47 55, 56-72, 73-104, 105-163, 163-188, 186-210 and 210-284 of the amino acid sequence disclosed in SEQ ID NO:2.

O Additional representative examples of polypeptide fragments of the mvention, include, 00 10 for example, fragments that comprise or alternatively consist of from about amino acid Sresidues: 1 to 143 1 150.47-143,47-150.73-143,73-150, 100-150, 140-145, 142 148.

140-150, 140-200, 140-225, and 140-266 of the amino acid sequence disclosed in SEQ ID NO'19 Moreover, polypeptide fragments can be at least 10, 20. 30.40. 50. 60, 90, 100. 110. 120, 130. 140. 150. 175 or 200 ammo acids in length. In this context, is about" means the particularly rected ranges and ranges larger or smaller by several, a few 5, 4, 3, 2 or I amino acid residues at either or both the amino- and carboxytermni. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

Additional preferred embodiments encompass polypeptide fragments comprsing, or alternatively consisting of. the predicted intraccllular domain of Neutroklne-alpha (amino acid residues 1-46 of SEQ ID NO:2), the predicted transmembrane domain of Neutrokme-alpha (amino acid residues 47 72 of SEQ ID NO:2), the predicted extiacellular domain of Neutrokmne-alpha (amrno acid residues 73-285 of SEQ ID NO:2). the predicted TNF conserved domain of Neuirokme-alpha (amino acids 191 to 284 of SEQ ID NO:2), and a polypeptide comprising, or alternatively consisting of the predicted intracellular domain fused to the predicted extracellular domain of Neutrokme-alpha (anuno acid residues 1-46 fused to amino acid residues 73-285 of SEQ ID NO;2). Polynucleotides encoding these polypeptldes are !siu :ncmpassa Cy te mvCnaUn.

Further additional preferred embodiments encompass polypcptide fragments comprising, or alternatively consistng of, the predicted intracellular domain of Neutrokine-alphaSV (amino acid residues 1-46 of SEQ ID NO-19). the predicted transmembrane domain of Neutrokme-alphaSV (amino acid residues 47-72 of SEQ ID COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRN LAWYERS 4 062837999 NO. 197 923 00 o 2 0 NO' 19), the predicted extracellular domain of Neutrokine-alphaSV (amino acid residues 73-266 of SEQ ID NO- 19) the predicted TNF conserved domain of Neutrokine-alphaSV (amno acids 172 to 265 of SEQ ID NO' 19), and a polypeptide comprising or alternomatively consisung of the predicted intracellular domamn fused to the predicted extracellular domain of Neutrolkine-alphaSV (ammo acid residues 1-46 fused to amino acid residues 73-266 of SEQ ID NO:19). Polynuleondes encoding these polypepudes are also encompassed by the mvention.

Certamn additional embodiments of the invention encompass polypeptide 0 fragments comprising, or alternatively consisting of, the predicted beta-pleated sheet 00 10 regions identified n Figure 7A. These polypeptid fragments of the invention comprise, or altematively consist of, anuno acid residues Gin-144 to Ala-151 Phe-172 to Lys-173. Ala-177 to Glu-179 Asn-183 to lie-185, Gly-191 to Lys-204, His-2 10 to Val-219 Leu-226 to Pro-237 Asn-242 to Ala-251 Gly-256 to lie 263 and/or Val-276 to Leu-284 of SEQ ID NO:2., In another, nonexclusive embodiment, these polypepude IS fragments of the invention also comprise, or altemrnatively consist of, amino acid residues Phe-153 to Lys-154. Ala-158 to Glu-160, Asn-164 to Ile-]66. Gly-172 to Lys- 185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile 244 and/or Val-257 to Leu-265 of SEQ ID NO-19- and amino acid residues Phe-42 to Lys- 43, Ala-47 to Glu-49 Asn-53 to lle-55, GIy-6 tto Pro-74 His-0SO to Val-89 Leu-96 to Pro-107 Asn-I 12 to Ala-121, Gly-126 to Ile-133 andlor Asp-146 to Lcu-154 of SEQ I) NO:23. In further nonexclusive embodiments, these polypeptide fragments of the invention also comprise, or alternatively consist of. aruno acid residues Gin-78 to Ala- Phe 106 to Lys-107 Ala- 111 to Glu-113 As- 17 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 le-197 and/or Val-210 to Leu-218 of SEQ ID NO;28; and amino acid residues GIn-78 to Ala- Phe-106 to Lys-107 Ala-I 111 to Gl- 113, Asn-117 to lIe 119 Gly-125 to Lys-138, His-144 to Val-153, Leu-160 to Pro-17 I Asn-176 to Ala-185. Gly-190 to lie-197 and/or Val-210 to Leu-218 of SEQ ID NO:30. Polynucleondes encoding these nnIvnn; -lr Igments are also pro-*dea.

A partial, non-limiting, and exemplary list of polypepudes of the invention which comprise, or altemrnatively consist of, combinations of amino acid sequences of the. invenution includes, for example. [Met-I to Lys-I 13] fused to [Leu- 114 to Thr 141] fused to [Ile-142 to Lys-160) fused to [Gly-161 to Gin-198] fused to (Val-199 to Ala- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008S 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D24 00 0 248] fused to [Gly-250 to Leu-285] of SEQ ID NO:2; Met-1 to Lys-113] fused to [Ile- 142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to S[Gly-250 to Leu-285] of SEQ ID NO:2; or (Met-I to Lys-113] fused to (Leu-114 to Thr-1411 fused to Ile- 142 to Lys-160] fused to [Gly-161 to Gln-198] fused to (Gly-250 s to Leu-285] of SEQ ID NO;2. Other combinations may include the polypeptide fragments in an order other than that recited above [Leu-l114 to Thr-141] fused to [Val-199 to Ala-248] fused to [Gly-250 to Leu-285] fused to [Iie-142 to Lys-160] of SEQ ID NO:2). Other combinations may also include heterologous polypeptide o fragments as described herein and/or other polypeptides or polypeptide fragments of 00 to the present invention [Met-l to Lys-I 13] fused to [Leu- 114 to Thr-141] fused to S(Ile-142 to Lys-160] fused to (Gly-161 to Gln-1981 fused to [Gly-250 to Leu-2851 of SEQ ID NO:2 fused to a FLAG tag). Polynucleoudes encoding any of these polypepudes are encompassed by the invention.

An additional parial, non-limiting, and exemplary list of polypeptides of the invention which comprise, or alternatively consist of, combinations of amino acid sequences includes, for example, [Met-l to Lys-1 13] fused to [Leu-1 14 to Thr-141] fused to [Gly-1 4 2 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu- 266] of SEQ ID NO'19- [Met-I to Lys-1 131 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-I to Lys- 113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gin-179] fused to [Gly- 230 to Leu.266] of SEQ ID NO: 19 Other combinations may include the polypeptide fragments in an order other than that recited above [Leu- 14 to Thr 141) fused to [Val-180 to Ala-229] fused to [Gly-230 to Lcu-266] fused to IGly-142 to Gin-179] of SEQ ID NO: 19). Other combinations may also include heterologous polypeptide 2s fragments as described herein and/or other polypeptides or polypeptde fragments of the present invention [Met- to Lys-113] fused to [Leu- 114 to Thr-141] fused to [Gly-142 to Gin-179] fused to [Gly-230 to Leu-266] of SEQ ID NO 19 fused to a FLAG tag). Polynucleotides encoding any of these polypeptides are encompassed by llte ivUention.

A further partal, non-limiting, and exemplary list of polypeptides of the invention which comprise, or alternatively consist of, combinations of amino acid sequences includes, for example, [Met-I to Lys-106] fused to [Lcu-107 to Thr-134] fused to [ile 167 to Lys-184] fused to [Gly-185 to Gln-22 4 1 fused to [Val-2 2 5 to Ala- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2009 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 00 0 9 0 272] fused to (Gly-273 to Leu-309] of SEQ ID NO:23. (Met-I to Lys-06] fused to [Glu-135 to Asn-1651 fused to Ile-167 to Lys- 1841 fused to IGly-185 to Gln-224] fused to fVal-225 to Ala-272] fused to [Gl1y-273 to Leu-309 of SEQ ID NO:23, or [Met- I to Lys-106 fused to [Leu-107 to Thr 134] fused to [Glu-135 to Asn-1651 fused s to [Ie-I 67 to Lys-184) fused to [Gly-S 85 to Gln-224] fused to [Gly-273 to Leu-309) of SEQ ID NO:23. Other comnbanations may include the polypeptide fragments min an order other than that recited above [Met-I to Lys- 106] fused to [Gly-185 to Gin- 224) fused to Ple-167 to Lys-184 fused to [Val-225 to Ala-272] fused to ILou-107 to o Thr-1341 fused to IGy-273 to Lcu-309] of SEQ ID NO;23). Other combminations may 00 In also include heterologous polypeptde fragments as described heremn and/or other O polypeptdes or polypeptide fragments of the present invention [Met-l to Lys- 106] fused to [Glu-135 to Asn-1651 fused to [lIe 167 to Lys-184] fused to [Gly-185 to Gln-224J fused to fVal-225 to Ala-272] fused to [Gly-273 to Leu-3091 of SEQ TD NO23 fused to a FLAG tag). Polynucleotides encoding any of these polypeptides are encompassed by the invention.

A further partial, non-limiting, and exemplary list of polypepudes of the invention which cornpnse, or alternatively consist of. combinations of amino acid sequences includes, for example, [Tyr- I to Lys-47] fused to [Leu-48 to Thr-751 fused to [Ile-76 to Lys-94] fused to [GIy-95 to Gln-132 fused to [Val-133 to Ala-182] fused to Gly-183 to Ala-219] of SEQ ID NO:28; [Tyr- I to Lys-471 fused to [Leu-48 to Thrfused to f11 76 to Lys-941 fused to [Val-133 to Ala-182] of SEQ ID NO:28; or [Tyr- I to Lys-47] fused to (Ile76 to Lys-94] fused to [Val-133 to Ala-182] fused to fGly- 183 to Ala-219] of SEQ ID.NO:28. Other combinations may include the polypeptade fragments in an order other than that recited above [Tyr- 1 to Lys-47] 23 fused to [Gly-183 to Ala-219] fused to [Val-133 to Ala-182] fused to [Leu-48 to Throf SEQ ID NO:28). Other combinations may also include heterologous polypepuide fragments as described herein and/or other polypeptides or polypeptide fragments of the present invention ILeu-48 to Thr 75J fused to [Ile-76 to Lys-94] fused to iGy-95 to GAt-- &32] ifiu t j'viai-433 so asa-itZj or SEQ ID IvO:28 rusco to an Fe receptor tag). Polynucleotides encoding any of these polypeptides are encompassed by the invenution.

A further partial. non-limuting and exemplary list of polypeprides of the invention which compoise, or alternatively consist of, combminations of amino acid COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P26 00 51 0 0 sequences includes, for example, [Tyr-1 to Lys-47) fused to [Leu-48 to Thr 75] fused to [Ile 76 to Lys-94] fused to [Gly-95 to Gln-132] fused to [Val-133 to Ala-182] fused Sto [Gly-183 to Ala-219] of SEQ ID N0:30; [Tyr-l to Lys-47] fused to [Leu-48 to Thr fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] ofSEQ ID NO:30; or S (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. Other combinations may include the polypeptlde fragments in an order other than that recited above [Tyr I to Lys-471 fused to [Gly-183 to Ala-219] fused to IVal-133 to Ala-182) fused to [Leu-48 to Thr- O 75] of SEQ ID NO:30). Other combinations may also include heterologous 00 io polypeptide fragments as described herein and/or other polypeptides or polypeptxde fragments of the present invention fLeu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to.Gln-132] fused to [Val-133 to Ala-182] of SEQ ID NO:30 fused to an Fe receptor tag). Polynucleotides encoding any of these polypeptides are encompassed by the invention.

Is Additional embodiments of the invention encompass Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide fragments comprsing, or alternatively consisting of, functional regions of polypeptldes of the invention, such as the Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions. Chou-Pasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Eminm surface-formnmg regions and Jameson-Wolf regions of high antigenic index set out in Figures 3 and 6 and i Table I and as described herein. In a preferred embodiment, the polypeptide fragments of the invention arc antigenc. The data presented in columns VIII, IX. XIII, and XIV of Table I can be used to routinely determine regions of Neutrokine-alpha which exhibit a high degree of potental for anigenicity Regions of high antgenicity are determined fiom the data presented in columns VIII. IX, XIII, and/or IV by choosinmg values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an Cvli-ozrumcrt In wllnn antigen recognlnon may occur In me process ot initiation ot an immune response. Among highly preferred fragments of the invention are those that comprise regions of Neutrokmnealpha and/or Neutrokine-alphaSV that combine several structural features, such as several 1. 2, 3 or 4) of the features set out above.

Polynucleotides encoding these polypeptides are also encompassed by the invention.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 127 00 0 In another embodiment, the invention provides a polypepude compnsing, or Salternatively consisting of, an epitope-barnng portion of a polypeptide of the invention.

Polynucleotides encoding these polypepudes are also encompassed by the invention.

The epitope of this polypepude portion is an immunogenic or anngenic eptope of a polypeptide of the invention. An "immunogenc epitope is defined as a part of a protem that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a proten molecule to which an antibody can bind is defined as an antgenic epitope. The number of immunogermc epiopes of a protein generally 0 is less than the number of antgenic epitopes. See, for Instance, Geysen et aL, Proc.

00 10 NatL Acad. Sc. USA 81.3998- 4002 (1983).

O As to the selection of polypeptides bearing an antgenic epitope that contain a region of a protein molecule to which an antibody can bind), it is well known an that art that relauvely short synthetic peputdcs that mimic part of a protein sequence are routinely capable of eliciting an antserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. Shmnnick. T Green, N. and Learner, R.

A. (1983) Antibodies that react with predetermined sites on proteins Science, 219:660-666. Peptides capable of eliciting proTeln-reactave scra are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins immunogenic epitopes) nor to the amino or carboxyl terminals.

Antagenic epltope-beanng peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptde of the invention. See, for instance, Wilson et aL, Cell 37-767 778 (1984) at 777 Antigemc epltope-beanng pepudes and polypeptdes of the invention preferably contain a sequence of at least 4, at least 5, at least 6, at least 7 more preferably at least 8, at least 9 at least 10, at least I1, at least 12, at least 13, at least 14.at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably between a3cut, to a-v-it 3V amain aci.s contained wiithet ihc aiuno aclu sequence o0 a polypeptde of the invention. Preferred polypeptides comprising immunogenic or antigenic epiopes are at least 10. 15, 20, 25, 30, 35, 40, 45. 50, 60. 65. 70. 75. 90, 95. or 100 ammo acid residues in length. Additional non-exclusive preferred COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 328 00 o 93 ianugenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.

SNon-limting examples of antgemc polypepdes or peptdes that can be used to 0 generate Neutrokme-alpha- and/or Neutrokine-alphaSV-specific antibodies include: a polypepude compnsing, or alternatively consisting of, amino acid residues from about Phe- 15 to about Leu-147 in Figures IA and IB (SEQ ID NO:2); a polypeptide compnsing, or alternatively consisting of, ammo acid residues from about le- 150 to about Tyr-163 in Figures IA and IB (SEQ ID NO;2); a polypeptide compnsing, or 0 alternatively consisung of. amnno acid residues from about Scr-171 to about Phe-194 in 00 1o Figures IA and 1B (SEQ ID NO:2); a polypepude compnsng. or alternatively consisting of, amino acid residues from about Glu-223 to about Tyr 246 in Figures 1A i and IB (SEQ ID NO:2); and a polypeptide comprising, or alternatively consisting of, anuno acid residues from about Ser-271 to about Phe-278 in Figures IA and IB (SEQ ID NO:2). In this context, about means the particularly recited ranges and ranges 1 larger or smaller by several, a few 5, 4, 3, 2 or I amino acid residues at either or both the ammino- and carboxy-termni. These polypepude fragments have been determned to bear antigemc epitopes of the Neutrokinc-alpha polypeptide by the analysis of the Jameson-Wolf antgenic index, as shown in Figure 3 and Table I, above.

Non-limnung examples of antlgenc polypepudes or peptides that can be used to generate Neutrokme-alpha. and/or Neutrokine-a]phaSV-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-32 to about Leu-47 m Figures 5A and 5B (SEQ ID NO 19); a polypeptde comprising, or alternatively consisting of, amino acid residues from about GluI 116 to about Ser-143 in Figures SA and 5B (SEQ ID NO:19); a polypeptude comprising, or alternatively consisting of, amino acid residues from about Phe-153 to about Tyr-173 in Figures 5A and 5B (SEQ ID NO; 19); a polypeptide comprising, or alternatively consisting of, ammo acid residues from about Pro-218 to about Tyr-227 in Figures and 5B (SEQ ID NO: 19); a polypeptlde compnsing, or alternatively consisting of, amino acid residues trom aoout Ala-t32 to avout Glin-2zi an Figures 5rn and 5B (EQ ID NO:19); a polypepide comprsing, or alternatively consistng of. amino acid residues from about Ile-244 to about Ala-249 In Figures SA and 5B (SEQ ID NO- 19); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-252 to about Val-257 in Figures 5A and 5B (SEQ ID NO:19). In this COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 929 00 S14) Scontext, about" means the particularly recited ranges and ranges larger or smaller by several, a few 5, 4, 3,2 or I ammo acid residues at ether or both the amrno- and Scarboxy-termni. Polynucleoudes encoding these polypeptides are also encompassed by the invenion. These polypeptide fragments have been determined to bear antgenmc epitopes of the Neutrokine-alphaSV polypeptide by the analysis of the Jameson-Wolf antigenic index. as shown in Figure 6 and a tabular representation of the data presented in Figure 6 generated by the Protean component of the DNA*STAR computer program (as set forth above).

o The eptope-beanng peptdes and polypeptides of the inventon may be 00 10 produced by any conventional means. See, Houghten, R. A. (1985) General O 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. Nail. Acad.

Sci. USA 82:5131 5135' this "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described m U S. Patent No. 4,631,211 to Houghten er al. (1986).

IS Eplope-beanng peptdes and polypeptides of the invention have uses that include, but are not limited to, to induce antibodies according to methods well known m the an. See. for instance. Sutcliffe et al., supra; Wilson et al., supra; Chow M. et al..

Proc. Natl. Acad, Sci. USA 82:910-914; and Bittle. F et al. J Gen. Virol.

66:2347-2354 (1985). Immunogenc epiope-beanng peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen. are identified according to methods known m the art. See, for instance, Geysen et al., supra. Further still, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amno acids or other compounds) which is a topological equivalent of the epitope a mtmotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site or a part:cular receptor of :ntrest. Similarly V.S. Patert Nu. 5,40,97 i tu Huughicu, R. A. ct al. (1996) on Peralkylated Oligopeptude Mixtures discloses linear Cl-C7-alkyl peralkylated oligopepudes and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptude that preferentially binds to an acceptor molecule of interest.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 030 00 0 Thus, non-pepide analogs of the epitope-beanng peptdes of the invention also can be made routinely by these methods.

SThe present mvention encompasses polypepudes comprising, or alternatvely Sconsstinmg of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleoude sequence contained m ATCC deposit No. 97768, or encoded by a polynucleoude that hybridizes to the complement of the sequence of SEQ ID NO: I or the cDNA sequence contained in ATCC deposit No. 97768 under hybndizauon conditions described herein).o The present invention further encompasses polynucleotide sequences comprsing, or 00 0o alternatvely consising of. a sequence encoding an epnope of a polypcptde sequence o of the invention (such as, for example, the sequence disclosed in SEQ ID NO- 1), ci polynucleotide sequences of the complementary strand of a polyoucleoude sequence encoding an epltope of the invention, and polynucleottde sequences which hybridize to the complementary strand under hybridization condiuons described herein).

Is The present invention also encompasses polypeptdes compnsing, or alternatively consisting of, an epitope of the polypeptide having an aruno acid sequence of SEQ ID NO- 19 or an epitope of the polypepude sequence encoded by a polynucleotlde sequence contained m ATCC deposit No. 203518. or encoded by a polynucleotide that hybndizes to the complement of the sequence of SEQ ID NO: 18 or the cDNA sequence contained m ATCC deposit No. 203518 under hybridization conditons described herein). The present Invention further encompasses polynucleotide sequences comprising, or alternatively consisting of, a sequence encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO: 18), polynucleonde sequences of the complementary strand of a polynucleotude sequence encoding an opitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under hybndization conditions described herein).

The term epltopes, as used herein, refers to portons of a polypeptide having anugenic or immunogenic acuviy m an animal, preieramy a mammaj, anu most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptde comprising an epitope, as well as the polvnucleotde encoding this polypeptide. An "immunogenic eptope, as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D31 00 C known an the art, for example, by the methods for generating antibodies described Sifra. (See, for example, Geysen et al., Proc. Natl. Acad. Sca. USA 81.3998- 4002 (1983)). The term antigenic epitope, as used herein, is defined as a portion of a Sprotein to which an antibody can immunospecifically bind Its antigen as determined by any method well known in the art, for example, by the Immunoassays described herein.

Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reacauviy with other antigens. Antigenie epitopes need not necessarily be immunogerc.

Ci Fragments which function as epitopes may be produced by any conventional 0 0 io means. (See, Houghten, Proc. Natl. Acad. Sci. USA 82:5131 5135 (1985), further 0 described in U.S. Patent No. 4,631.211).

In the present mvenuton, anugenic epnopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7 more preferably at least 8, at least 9 at least 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, at least 50, and, most preferably between about 15 to about 30 amuno acids. Preferred polypeptides comprnsng immunogenic or antgemc epitopes are at least 10, 15, 20, 25, 30, 35.40,45. 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusave preferred anagenic epnopes include the anugenic epitopes disclosed herein, as well as portions thereof. Antgenrc epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigemc epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigemc epatopes. Antagemc epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37-767-778 (1984): Sutcliffe et al., Science 219:660-666 (1983)).

Similarly immunogenic epitopes can be used, for example, to induce antibodies according to methods well known m the art. (See, for instance, Sutcliffe et al., supra: Wilson et al., supra: Chow et al., Proc. Natl. Acad. Scs. USA 82:910-914: and Battle et zcn. "'iro. =:2347-235-, 9t Prefrred iiimiunugenm epitopes include the immunogenic epitopes disclosed herein, as well as any combiauon of two, three, four, five or more of these immunogenc epitopes. The polypepttdes comprising one or more mumunogemc cpitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albuumn, to an animal system (such as rabbit or COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D32 0 mouse), or, if the polypepude is of sufficient length (at least about 25 armno acids), the polypptade may be presented without a carrer. However, immunogenic epitopes Scomprising as few as 8 to 10 ammno acids have been shown to be sufficient to raise antibodies capable of binding to. at the very least, linear epitopes in a denatured polypeptide n Western blotting).

Epitope-beanng polypeptudes of the present invention may be used to induce antibodies according to methods well known n the art including, but not limted to, i vivo immunization, m vitro mmunization, and phage display methods. See, e.g., o Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 00 t0 2354(1985). If in vivo Immumzaton is used, animals may be immumzed with free pepude; however, ant-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrer, such as keyhole limpet hemacyanm (KLH) or tetanus toxoid.

For instance, peptides containing cysteme residues may be coupled to a earner using a linker such as malecmldobenzoyl-N-hydroxysuccnimde ester (MBS). while other pepudes may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and rmce are immunized with either free or carrier-coupled peptides, for instance, by mtrapentoneal and/or intradennal injection of emulsions containing about 100 micrograms of peptide or carner protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.

Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anu-peptde antibodies in serum from an immunzd animal may be increased by selection of anti- pepude antibodies, for instance, by adsorption to the pepide on a solid support and elution of the selected antibodies according to methods well known in the an.

As one of skill in the an will appreciate, and as discussed above, the polypeptides of the present mvention comprising an immunogenic or antigenic epltope can be fused to other polypeptide sequences. For example, the polypeptides of the present !n.f on ma y be fued with the constant uu~iraus i mununogmouiin.s (igf, IgE, IgG, IgM), or portions thereof (CH I, CH2. CH3, or any combination thereof and portions thereof) resulting in chimeric polypepttdes. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimenc proteins consisting of the first two domains of the human CD4-polypeptde COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 33 00 0 l and varous domains of the constant regions of the heavy or light chains of mammalian C immunoglobulins. See, EP 394.827- Traunecker et al., Nature, 331:84-86 (1988).

Enhanced delivery of an antigen across the epithelial barrer to the immune system has been demonstrated for antigens insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, PCT Publicatons WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimenc structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof C alone. See, Fountoulakls et al., J. Biachem., 270:3958-3964 (1995). Nucleic 00 o to acids encoding the above epitopes can also be recombined with a gene of interest as an Sr epitope tag the hemagglutinn tag or flag tag) to aid in detection and purification of the expressed polypeptude. For example, a system described by Janknecht et al. allows for the ready punficaton of non-denatured fusion proteins expressed m human cell lines (Janknecht et al., 1991, Proc. Nail. Acad. Sci. USA s1 88:8972 897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amno-terminal tag consistig of six histidine residues. The tag serves as a matrix-bmding domain for the fusion protein. Extracts from cells infected with the recombinant vaccina virus are loaded onto Ni* nitriloacetic acid-agarose column and lustidine-tagged proteins can be selectively eluted with iumdazole-contanig buffers.

In another embodiment, the Neutrokmne-alpha and/or NeutrokinealphaSV polypeptides of the present invention and the epuiope-beanng fragments thereof arc

C--

fused with a heterologous antigen polypcptide. carbohydrate, phospholipid, or nucleic acid). In specific embodiments, the heterologous antigen is an immunogen.

2a In a more specific embodiment, the heterologous antigen is the gpl20 protein of HIV or a fragment thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alphaSV polvpeptides of the present nveton and the epiLope-bcarins fagusis iiereol are fused with polypeptide sequences of another TNF ligand family member (or biologically active fragments or variants thereof). In a specific embodiment, the Neutrokme-alpha and/or Neutrokme-alphaSV polypepudes of the present nvention are COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 934 00 Sfused with a CD4L polypepude sequence. In a preferred embodiment, the polypepude sequence as soluble.

The techniques of gene-shuffling, motif-shuffling, exon-shuffling, andlor codon-shuffling (collectively referred to as "DNA shuffling") may be employed to modulate the activities of Ncuntrokmne-alpha and/or Nutrokine-alphaSV thereby effectively generating agonsts and antagonists of Neutrokme-alpha and/or Neutrokine alphaSV See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830.721,5,834,252, and 5,837 458, and Patten, P el aL. Crr Opinion Biotechnol. 8:724-33 (1997); o Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. et at., J. MoL.

00 o Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco. R. Biotechrques 24(2):308- 13 (1998) (each of these patents and publicaous are hereby incorporated by reference).

In one embodiment, alteration of Neutrokmne-alpha and/or Neutrokme-alphaSV polynucleotdes and corresponding polypepudes may be achieved by DNA shuffling.

DNA shuffling minvolves the assembly of two or more DNA segments into a desired Neutrokme-alpha and/or Neutrokine-alphaSV molecule by homologous, or sitespecific, recombmination. In another embodiment, Neutrokane-alnipha and/or Neutrokme.

alphaSV polynucleoudes and corresponding polypepudes may be altered by being subjected to random mutagenesis by error-prone PCR. random nucleotide minsertion or other methods pnror to recombinaton. In another embodiment, one or more components, motifs, sections, parts. domaims fragments, etc., of Neutrokmne-alpha and/or Neutrokne-alphaSV may be recombined with one or more components. motifs, sections. panrts, domamns, fragments, etc. of one or more heterologous molecules, in preferred embodiments, the beterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterorner LT-alpha2-beta), OPOL, FasL, CD27L, CD30L, CD4DL, 4-iBBL, DcR3 TNF-gamma (International Publication No. WO 96/14328). AIM-i (International Publication No. WO 97/33899), AIM-Il (International Publication No.

WO 97/34911), APRIL Exp. Med. 188(6):1 185-1190). endokne-alpha (Intemratina! Dnblicatt. No. WO 98/0 7 8S0), OX, 0X0 uua am;' vc guwtb iutuor and soluble forms of Fas, CD30, CD27 CD40 and 4-IBB, TR2 (Intemational Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904) DR4 (International Publication No. WO 98/32856), TR5 (International Publication No.

WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 00 c Publication No. WO 98/41629). TRANK, TR9 (International Publicaton No. WO S98156892), TRIO (International Publication No. WO 98/54202),312C2 (International SPublication No. WO 98/06842), TR12, CAD and v-FLIP In further embodiments, the heterologous molecules are any member of the TNF family In a preferred embodiments, Neutrokane-alpha and/or Neutrokzne-alphaSV polypeptides of the invention (inicuding biologically active fragments or varants thereof), are fusedwith soluble CD40L polypeptdes, or biologically acitve fragments or variants thereof.

o To improve or alter the characteristics of Neutrokme-alpha and/or 0 0 10 Neutrokme-alphaSV polypeptdes, protean engineering may be employed.

op Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "mutins including single or multiple armno acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show enhanced activity or increased stability In addition, they may be purified in is higher yields and show better solubility than the corresponding natural polypepide, at least under certain purification and storage conditions. For instance, for many proteins.

including the extracellular domain or the mature form(s) of a secreted protein. n is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance. Ron et al., J.

Biol. Chem., 268.2984-2988 (1993) reported modified KGF proteins that had heparm binding activity even f 3. 8, or 27 amino-terminal amino acid residues were missing.

In the present case. since the protein of the invention is a member of the TNF K polypeptade family deletions of N-termnal ammno acids up to the Gly residue at position 191 an Figures IA and IB (SEQ ID NO:2) may retain some biological activity such as, for example, the ability to stimulate lymphocyte B cell) proliferation, differentation, and/or activation, and cytotoxlcity to appropriate target cells.

Polypepudes having further N-terminal deletions including the Gly residue would not be expected to retain biological activities because it is known that this residue in TNF-related polypepti:ds is n te beginnig of the uLwLvcu uuiiia requiro lor biological activities. However, even if deleton of one or more amino acids from the N-temunus of a protein results mn modification or loss of one or more biological functions of the protein, other functional activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 197 P36 00 o 401 0 complete or extracellular domain of the protein generally will be retained when less than the majority of the residues of the complete or extracellular domain of the protein arc removed from the N-termnus. Whether a particular polypeptide lacking O' N-terminal residues of a complete protein retains such immunologic activities can S readily be determined by routne methods described herein and otherwise known m the art.

Accordingly the present invention further provides polypeptides having one or more residues deleted from the ammo terminus of the anuno acid sequence of the 0 Neutrokme-alpha shown m Figures IA and IB (SEQ ID NO:2). up to the glycine 0 0 10 residue at position 191 (Gly-191 residue from the ammnuno terminus), and polynucleoldes O encoding such polypeptudes. In particular, the present invention provides polypepudes comprising, or alternatively consisting of. the ammno acid sequence of residues n' 285 of SEQ ID NO:2. where n' as an integer in the range of the amino acid position of amno acid residues 2 190 of the amino acid sequence in SEQ ID NO:2.

Is Polynucleotides encoding these polypeptdes are also encompassed by the invention.

More in particular the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of. an aminuno acid sequence selected from the group consisting of residues 2 285. 3-285, 4-285. 5-285, 6-285, 7 285, 8-285, 9-285, 10-285, 11 285, 12 285. 13-285, 14-285, 15-285, 16-285. 17-285, 18-283. 19-285.

20-285, 21 285, 22 285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285.

30-285, 31 283. 32 285, 33-285, 34-285, 35-285, 36-285, 37-283. 38-285. 39-285.

40-285.41 285,42 285, 43-285, 44-285, 45-285,46-285,47 285,48-285,49-285, 50-285, 51 285, 52 285, 53-285, 54-285, 55-285, 56-285.57 285, 58-285, 59-285. 60-285. 61 285. 62-285. 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71 285, 72 285, 73-285, 74-285, 75-285, 76-285, 77-285. 78-285, 79-285, 80-285, 81 285, 82 285 83-285. 84-285. 85-285, 86-285. 87 285. 88-285. 89-285.

90-285, 91 285,92 285, 93-285,94-285,95-285,96-285,97 285,98-285,99-285 100-285, 101 285. 102 285, 103-285, 104-285 105-285, 106-285, 107-285. 108-283 10Q-2S5. 0-285, 'I 285 '1285, 85, 5, 2 8v-285, 5-S-285 i. o-285. 7 283.

118-285. 1 19-285. 120-285, 121-285. 122 285, 123-285, 124-285, 125-285, 126-285.

127-285, 128-285, 129-285, 130-285, 131 285, 132 285. 133-285. 134-285. 135-283.

136-285, 137-285. 138-285, 139-285, 140-285, 141 285. 142 285. 143-285, 144-285, 145-285, 146-285, 147.285, 148-285. 149-285, 150-285. 151 285, 152 285, 153-283, COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 937 00 N 154-285, 155-285, 156-285, 157-285, 158-285, 159-285. 160-285. 161 285, 162 285, C 163-285. 164-285. 165-285. 166-285, 167-285. 168-285. 169-285. 170-285. 171 285.

172 285, 173-285. 174-285, 175-285, 176-285, 177-285. 178-285, 179-285 180-285.

S181 285, 182 285, 183-283. 184-285, 185-285. 186-285, 187 285, 188-285, 189-285, and 190-285 of SEQ ID NO:2. Polypeptides encoded by these polynucleondes are also encompassed by the invention. The present invention is also directed to nucleic acid molecules comprsing, or alternatively consistng of, a polynucleoude sequence at least _1 80%. 85%. 90%, 92%. 95%. 96%. 97%. 98% or 99% identical to the polynucleotide O sequence encoding the Neu rokine-alpha andlor Neutrokne-alphaSV polypeptides 00 in described above. The present invention also encompasses the above polynucleotide o sequences fused to a heterologous polynucleon de sequence. Polypeptdes encoded by C these nucleic acids and/or polynucleoude sequences are also encompassed by the invention, as are polypepudes comprising, or alternatively consistng of. an aimno acid sequence at least 80%, 85%, 90%, 92%, 95%. 96%, 97%, 98% or 99% identical to the Is ammno acid sequence described above, and polynucleotides that encode such polypeptides.

Furthermore, since the predicted extracellular domain of the Neutrokme-alpha polypepudes of the invention may itself elicit biological activity deletions of N- and C-ternunal amino acid residues from the predicted extracellular region of the polypepude (spanning positions Gln-73 to Leu-285 of SEQ ID NO:2) may retain some biological activity such as, for example, ligand binding, stimulation of lymphocyte B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities. However, even if deletion of one or C more amino acids from the N-terminus of the predicted extracellular domain of a Neutrokne-alpha polypeptide results m modification of loss of one or more biological functions of the polypeptide. other functional activities may still be retained. Thus, the ability of the shortened polypeptdes to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generaliv will hA retained when less than the m-jory of the residui u tilie compete or mature Or extracellular domains of the polypeptides are removed from the N-terminus.

Whether a particular polypeptlde lacking N-terminal residues of a complete polypepude retains such immunologic activities can readily be deternuned by routine methods described herein and otherwise known in the an.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/20oe 19/032809 16:09 BLAKE DAWJSON WAPLDRON LAWJYERS 4 062837999 h~S 3 NO. 19? P3e 00 o A101 Accordingly the present invention further provides; polypeptades having one or Ct more residues deleted from the amino terminus of the aminro acid sequence of Neutrokmne-alpha shown in SEQ ID NO:2, up to the glycinie residue at position number O 280, and polynuicleoudes encoding such polypepuides. In particular, the present itnvention provides polypptds comprising. or alternatively consisting of, the AMino acid sequence of residues n- 295 of SEQ ID NO:2, where n- is an integer in the range of the amiuno ncid position of amino acid residues 73-280 in SEQ ID NO:2, and 73 is the position of the first residue from the N-termnus of the predicted extracellular o doma of the Neurokine-aipha polypepuide (disclosed in SEQ I) NO:2).

00 10 Polynucleotides encoding these polypeptdes are also encompassed by the inventiono More in particular. in certin embodiments, the invention provides potynucleotides Clencoding polypeptides com npising, or alternatively consisting of, an amino acid sequence selected fromn the group consisting of residues of Q-73 to L 285 G-74 to L 285;1 D-75 to L 285; L 76 to L 285- A-77 to L 28za: S-78 to L 285; L 79 to L 285:.

k-8O 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 285: H-86 to L 285; H1-87 to L 285; A-BR 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 totL 285; G-94 to L 285' A495 to L 285- G-96 to L 285: A-97 tot. 285; P-98 to L 285- K-99 to L 285; A-lO00 to L 285: G-1IOf to L L 102 to L285; E-103to L285; E-104to L 2S;A-O0mo L285; P 106 toL 285; A107 to L 25; V 10 toL 285; T109 to L 285,A-lI o L 285- -1l11toL25; L 112 to L 2S:K 113 to L285;I1-114 to L285;F-15 to L285E16 toL 285; Pl117 o L 285:Pl118to L 293A-1 19to L 25;P120 to L2850-12Ito LZ28; E-12lto L 285-,G-I23 toL 285; N-124to L 285; S-12S tot. 285, S-l26 toL 285; Q-l127 toL 285- N-12B to L 285;, S-I129 toL 285; R-13Oto L 2S5;N- 131 to L 285; K 132to L 285; R-133 to L 285,;A- 134 to L285: V 135 to L 285 Q-l136 toL 285; (3-137to L 285, P 138 to L 285; E- 139 to L 285; E-140 totL 285-;T 141 to L 285- V 142to L 2S5;:T 143 toeL 285- Q-144 to L 285; D- 145 totL 295- C I46 to L 285: L 147 coaL 285; Q-l48to L 285; L 149to L 285;I-l SO totL 285- A-151 to L 283; V-I152 to L 285: S- 153 to L 285- F-i I54:n L 285-? T355 to L 2 85;C0 15610o L T 157 to1285;1-I5Sto L 285-Q-159to L 2S5;K 160 to L285 G-161 to L 285; S-162ito L285 Y 163 to1L285:1T 164to L285F-l65 to L285; V-166to L 285- Pl167to L 285;W- 168to L285;:L169 to L28; L 17O0 oL 285-S-1711toL 285 F l72 toL 2S5; K 173 to L 285; R 174 to L285; G-l17Sto L 285;:S-I176 toL 285' COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P39 00 o -4o 4 A04 cN~ ~A-177 to L285; L 178 to L285;E-179 to L285: E 180 to L285; K 181 to L285- SE-182 to L285: N-183 to L 285;K 184 to L285;1-185 to L285;L 186 to L. 285; V 187 to L 285; K 188 to L 285; E 189toL 285: T 190 to L 285; 0-191 to L 285; Y 192 to L 285; F 193 to L 285- F-194 to L 285- 1-195 to L 285; Y-196 to L 285; s 0-197 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- 0-209 to L 285; H-210 to L 285; L 211 to L 285; 1-212 to L 285 Q-213 to L 285: R-214 to L 28S5: K 215 to L 285: K 216to L 285; o V 217 1o L 285- H-218 to L 285- V 219 to L 285; F-220 to L 285; 0-221 to L 285' 00 io D-222 to L 285- E223 to L 285- L 224 to L 285: 5-225 to L 285; L 226 to L 285- SV 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- 1-233 to L 285; Q-234 to L 285; N-235 to L 28D; 1-236 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 285is S-247 to L 285- A-248 to L 285- 0G-249 to L 285:1-250 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 285:0 G-256 to1 L 285; D-257 to L 285' E 258 to L 285' L 259 to L 285: Q-260 to L 283; L 261 to L 285: A-262 to L 285' 1-263 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; 1-270 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' F-279 to L 285; and G-280 to L 285 of SEQ 1D NO2. Polypeptdes encoded by these polynucleotdes are also encompassed by the invention. The present invention is also directed to nuclete acid molecules comrnpnsng.

or alternatively conssting of, a polynucleoude sequence at least 80%. 85%.90%, 92%.

95%. 96%, 97%, 98% or 99% idenucal to the polynucleotide sequence encoding the Neuurokane-alpha and/or Neutrokline-alphaSV polypepudes described above. The present invention also encompasses the above polynucleoude sequences fused to a heterologous polynucleoude sequence. Polypepudes encoded by these nucleic acids and/or nolynucleotde seauencet ari also encompassed by the :nvenuon. as are 3o polypepudes comprising, or alternatively consistmng of, an amino acid sequence at least 85%, 90%. 92%, 95%.96%. 97%. 98% or 99% identical to the amino acid sequence described above, and polynucleotudes that encode such polypeptides.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 052837999 NO.197 00

N

c Highly preferred embodiments of the invention are directed to nucleic acid C3 molecules comprising, or alternanvely consisung of a polynucleotide having a nucleotde sequence at least 80%, 85%, 90% dentical and more preferably at least 96%, 97%. 98%. 99% or 100% identical to a polynucleotde sequence encoding the Neutrokme-alpha polypeptde having the ammno acid sequence at positons 134-285 m in Figures IA and 1B (SEQ ID NO;2). Preferred embodiments of the invenuon are directed to nucleic acid molecules comprising, or altematively consisting of a polynucleotide having a nuclcoudc sequence at least 90% identical to a polynucleoude 0 sequence encoding the Neutrokin-alpha polypeptide having the amin acid sequence 00 to at positions 134-285 in Figures IA and IB (SEQ ID NO:2). More preferred Oembodiments of the invention are directed to nucleic acid molecules comprsing, or alternatvely consisting of a polynucleoude having a nucleotide sequence at least 95% identical to a polynucleotide sequence encoding the Neutrokme-alpha polypepude having the anuno acid sequence at positions 134-285 in Figures IA and IB (SEQ ID is NO:2). More preferred embodiments of the invention are directed to nucleic acid molecules comprising, or alternatvely consisting of a polynucleotade having a nucleoude sequence at least 96% identical to a polynucleoude sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134-285 in Figures IA and IB (SEQ ID NO:2).

Additionally more preferred embodiments of the inventon are directed to nucleic acid molecules comprising, or alternatively consisung of a polynucleoude having a nucleoude sequence at least 97% to a polynucleoude sequence encoding the Neutrokine-alpha polypepttde having the amino acid sequence at positions 134-285 in Figures IA and I B (SEQ ID NO:2). Additionally more preferred embodiments of the invention are directed to nuclei acid molecules compnsmg, or alternatively consistinmg of a polynucleotide having a nucleoude sequence at least 98% to a polynucleotide sequence encoding the Neutrokine-alpha polypeptide having the amino acid sequence at positions 134.285 m Figures 1A and IB (SEQ ID NO:2). Additionally more preferteu enibudinciis the Iuventliun aie ditetcu to tucciC accu muteCUlc comprising, or alternatively consisting of a polynucleotde having a nucleotde sequence at least 99% identical to a polynucleotide sequence encoding the Neutrokne alpha polypepude having the amino acid sequence at positions 134-285 an Figures 1A and IB (SEQ ID NO:2).

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D41 00 ob 0 6 C( In specific embodiments, a polypeptide comprising, or alternatvely consisting ct of, one of the following N-terminally deleted polypeptide fragments of SNeutrolune-alpha and/or Neutrokne-alphaSV are preferred; amino acid residues Ala-71 through Leu-285, anuno acid residues Ala-8 I through Len-285, amnno acid residues Leu-l 12 through Leu-285. amino acid residues Ala-134 through Leu-285, anuno acid residues Leu-147 through Leu-285, and amino acid residues Gly-161 through Lou-285 of SEQ ID NO.2. Polynucleoudes encoding these polypeptides are _also encompassed by the invention.

O Similarly many examples of biologically functional C-terrmnal deletion 00 oi muteins are known. For instance, Interferon gamma shows up to ten times higher Sactivities by deleting 8-10 ammo acid residues from the carboxy terminus of the protein (Dbeli at J. Biotechnology 7-199-216 (1988). Since the present protein is a member of the TNF polypeptde family deletions of C-ierminal amino acids up to the leucne residue at position 284 are expected to retain most if not all biological acinvty is such as, for example, ligand binding, the ability to stimulate lymphocyte B cell) proliferation, differentiation, and/or actvation, and modulation of cell replication.

Polypeptides having deletions of up to about 10 additional C-terminal residues up to the glycine residue at position 274) also may retain some activity such as receptor binding, although such polypeptides would lack a portion of the conserved TNF domain which extends to about Leu-284 of SEQ ID NO:2. However, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activites may still be retained. Thus, the ability of the shorened protein to induce and/or bind to C antibodies which recognize the complete or mature protein generally will be retained when less than the majonty of the residues of the complete or mature protein are removed from the C-terminus. Whether a particular polypeptde lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known m the art.

Accrdigly the present invention ftrth'i pirviucs puiypcpLiuLc nvadi;n ue or more residues deleted from the carboxy terminus of the amino acid sequence of the Neutrokine-alpha polypeptlde shown m Figures IA and IB (SEQ ID NO:2), up to the glycine residue at position 274 (Gly-274) and polynucleotides encoding such polypepudes. In particular, the present invention provides polypeptides comprising, or COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P42 00 ahemrnatively consisting of, the amino acid sequence of residues I-m' of the amino acid sequence in SEQ ID NO:2, where m' is any integer in the range of the armno acid Sposiion of amino acid resrdues 274-284 in SEQ ID NO:2. Polynucleoudes encoding these polypeptades are also encompassed by the mvnetion. More in partcular, the invention provides polynucleotides encoding polypeptides comprising. or alternatvely consisting of, an ammo acid sequence selected from the group consisting of residues l1 274, 1 275. I 276. 1 277 1-278, 1 279 1-280, 1 281, 1 282. 1-283 and 1-284 of SEQ ID NO:2. Polypeptdes encoded by these polynucleotides are also encompassed O by the invention. The present invention is also directed to nucleic acid molecules 00 10 compnsing, or alteratively conssting of, a polynucleotude sequence at least 0 85%, 90%. 92%, 95%, 96%. 97%, 98% or 99% identical to the polynucleotde l sequence encoding the Neutrokine-alpha and/or Neutrokine-lphaSV polypeptides described above. The present invention also encompasses the above polynucleolide sequences fused to a helerologous polynucleotide sequence. Polypeptides encoded by s1 these nucleic acids and/or polynucleotde sequences are also encompassed by the invention, as are polypeptides comprsing, or alternatively consisting of, an ammo acid sequence at least 80%, 85%, 90%. 92%. 95%, 96%. 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotides that encode such polypeptides.

Also provided are polypepudes compnsing, or alternatively consisting of, one or more amino acids deleted from both the ammo and the carboxyl termini, which may be described generally as having residues of SEQ ID NO:2, where n' and m' are integers as defined above. Also included are a nucleotlde sequence encoding a polypeptide comprising, or alternatvely consisting of, a portion of the complete Neutrokine-alpha amino acid sequence encoded by the deposted cDNA clone contamed in ATCC Accession No, 97768 where this portion excludes from I to 190 amino acids from the amino terinnus or from I to II amino acids from the C-temunus of the complete amuno acid sequence (or any combinaton of these N-terminal and C-term na dclu ons) cucodcd .b the cDNr clone in the depusiueu plasriu.

Polynucleotides encoding all of the above deletion polypepudes are encompassed by the invention.

Similarly deletions of C-terminal amino acid residues of the predicted extracellular domain of Neutrokme-alpha up to the leucine residue at position 79 of COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 197 943 00 8 g SSEQ ID NO:2 may retain some biological activity such as, for example, ligand t binding. stimulation of lymphocyte 8 cell) proliferation, differentuaion. and/or activation, and modulation of cell replication or modulation of target cell activities.

Polypeptides having further C-terrmnal deletions including Leu-79 of SEQ ID NO:2 s would not be expected to retain biological activities.

However even if deletion of one or more amno acids from the C-termmns of a polypeptide results in modification of loss of one or more biological functions of the polypeptde, other functional activities may still be retained. Thus, the ability of the 0. shortened polypeptade to induce andlor bind to antibodies which recognize the 00 to10 complete, mature or extracellular forms of the polypeptide generally will be retained O when less than the majority of the residues of the complete, mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypepude lacking C-terminal residues of the predicted extracellular domain retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the ant.

Accordingly the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the arrino acid sequence of the predicted extracellular domain of Neutroklne-alpha polypepude shown in SEQ ID NO:2, up to the leucine residue at position 79 or SEQ ID NO:2. and polynucleotides encoding such polypeptides. In particular, the present invention provides polypepudcs comprising, or alternatively consisting of, the amino acid sequence of residues 73-m- of the amino acid sequence in SEQ ID NO:2, where m- is any integer in the range of the amno acid position of amino acid residues 79 o 285 in the amino acid sequence m SEQ ID NO:2, and residue 78 as the position of the first residue at the C terminus of the predicted extracellular domain of the Neutrolone-alpha polypepude (disclosed in SEQ ID NO:2). Polypeptides encoded by these polynucleotides are also encompassed by the invention. More i particular, in certain embodiments, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of. an ammo acia sequence ssctea rom tne group consisung o0 rst'aues Q-73 tu Ltu-285, 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 1-270; 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 1-263.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO.197 944 00 co C' 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 0-256; Q-73 to E-255; Q-73 to E-254. Q-73 to L 253 Q-73 to K 252: C Q-73 to A-251 Q-73 to 1.250; Q-73 to G-249' Q-73 to A-248, 0-73 to 5-247- Q-73 to Y 246; Q-73 to C 245' Q-73 to 5-244; Q-73 to N-243, Q.73 to N-242; Q-73 to P 241 s 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 C 232; Q-73 to R 231 Q-73 to F-230; Q-73 to L 229- Q-73 to T 228; 0-73 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-22: Q-73 to G-221 Q-73 to F-220 Q-73 to V 219o Q-73 to H-218; Q-73 to V 217- Q-73 to K 216; q73 to K 215; Q-73 to R 214 Q.73 to 00 io Q-213 Q-73 to 1.212: Q-73 toL 211 Q-73 to H-210 Q-73 to 0-209- Q-73 to M-208: o Q-73 toA-207* Q-73to Y 206; Q73to T 205; Q-73to K 204 Q-73 toD-203 Q-3 to C']T 202; Q-73 to Y 201 0-73 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 1-195: Q-73 to F-194; Q-73 to F-193 Q-73 to Y 192; Q-73 to 0-191 Q-73 to T 190; Q-73 to E-t89- Q73 to K 188; Q-73 to V 187' Q-73 to L 186; Q-73 to 1-185; 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 5-176: Q-73 to G-175; Q-73 to R 174; 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 16; Q-73 to F-165; Q-73 to T 164; Q-73 to Y 163, Q-73 to S-162; Q-73 to 0-161 Q-73 to K 160; Q-73 to 0-159- Q-73 to 1- 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 1-150; Q-73 to L 149' Q-73 to Q-148, Q73 to L 147' Q-73 to C 146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T 143.0 -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; Q73 to A-134: Q-73 to R 133; 0-73 to K 132; 0-73 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; 0-73 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-lI15; 0-73 to 1-114: Q-73 to K 1l3, Q-73 to L 112; Q-73 toG-111 Q-73 to A-110; Q-73 to T 109, 0-71 tn ll IS, Q73 to A -107- Q-73 to D '1n; Q-"3 to P-105 Q-73 tu E AUQ, Q-73 to E-103; Q-73 to L 102; Q-73 to G-101 Q-73 to A-100O; 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 0-73 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 COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2009 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 00 l and Q-73 to L 79 of SEQ ID NO:2. Polypepuldes encoded by these polynucleotides are also encompassed by the invention. The present mvention is also directed to nucleic Sacid molecules compnsmg, or alternatively consisting of, a polynucleotide sequence at 0least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Neutrolme-alpha and/or Neutrolune-alphaSV polypeptdes described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence.

Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also O encompassed by the Invention, as are polypeptides comprising, or alternatively 00 10 consisting of, an amino acid sequence at least 80%. 85%, 90%, 92%, 95%. 96%, 97%.

0 S98% or 99% identcal to the amino acid sequence described above, and polynocleotides (C that encode such polypepudes.

The invention also provides polypepides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular is domain of Neutrokine-alpha, which may be described generally as having residues of SEQ ID NO:2 where n- and mn are integers as defined above.

In another embodiment, a nucleoude sequence encoding a polypeptide consisting of a portion of the extracellular domain of the Neutrokmn-alpha amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC accession no. 97768, where this portion excludes from I to about 206 ammino acids from the amino termunus of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC accession no. 97768, or from I to about 206 amino acids from the carboxy terminus of the extracellular domain of Q the amuno acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC accession no. 97768, or any combination of the above armno terminal and carboxy termnal delctions, of the entire extracellular domain of the ammo acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC accession no. 97768.

As mentioned abnve, even if deletion of one or more ariiu uads f1roni tlt N-terminus of a polypepude results in modification of loss of one or more functional activities biological activity) of the polypeptde. other functions or biological activiies may still be retained. Thus. the ability of a shortened Neutroklne-alpha mutein to induce and/or bind to antibodies which recognize the full-length or mature COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 197 D46 00 C forms or the extracellular domain of the polypeptide generally will be retained when Sless than the maornty of the residues of the full-length or mature or extracellular domain of the polypeptide are removed from the N-termnus. Whether a parucular Spolypeptide lacking N-terminal residues of a complete polypepntde retains such immunologic activities can readily be determined by routine methods described herein and otherwise known m the art. It is not nlikely that a Neutrokine-alpha mutein with a large number of deleted N-termnnal amino acid residues may retain some functional biological or unnunogenic) activities. In fact, pepudes composed of as few as 0 six Neutrolone-alpha amino acid residues may often evoke an immune response.

0 0 o Accordingly the present mvention further provides polypeptides having one or 0 more residues deleted from the amino terminus of the predicted full-length ammro acid sequence of the Neutrokne-alpha shown in SEQ ID NO:2, up to the glycne residue at position number 280 of the sequence shown SEQ ID NO:2 and polynucleoudes encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n' 285 of the sequence shown In SEQ ID NO:2, where n is an integer m the range of the amino acid position of ammo acid residues 1 to 280 of the amino acid sequence in SEQ ID NO:2.

More in particular, the invention provides polynucleotides encoding polypepudes comprising, or alternatively consisting of, an armno acid sequence selected from the group consisting of residues of D-2 to L 285; 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; 1-30 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 2 5' to L 283.. 52 w 285; 53 to t, 283: Lt 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 285to L 285: L-61 to L 285: T-62 to L 285; V-63 to L 285: V-64 to L 283; S-65 to L 285: F-66 to L 285: Y-67 to L 285; Q68 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 28. L 76 to COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DLAWSON UWALDRON LLAWYERS 4 062837999 NO.19? P4? 00 0 -4.

0

CI

L 285; A-77 to L 285- 5-78 to L 285 L 79 to L 285' R-80 to L 285- A-S1 toL 285 c E-82 to L 285:1-83 to L 28a; Q-84 to L 285: G-5 to L 285; H-86 to L 285; H-87 to SL-285; A-88 to L 285' E-89 to L 285: K-90 to L 285- L91 to L 285- P-92 to L 285: A-93 to L 285; G-94 to L 285; A-95 to L 285: 0-96 to L 285- A-97 to L 285; P-98 to S L 285; K-99 to L 285' A-100 to L 285' G-I01 to L 285- L 102 to L 285; E.103 to L 285; E- 104 to L 285; A-0lS 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 io L 285;G-1 to L 285 L 112 to L 285; K 113 to SL 285; 1-114 to L 285; F- 115 to L 285- E- 16 to L 285- P 117 to L 285- P 118 to SL 285;A-119to L 285;P 120to L 285; G-121 toL 285; E- I22 toL 285:0-123 to 00 10 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 toL 285-N-131 to L 5;K 132 toL 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 283; V 142 to L 285- T 143 to L 285; Q-144 to L 285; D-145 to L 283- C 146 t L 285; L 147 to L 285: Q-148 to L 285; L 149 to L 285- 1-150 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 28t; P 156 to L 285- T 157 to L 285; 1-158 to L 285;Q-159 to L 285- K 160 to L 285- 0-161 to L 28; S-162 to L 285; Y 163 to L 285; T 164 to L 285; F-165 to L 285; V- 166 to L 285; P 167 to L 285; W-168 to L285;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 L285; E-179to L 285 E-180 to L 285 K 18lI toL285;E-182 to L 285:N-183 to L 285;K 184 to L 285; 1-185 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- 1-195 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 285:1-212 to L 285; Q-213 to L 285: R 214 o L 285- K 215 toL 285- K 216toL 285- V 217 to L 285; H-218 to L 285; 2 9 to 285 F-220 u Z85' G-:2ji 10 L 28a; D-2:2 to L 2 3- E-ZJ to L 285; L 224 t L 28- S-225 to L 28a- L 226 to L 285; V 227 to L 285- T 228 to L 285; L 229 to L 285- F-230 to10 L 285- R-231 to L 285: C 232 to L 285; 1-233 in 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 to1 L 285: P 24 110 L 285: N-242 to L 285; N-243 to COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D48 00 C L 285; S-244 to L 285; C 245 to L 285; Y 246 to L 285; 5-247 to L 285; A-248 to C L 285; G-249 to L 285; 1-250 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- -256 to L 285: D-257 to L 285; E-258 to O L 285: L 259 to L 285; Q-260 to L 285; L 261 to L 285; A-262 to L 285; 1-263 to SL 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- 1-270 to L 285; S-271 to L 285; L272 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- F-279 to L 285; and G-280 to L 285 ofSEQ ID NO:2. The present application O is also directed to nucleic acid molecules compnsing, or alternatively consisting of, a 0O 10 polynucleotide sequence at least 80%, 85%. 90%. 92%. 95%, 96%. 97%. 98% or 99% Sidentical to the polynucleotide sequence encoding the Neutrokme-alpha and/or Neutrokme-alphaSV polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleoude sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the inventon, as are polypeptides comprising an amino acid sequence at least 80%, 85%, 90%, 92%, 95%. 96%, 97%, 98% or 99% identical to the amino acid sequence described above, and polynucleotdes that encode such polypeptides.

Also as mentioned above, even if deletion of one or more amino acids from the C-tcrmnus of a protein results in modification of loss of one or more functional activities biological activity) of the protein, other functional activities may still be retaned. Thus, the ability of a shortened Neutrokme-alpha mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptde generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-termmus. Whether a particular polypeptlde lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It as not unlikelv that a Ni-trokine-alpha mutein w'h large nw:ber of deleted C-terminal amino acid residues may retain some funcuonal biological or immunogenic) activities. In fact, pepudes composed of as few as six Neutrokne-alpha amino acid residues may often evoke an immune response.

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LIAWYERS 4 092837999 NO.197 949 00 o ~444 0 cl Accordingly the present invention further provides min another embodiment, polypeptides having one or more residues deleted from the carboxy termnunus of the amino acid sequence of the Neutrolune-alpha shown in SEQ ID NO:2, up to the glutamic acid residue at position number 6, and polynncleotdes encoding such s polypeptides. In particualar, the present minvention provides polypepudes comprisimg the amino acid sequence of residues 1-m' of SEQ ID NO:2, where n 3 is an integer in the range of the amino acid position of ammino acid residues 6-284 of the ammno acid sequence m SEQ ID NO:2.

More In particular, the invention provides polynucleotidcs encoding 00 to polypepudes comprismg or alternatively consisting of, an amino acid sequence 0 o selected from the group consisting of residues M-1 to L 284; M-1 to K 283 M-1 to c L 282; NM-1 toA-281 M-L to G-28 M-i toF-279, M-I toF 278. M-1 to T 277- M-1 to V 276; M. to D-275: M-I to G-274, M- I to D-273, M- I to L 272: M-1 to S-271 M-1 to l-270; M-I LoQ-269* M-I to A-268, M- toN-267- M- to E-266; M-i to R 265; M-1 toP 264: M-I to1-263, M-1 to A-262; M-I toL 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; N-1 to E 255- M-1 to E-254, M-l to L 253, M- I to K 252; M-1 to A-25I M-I to I-250; M- I to G-249- M- I to A-248; M-I to 5-247- 4-1 to Y 246: M-1 to C 245; M-I to 5-244; M-I to 14-243, Ml to N-242; M-I to P 241, M-I to L 240; M-I to T 239- M-I to E-238. M- I to P 237 M-1 to M-236; M-I to N-235- M-I to Q-234; M-I ic 1-233 M-1 to C 232; M-I to R 231 M-1 to FP.230; M-I to L 229- M-i toT 228; M- to V 227' M-l to L 226; M-i to 5-225; M- to L 224; M- I to E-223, M- I to D-222; M-I to G-22 I M-1 to F-220; M-I to V 219- M-I to H-218 M- to V 217- M-I to K 216; M- to K 215; M-1 to R 214:; M- to Q-213; M- to I-212; M-I to L 211t M-I to H-210; M-I to G-209- M-1 2s to M-208: M-l to A-207- M-I to Y 206; M-1 to T 205; M-1 ro K 204: M-I to D-203.

M-I toT 202: M-I to Y 201 M-l to L 200; M-I to V 199- M. I to Q-198; M- I to G-197 M-1 to Y 196; M-I to 1-195; M-1 to F-194: M-1 to F-193. M-1 to Y 192; M-I to G-191.IM-I to T 190- M-I to E-189- M- Ito K 188; M-I to V 187- M-I to L 186; M-I tot-185:M- I toK 184,1M-1 tofl-182w-r toK S 8 Iv- tu E-180; M-I to E-179- Mi-I to L 178; M-1 to A-177- M-1 to 5-176; M-l to G-175- M-I to R 174: M-I to K 173; M-I to F-172; M-I to S-171 M-I to L 170; M-1 to L 169- M-I to W 168; M-I to P 167 M4-1 to V 166; M-I to F-165- M- to T 164 M-L to Y 163. M-I to S-1 62; M-1 to G-161 M-1 roK 160; M-l toQ-159- M-I to 1-158: M-1 COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRDN LAWYERS 4 062937999 NO. 197 00 Ai;: 00 Ot oT 157-NM-1 to P 156;M-1 to T 155;M-1 to E-154;M-i to S-153,M-1 to D-152-; M-1 to A-151 M-1 to 1-150 M-i to L 149- M-1 to Q-148; M-I to L 147- M-l to C 146: M-I to D-145; M-I toQ-144:M-I toT 143.M-I to V 142; M-1 to T 141 M-I to E-140; M-I to E-139- M- Ito P 138; M-I to G-137' M-1 to Q-136; 44 to V 135 M-1 to A-134; M-1 to R 133, M-1 to K0 132; M-1 to N4-131, M-1 to R 130, M-1 to S-129- M-I to N-128; M-J to Q-127' M-I to 5-126; M-I to S- 125; M-I to N-124; M-I toG-123,M-i toE-122;M-1 to 0-121 J -1 toP 120: M-1 to A-I 19' M-i zaP 118g: 4-1oP 117- M-I toE-116:M-1 to 115;M-1to-114; M-1 K 113.M-I to L 112; M- to G-1 II M-1 to A-1l10; M-1 toT 109 M-1 to V 108; M-1 to A-1071 M-1 0 010 to P 106; M-i to A-105; M-I to E-104- M- to E-103; M-I toL 102: M-I to -101, o M-I to A-iO0; W- to K-99 M-1 to P-98, M-1 to A-97- M-1 to 0-96; -1 to 0 M-I toG-94- M-toA-93 M-1toP-9;M- to -91 M-I toK-90:M-l to 2-89- M-l to A-88. M-1 to H-87- 4-1 to 86; M- to G-85; N- ito Q-84- M-1 to L-3. M-I to E2-92 M-1 to A-81 M-1 to R-80; N- I to L 79- M-1 to S-78: M-1 to A-77, i-1 to is L 76; NI-I to D-75; M-1 to G-74; 4-1 to Q-73: M-I to L 72; M-I to A-71 4-1 to A-7O; 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-I to V-63, M-I to T-62; M- I to L-61 NI-I to C-6O; M- I to C 59- M-I to S-58; M-I to L 57- M-1 to L 56; M-I to A-55; I-I to L 54- M-I1 toL 53- M-1 to L 52: M-I toT 51 M-1 to A-SO; M-i toA-49- NI-I toL48;M-I roL-47' M-I toK-46:M-1 2o to G-45- M- I to D-44 4-1 to K-43 M- 11o S42; M-I to S-41 M-I to R-40; M-I to V 39- M-1 0to -33; M-1 to P 37- M-1 to S-36; M-I to E-35; M-I to K 34: M-I to R 33, NI-I toP 32; NI-I to 1 31 N-i 10 1-30; NI-I to S-29 4-1to V 28; M-I to C M-i to -26; M-I toK 25; 4-I toL 24; M-1 to K 23:4-1 to 4-22; N-lIto E-21. NI-I to 2-20; M-I to R 19 l4- toK 18; M-toK 17- M- toL 16: M-1 toC 15; M-I to S-14; M-1 toT 13M-I to L 12; to 11 I-I boS-10;M-I toQ-9 M-I to E-8.

M-1ito R 7- and M-1Ito E-6 of SEQ ID N0.2. The present application is also directed to nucleic acid molecules compnsng, or altenaively consisting of, a polynucleotide sequence at least 80%. 85%. 90%, 92%, 95%, 96%, 97%. 98% or 99% identical to the Polynucleou:;de s=;equcr= XTcodin :hc Ncutrokmc-apha and/cul Ile polypepudcs described above. The present inventin also encompasses the above polynucleotide sequences ffied to a heterologous polynucleonide wquence.

Polypcptides encoded by these nucleic acids and/or polynucleouide sequences are also encompassed by the invenuon, as are polypeprides compnsing an amino acid sequence COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 P51 00 c at least 80%, 85%, 90%, 92%, 95%, 96%, 97%. 98% or 99% identical to the aruno C acid sequence described above, and polynucleoudes that encode such polypepudes, The invention also provides polypeptides havmg one or more ammno acids deleted from both the amino and the carboxyl termni of a Neutrokme-alpha polypepude, which may be described generally as having residues n -m of SEQ ID NO:2, where n 3 and m 3 are integers as defined above.

Furthermore, since the predicted extracellular domain of the Neutrokmin-alphaSV polypeptides of the mvention may itself elicit functional actvnty O biological activity), deletions of N- and C-termnal amno acid residues from the 00 10 predicted extracellular region of the polypeptide at positions Gln-73 to Leu-266 of SEQ 0 o ID NO 19 may retain some functional activity such as. for example, ligand binding, to r- stamulauon of lymphocyte B cell) proliferation, diffrentiation, and/or acuvation.

modulation of cell replication, modulation of target cell acuvities and/or immunogemcity However, even if deletion of one or more arruno acids from the is N-termnus of the predicted extracellular domain of a Neutrokine-alphaSV polypeptide results in modification of loss of one or more functional activities of the polypepude, other functional activities may still be retained. Thus, the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptldes generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activites can readily be determined by routine methods described herein and otherwise known in the art.

2s Accordingly the present invention further provides polypeptudes having one or more residues deleted from the amino terminus of the amino acid sequence of Neutrokine-alphaSV shown in SEQ ID NO' 19 up to the glycine residue at position number 261, and polynucleotdes encoding such polypepudes. In particular, the present iment on prouides polypept:de compnsnag the amiri acid s.querice Q& residues n 4 266 of SEQ ID NO: 19 where n 4 is an integer in the range of the amino acid posttion of amino acid residues 73-261 of the amino acid sequence in SEQ ID NO' 19 and 261 is the position of the first residue from the N-terminus of the predicted extracellular domain Neurrokine-alphaSV polypeptide (shown in SEQ ID NO 19).

COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON UWALDRON LAWYERS 4 062837999 NO.197 P52 00 o 7 More in paruticular, in certain embodiments, the invention provides i~ polynucleotdes encoding polypepudes comprising, or alternatively consisting of. an Samino acid sequence selected from the group consisting of residues of Q-73 to L 266: S(3-74 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 s L 266; R-80 to L 266; A- I 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 0 to L 266: L 102 to L 266; E-103 to L 266; E-104 in L 266; A-105 to L 266; P 106 to 0 0 10to L 266; A-107 to L 266; V 108 to L 266; T 109 to L 266; A-110 toL 266; G-111 to SL266;L 112 to L 266;K 113 toL266; -114 toL266:-F-115to L 266 Ell6to L266;P 117toL266;P ll8toL 266;A-119toL 266;P 120to L 266 G. 121 to L 266; E-122 to L 266; G-123 1 L 266; N-124 to L 266; S-125 to L 266; S-126 to L 266;Q-127toL 266;N-128toL 266;S-129toL266:R-130toL266;N-131 to Is L 266; K 132 to L 266; R 133 to L 266; A-134 to L 266; V 135 toL 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 toL 266: F.146 to L 266: V 147 to L 266; P 148 to L. 266; W-149 to L 266: L 150 toL 266: L 151 to L 266; S-152 to L 266; F-153 to L 266; K154 to L 266; R-155 to L 266; 0-156 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; 1-166 to L 266; L 167 to L 266; V 168 to L 266: K 169 to L 266; E-170 o L 266; T 171 to L 266; G-172 t10o L 266; Y 173 to L 266; F-174 to L 266; F-175 to L 266; 1-176 to L266;Y 177 toL266;G-178toL266;Q-179toL 266;V 180to 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; 1-193 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 o L 266; F-201 to i 200oo; 0-20: Lo L Zoo: D-t03 to L 200; E-U204 to L 2ob: L Wu to L £01; S-Zuo to L 266; L 207 to L 266; V 208 to L 266; T 209 to L 266; L 210 to L 266:F-21 !to L 266; R 212 to L 266; C 213 to L 266; 1-214 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 COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 U53 0 0 CN L 266; Y 227 to L 266; S-228 to L 266; A-229 to L 266; G-230 to L 266; 1-231 to t 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;1-244 to L 266; P 245 to L 266:R-246 to Ls 266; E-247 to L 266: N-248 to L 266; A-249 to L 266; Q-250 to L 266; 1-251 to L 266; S-252 to L 266; L 253 to L 266; D-254 to L 266; 0-255 to L 266; D-256 to L 266; V 257 to L 266; T 258 to L 266; F-259 to L 266; F-260 to L 266; and G-261 to L 266 of SEQ ID NO: 19 The present application is also directed to nucleic acid O molecules comprising, or alternatively consisting of, a polynucleoude sequence at least 00 10 80%, 85%, 90%, 92%. 95%, 96%. 97%. 98% or 99% idenucal to the polynucleotide o sequence encoding the Neutrokme-alpha and/or Neutrokine-alphaSV polypepuldes C, described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleoide sequence. Polypeptides encoded by these nucleic acids and/or polynucleoude sequences are also encompassed by the la invention, as are polypeptides comprising an ammo acid sequence at least 80%, 92%, 95%, 96%, 97%. 98% or 99% identical to the anuno acid sequence described above, and polynucleotides that encode such polypeptides.

Similarly deletions of C-terminal amino acid residues of the predicted extracellular domain of Neutrokmie-alphaSV up to the leucine residue at position 79 of SEQ ID NO' 19 may retain some functional activity such as. for example, ligand binding, the ability to sumulate lymphocyte B cell) proliferation, differenuiaton, and/or activation, modulation of cell replication, modulation of target cell activities and/or iimmunogenicty Polypepudes having further C-tenmnal deletions including Leu-79 of SEQ D NO 19 would not be expected to retain biological activities.

However, even if deletion of one or more amino acids from the C-terminus of a polypeptde results in modification of loss of one or more functional activities biological activity) of the polypeptide, other functional activities may still be retained.

Thus, the ability of the shortened polypepude to induce and/or bind to antibodies which recogmnze the complec, mat.re cr ext lu. forms of the polypeptide generally will be retained when less than the majority of the residues of the complete. mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of the predicted extracellular domain COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D54 O i 0 LCA retains such immunologic activities can readily be determined by routine methods described herein and otherwisc known in the art.

Accordingly the present invention further provides polypeptides having one or Smore residues from the carboxy terminus of the amino acid sequence of the prcdicted extracellular domain of Neutrokine-alphaSV shown in SEQ ID NO: 19 up to the leucme residue at position 79 of SEQ ID NO:19 and polynucleoudes encoding such polypeptdes, In particular, the present invention provides polypeptides having the anmuno acid sequence of residues 73-m 4 of the aminuno acid sequence in SEQ ID NO: 19 O where m 4 is any integer in the range of the amino acid position of amino acid residues 00 1to 79-266 of the amnuno acid sequence in SEQ ID NO: 19 More in particular, in certain embodiments, the invention provides C. polynucleoudes encoding polypeptades comprising. or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to L 265; 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 1-251 Q-73 to Q-250; Q-73 to A-249" Q-73 to N-248; Q-73 to E-247- 0-73 to R 246; Q-73 to P 245- Q-73 to 1-244, 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 1-231, 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; 0-73 to Q-215- Q-73 to1-214; 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 1-193, Q-73 to L 192; Q-73 to H-191 Q-73 to G-190; Q-73 to Q-7389- 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 toT 183; Q-73 to v '12; Q-73 to L S Q-'13 to 80; Q-"3 to Q-79 Q-73 to G-i78. Q-73 to Y 177- Q-73 to 1-176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y 173, Q-73 to 0-172; Q-73 to T 171 Q-73 to E-170; Q.73 to K 169 0Q-73 to V 168. Q-73 to L 167- Q-73 to 1-166; 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' COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 16:09 BLAKE DAWJSON WALDRON LAWYERS 4 062837999 NO.197 00 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 Ct W 149- Q-73 to P 148. Q-73 to V 147' Q-73 to F-146; Q-73 to T 145; Q-73 toY 144; Q-73 to S-143; Q-73 to0-142; Q-73 to T 141 Q-73 to E-140; Q-73 toE- 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, s 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 8-126; Q-73 to S-125- Q-73 to N-124; Q-73 to G-123, Q-73 to E-122; Q-73 to 0-121 Q-73 to P 120; Q-73 to A-119- -73 to P 118; Q-73 to P 117 0-73 to E-116; Q-73toP-115; Q-73to 1-114;Q-73toK 113,Q-73toL 112;Q-73to G-I1I Q P-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 00 10 A-lOS; 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-99, Q-73 to A-97- Q-73 to G-96; Q473 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 0-85; 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; Q-73 to L 79 and Q-73 to S-78 of SEQ ID NO*19 The is present application is also directed to nucleic acid molecules compnsing, or alternatively consisting of, a polyniueleotide sequence at least 80%, 85%, 90%, 92%.

96%. 97%, 98% or 99% Identical to the polynucleotide sequence encoding the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides described above. The present invention also encompasses the above polynucleoide sequences fused to a heterologous polynucleotide sequence. Polypepudes encoded by these nucleic acids andlor polynucleotide sequences are also encompassed by the invention, as are polypeptides comprising an amino acid sequence at least 80%. 85%, 90%, 92%. 96%, 97%. 98% or 99% Identcal to the amnno aid sequence described above, and polynucleotides that encode such polypeptides.

The invention also provides polypeptides having one or more amino acids deleted from both the armno and the carboxyl termina of the predicted extracellular domain of Neutroklune-alphaSV which may be described generally as having residues n 4 of SEQ ID NO- 19 where n 4 and Wf are mintegers as defined above.

In another embodirntw, a nucleo::de sequcnc cn udim 5 a puolypupt-ue consisting of a portion of the extracellular domian of the Neutrokne-alphaSV amino acrid sequence encoded by the cDNA clone contained in the deposit havming ATCC Accession No. 203518, where this portion excludes from 1 to about 260 amuno acids from the amino atermunus of the excracellular domain of the ammino acid sequence COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 956 00 o 42-4 c encoded by cDNA clone contained in the deposit having ATCC Accession No. 203518.

c or from I to about 187 anuno acids from the carboxy terminus of the extracellular Sdomain of the amino acid sequence encoded by cDNA clone contamned m the deposit having ATCC Accession No. 203518, or any combination of the above amino termnal s and carboxy temunal deletions, of the entre extracellular domain of the ammo acid 1 sequence encoded by the cDNA clone contained m the deposit having ATCC Accession No. 203518.

As metioned above. even if deleton of one or more amino acids from the C N-termnus of a polypepude results in modificaton of loss of one or more functional activities biological actuvity) of the polypepude. other functional activities may Sstill be retained. Thus, the ability of a shortened Neutrokine-alphaSV mutem to induce and/or bind to antibodies which recognize the full-length or mature forms or the extracellular domain of the polypepttde generally will be retained when less than the majorty of the residues of the full-length or mature or extracellular domain of the is polypeptide are removed from the N-termnus. Whether a paricular polypepude lacking N-termmnal residues of a complete polypepude retains such immunologic activities can readily be determined by rouune methods described herein and otherwise known un the art. It is not unlikely that a Neutrokme-alphaSV mutem with a large number of deleted N-terminal anuno acid residues may retain functional immunogenic) activiues. In fact. pepudes composed of as few as six Neutrokine-alphaSV anuno acid residues may often evoke an immune response.

Accordingly the present mventon further provides polypeptides having one or more residues deleted from the armno terminus of the predicted full-length amino acid sequence of the Neutrolkne-alphaSV shown m SEQ ID NO: 19 up to the glycime residue at position number 261 of the sequence shown SEQ ID NO 19 and polynucleotides encoding such polypepudes. In particular, the present invention provides polypepudes comprising the anuno acid sequence of residues n' 266 of the sequence shown in SEQ ID NO- 19 where n' is an integer in the range of the anuno acid noitnn of ammo acid residues 1 to 26 1 of the a ano acid sequerice ir SEQ ID NO-19 More in particular, the invention provides polynucleotades encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consising of residues of D-2 to L 266; D-3 to L 266: S-4 to COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 19? 057 00 L 266: T 5 to L 266;E-6toL266; R 7 to L 266; E-8 to L 266; Q-9 to L 266; S-10 to Ct L 266;R 11 toL 266;L 12toL 266;T 13toL 266; S-14 toL 266;C 15 toL 266; L 16 toL 266; K I7 toL 266; K 18 to L-266; R 19 to L 266; E 20 to L 266; E-21 to ON L 266; M-22 to L 266; K 23 to L-266; L 24 io L 266;K 2: to L 266; E-26 to L 266; s C 27 to L 266; V 2S to L 266: S-29 to L 266;1-30 to L 266; L-31 to L 266: P 32 to L 266; R 33 to L 266; K 34 to L 266; 2-35 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; 5-42 to L 266; K-43 to L 266;D-44 to L 266 G-45 to L 266; K-46 to L 266; L47 to L 266; L-48 to L 266; 0 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 O 10 L 266: A-$5 to L 266; L 56 to L 266; L 57 to L 266; S-58 to L 266; C 59 to L 266; 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-7 to L 266; L 79 to L 266: R-80 to L 266; A-SI to L 266; is E-82 to L 266; L-3 to L 266; Q-84 to L 266; (3-5 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; 0-96 to L 266; A-97 to L 266; P-98 to L 266; K-99 to L 266; A-100 to L 266; -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 toL 266; A-1I0toL 266;G-1 II toL 266, L 112toL 266;K 13to L 266;I-114 to L 266; F- 115 to L 266; E- 116 to L 266; P 117 to L 266; P I 18 to L 266; A- 119 to L 266- P 120 to L 266- G0-121 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- 12 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 135toL 266;Q-136toL 266;G-137toL 266;P 138to L 266; E 139 to L 266: E- 140 to L 266; T 141 to L 266; G-342 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 26,6; R-,55 266; G-,56 to 266, S-157 W L- 266, ^-t53 aU 3n L 266;L 159 to L 266;E-160toL 266: E-161 toL 266; K 162 toL 266;E 163to L 266; N-164 to L 266; K 165 to L 266; 1-166 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; 0-172 to L 266: Y 173 to L 266: F-74 to L 266; F-175 to L 266; 1-176 toL 266: Y 177 to L 266: G-178 to COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D58 00 L 266; Q-179 to L 266; V 180 to L 266; L 181 o L 266; Y 182 to L 266; T 183 to t L 266; D-14 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 toL 266; -190 to L 266; H-191 to L 266; L 192 to L 266; 1-193 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 s 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; 1-214 to L 266; Q-215 to L 266; N-216 to L-266; M-217 to L 266; P 218 to- 0L 266; E-219 to L 266; T 220 to L 266; L 221 to L 266: P 222 to L 266: N-223 to 00 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 0 L-266; A-229 to L 266; G-230 to L 266; 1-231 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; 1-244 to L 266; P 245 to L 266; R-246 to L 266; E-247 to L 266; N-248 to IS L 266; A-249 to L 266; Q-250 to L 266; 1-251 to L 266; S-252 to L 266; L 253 to L 266; 0-254 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; F-260 to L 266; and G-261 to L 266 of SEQ ID NO-19 The present application is also directed to nuclei acid molecules comprising, or alernatively consistng of, a polynuclcoude sequence at least 80%, 85%, 90%. 92%, 95%. 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding the Neurokine-alpha and/or Neutrokine-alphaSV polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acids and/or polyncleotide sequences are also encompassed by the mvention, as are polypeptides compnsing an amino acid sequence at least 80%, 85%, 90%, 92%. 96%, 97%, 98% or 99% identical to the aruno acid sequence described above, and polynucleoudes that encode such polypeptides, Also as mentioned above, even if deletion of one or more amino acids from the C-termiunw of a nrmte resu!ts i modificat:on of loss of onr or mTre fntioal activites biological activties) of the protein, other funcuonal activiies may still be retained. Thus, the ability of a shortened Neuirokmne-alphaSV mutem to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypepude generally will be retained when less than the COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 15:09 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO.197 P59 00 o~ AzA majority of the residues of the complete or mature form or the extracellular domnam of the polypepude are removed from the C-terminus. Whether a parcular polypepude lacking C-termnal residues of a complete polypepude retains such immunologic activates can readily be determined by routine methods described herein and otherwise s known in the art. It is not unlikely that a Neutrokine-alphaSV muten with a large number of deleted C-termmial ammo acid residues may retain some functional immunogenc) actvitries. InT fact. pepndes composed of as few as six Neutrone-alphaSV ammino acid residues may often evoke an immune response.

o Accordingly the present nvention further provides mn another embodiment, 00 10 polypeptides havig one or more residues deleted from the carboxy terminus of the o armno acid sequence of the NeutrokuncalphaSV shown in SEQ ID NO: 19 up to the C glutamac acid residue at position number 6, and polynucleoudes encoding such polypeptides. In particular. the present minvention provides polypeptades comprisinmg the armno acid sequence of residues 1-m' of SEQ ID NO: 19 where ms as an integer in the I, range of the amino acid position of amuno acid residues 6 to 265 in the anno acid sequence of SEQ ID NO: 19 More an particul ar, the minvention provides polynucleotides encoding polypeptides comprising, or alternatively consastung of, an amino acid sequence selected from the group consisting of residues M-1 to L 265; M-l to K 264; M-1 to L 263, M- to A-262; M-I to G-261 M-i to PF260; M-I to F-259- M- to T 258; M-I to V 257' M- I to D-256; M- Ito 0-255; M-1 to D-254; M- 1 to L 253; M- I to S-252; M-1 to 1-251 M-I to Q-250; M-i1 to A-249- M- I to N-248; M-I to E-247 M-I to R 246; M-I to P 245' M-I to 1-244; M-I to A-243, M-I to L 242; M-I to Q-241, M-I C to L 240; M-I to E-239- M-I to D-238; M-I to 0-237 M-I to E 236; M- to E 235; M-l to L 234; M-I to K 233, M-i toA-232; M- to l-231 M-1 toG-230; M1- to0 A-229- M- to S-228, M-I toY 227' M-I to C 226: M-I toS-225; M-I to N-224; M-1 to N-223; M-1 to P 222; M-I to L 221 M-I to T 220; M-I to E-219- M- to P 218, M-I to M-217M- I to N-216; M-1 to Q-215; M-l tol-214; M-I to C 213, M-I to R 212: M-I to F 211 M-I 'nL 210; M-I to 209' to 208; M- to 20 m-i 3o to S-206; M-I to L 205: M-I to E-204, M-I to 0-203;M-I to 0-202: M-1 to F-201 M-I to V 200; M-I to H-199* M-l to V 198; M- to K 197- M-I to K 196; M-I to R 195; M-I to Q-194; M-I to 1-193; M-I to L 192; M-I to H-191. M- to 0-190; M-I to M-189- M-I to A-I8; M-I to Y 187' M- to T 186; M-l to K 185; M-1 to D-184- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 19/032008 1:09 BLPKE DAWSON WAPLDRON LPAYERS 4 082837999 El? [6 NO. 197 IPGO 00 Cl M-1 toT 183; M-1 to Y 182; NI-1 to L 181 M-1 to V 180; M-1 toQ-179- NI-I to Ct ~~G-178; M- I to Y 177- M-lI to 1- 176:. M-Lt to F-i175. M-1I to P-474, M-I to Y 173; MI rofl-172; M-J to T 171, M-1 to E-170:M4-1 toKX 169-M-1 to V 168.;M-1 to L 167- M- NII tol1- 166-:M- I to K 165:,M-lI to N- 164: M-I to E- 163, M-lI to K 162; M-1I to SE- 161 M-I to E- 160; M-I to L 159- M- I boA- 158, I- I to S5- M-I to a- 156; MI toR-l55;.M-ltoK 154;MIWItoF-153,M-ltoS-152;,M-I toL 151 M-I toL 150;, M-1 toW149-tcoP 148: t-I toy 147-MN-lI o-146;MN-I toT 145;MN-lI I Y 144; M- I to S- 143, M- 1 to C- 142; M-I to T 141 M- I to E- 140; M-I to E- 139- NI-I ic tP 138; M-Ii to 0- 137' M-1 to Q- 136; MI-I to V 135; M-I to A-1 34; M- I to Rt 133; 00io M-1 zoK 132,;M-1toN-131 I-I taR 130;lv-i coS-129 M-1 toN-128; M-ito O 04~Q-27-I-li oS-126:M-I toS-125- N-I toN-124, M-l ;oG-123.M-I toE-122;M-1 Cl ~~toG-iZI Nt-ItoP 120;N-I toA-119-M-i toPIB;M-i toP 117- M-I toE-116:.

NI-i wP-115;,M-1 tol-114;M- toK 113.M-I toL II2:i-i coG-Ill M-1 A-II0kM-1 roT 109'N-I toV 108, N-I toA-I07-MN-i coP 106:-Iito A-lOS' N-i is to E- 104; M- I to E- 103; M-I to L 102; Nt-I to a- 10 1 M-I to A-100; M- to K-99- NI-1 to P-98; M-1 to A-97- M-1 to G-96; M-1 to A-95; M-1 To 0-94, M-1 to A-93 M-1 to P-fl: N-Ito L-91 NI-Ito K-90: N-I to E-89'N-I to A-SB;N-lIto H-ST t-I to H-86: N-I to 0-85; N-I to Q-84, N-I to L-83,MN-lI oE-82;-I to A-i NI-I to R-RO;MN-i to L79-i to S-78, -I toA-fl- M-1to L76; N-i to 175; NIito 2o G-74;MN-i to Q-73; M-i toL 72;MN-I toA471 I-I to A-70: N-I to V-69-MN-i To Q-68; M-I to Y-67- M-1I toF-66; M-I to S-65; I- I to V-64; M-I to V-63, M- I to T-62;N-I to 1-61 NIto C-60;MIito C 39'N- ItoS-58; M- to151- M-I to L56:, MNt oA-55:M-1 tol54;M-l to L53, M-tioL52;M.1 coT 51 M-1to coDA-49-MN-I toL-48;N4-1 oL-47-I-ItoK-46;M-i boG-45;M-I :oD-44,M-1 to K-43: N-I to -42;MN-I to -41 I-I to R40;N-I zoV 39-N-I to S-38; M-i to P3 N-IioS-36; N-I toE-35;MN-I to K34;MN-I to ft33. N-IltoP 32; Mn!toL 31 N-I :01-30;N-I to S-29-MN-ItoV 28;MN-lIto C27 I-Ito 2-26;N-I 1K 25; M-I to L 24:N-i to K23,Iito N-22;MN-I to E-21 t-I toE 20;MN-I toR 19-MN-Ito L 12; M-I to R I I M-I to S-JO; NI-1 to Q-9- M-1 to E-8; M-I to Rt 7-a4nd M-1 to E-6 of SEQ IDNO:19 The present application is also directed to nucleic acid molecules compnsrng, or altemativoly consisting of. a polynucleotida sequence at leas 90%, 92%. 95%, 96%, 97%, 98% or 99% identical to the polynucleotide COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 P61 00 0 sequence encoding the Neutrolkne-alpha and/or Neutrokme-alphaSV polypepudes described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeputides encoded by these nuclew acids andlor polynucleoude sequences are also encompassed by the invention, as are polypephtdes comprstnag an amino acid sequence at least 80%, 92%, 95%, 96%. 97%, 98% or 99% identical to the armno acid sequence described above, and polynucleodes that encode such polypeputides.

The minvention also provides polypeptudes havming one or more amino acids 0 deleted from both the amino and the carboxyl termuni of a Neutrokne-alpbaSV polypeptide. which may be described goncrally as having residues n'-ms of SEQ ID o NO- 19 where n' and nm' are Integers as defined above.

In additional embodiments, the present invention provides polypeprides corpnsing the anmino acid sequence of residues 134-0' of SEQ ID NO:2, where m6 is an minteger from 140 to 285, corresponding to the position of the ammino actd residue in SEQ ID NO:2. For example, the invention provides polynucleotides encoding polypcptides comprisming. or alternatively consisting of. an amino acid sequence selected from the group consisting of residues A-134 to Leu-28a; 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 0-274; A- 134 to D-273; A-134 to L 272; A-134 to S-271 A-134 to 1-270; 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 1-263, 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 toD-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 1-250; A-134 to G-249' A- 134 to A-248. A-134 to S-247- A-134 toY 246: A-134 to C 245; A-134 to S-24: A- 134 to N-243; A-134 toN-242; A-134 toP 241 A-134 to L 240; A-134 toT 239- A- 134 to E-238, A-134 to P237' A-134 to M1-236 A-134 to N-235; A134 to Q-234: A- 134 to 1-233; A-134 to C 232: A-134 to t 231 A-134 to F-230: A-134 to L 229- A- 1Atr1 ,A 1-i4 ti nt A..124'I.-.I '6I 1 13 te l 228: 134 :o t' 22'- to L 226; 3, t s 3o 22P1. t- 134 to E-223, A-134 to D-222; A-134 to G-221 A-134 to F-220; A-134 io V 219' A- 134 toH-218; A-134to V 217 A-134to K 216;A-134to K215; A-134 to R 214; A- 134 to Q-213.A-134 t11-212;A.134to L 21 A-134toH-210 A-134to0-209- A- 134 to M-208: A-134 to A-207- A-134 to Y 206; A-134 to T 205; A-134 toK 204 A- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/20e8 19:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 D62 00 0 t 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 0-198; A-134 to G-197- AL-134 to Y 196; A-134 to 1-195- A-134 to F-194. A- 134 toF 193; A-134 to Y 192: A-134 to 0-191 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-134to I-183; A-134 to K 184, As 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 174; 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-34 to V 166; A-134 to F-165' A-134 to T 164; A S134 to Y 163. A-134 to 5-162; A-134 to 0-161. A-134 to K 160; A-134 to Q-159- A- 00 10 134 tol-158; A-134 to T 157' A-134 to P 156; A-134 to T 155' A-134toE-154 A- 134to $53, A-134 to D-152; A-134 to A-iSI A-134 to 1-50; A-134 to L 149- A- 134 to0-148; A-134 toL 147- A-134toC 146; A-134 to D-145;A-134 toQ-144 A- 134 to T 143. A-134 to V 142; A-134 to T 141 and A-134 to E-140 of SEQ ID NO:2.

The present applicauon is also directed to nucleic acid molecules compnrising, or is alternatively consisling of, a polynucleotide sequence at least 80%, 85%. 90%, 92%.

96%, 97%, 98% or 99% idenucal to the polynucleoide sequence encoding the Neutrokine-alpha andlor Neutrokne-alphaSV polypeptides described above. The present invention also encompasses the above polynuicleotide sequences fused to a heterologous polynucleotide sequence. Polypeprides encoded by these nucleic acids and/or polynucleotide sequences arc also encompassed by the invennon, as are polypeprdes comprising an amino acid sequence at least 80%, 85%, 90%, 92%, 96%, 97%, 98% or 99% identical to the amuno acid sequence described above, and polynucleolides that encode such polypeptudes.

Additional preferred polypeptade fragments of the invention comprise, or 23as alternatively consist of, an amino acid sequence selected from the gmroup consisting of residues: M-1 to C 15; D-2 to L 16; D-3 to K 17- S-4 to K 18; T 5 to R 19- E-6 to E R 7 to E-21 E-8 to M-22; Q-9 to K 23; S-10 to L 24; RII 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 to1-30; 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- MI-22 to S-36; K 23 ;o P 3T 2-t io 5-38. 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, 1-30 to D-44; L 31 to 0-45; P 32 to K-46; R 33 to L-47- K 34 to L-48; E-35 to A-49, 5-36 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; S-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 L61 L-48 to T-62; A- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WAlLDRON LAWYERS 4 062837999 NO.197 P63 00 c 49 to V-63. A-50 to V-64: T 5) to S-65; L 52 to F-66; L 53 to Y-67- L 54 to Q-68; Ato V-69- L 56 to A-70; L 57 to A-71 SS-58 to L-72; C 59 to Q-73. C-60 to 0.74; 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 0-68 to E-82; V-69 to L-83 A-70 to 0-84; A-71 to G-85; L 72 to H-86; Q- 73 to H-87 0-74 to A-8; 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-8 I to A-95; E-82 to 0-96; L-83 to A-97' Q-84 to P-98; Gto K-99' H-86 to A-100; H-B7 to 0-101 A-8 to L 102; E-89 to E-103, K-90 toE- 104; L-91 to A-105;P-92 toP 106; A-93 to A-107 G-94 to V 108: A-95 toT 109- G- 96 to A-110;A-97 toG-111I P-98 toL I12; K-99 toK I13: A-100 to I-114;G-101 to O ~~10 F-115: L 102to E16:E-103to P 17' E 104 to P 18; A-105 to A-119. P 106to P 120; A-107 to G-121 V 10B to E-122 T 109 to 0-123; A- 110 to N-124; 0- 11I to S.

125; L 1l2 toS-126;K 13 toQ-127- 114toN-128; F-11 to S-129- E-116 to R 130;P il7 toN-131 P 118 toK 132; A- 119 to R 133, P 120 to A-134: G-12L toV 135; E-122 to Q-136; G-123 to G-137' N-124 to P 138: S-125 to E-139- 5-126 to E 140: Q-27 to T 141. NI28 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 150; 0-137 to A-151 P 138 to D-152; E-139 to 5-153; E-140 to E-154, T 141 to T 155; V 142 to P 156; T 143 to T 157 Q-144 to 1-158, D-145 to Q-159- C 146 to K 160; L 147 to G- 161 Q-148 to 5-162: L 149 to Y 163, 1-150 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 174' G-161 to G- 175; 5-162 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 toN-183,L 170to K 184; S-171 to I- 185: F-172 to L 186; K 173 to V 187- R 174 to K I1; G-175 to E-189- S-176 to T 190 A-177 to 0-191 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; 1-185 to V 199- L 186 to L 200; V 187 to Y 201 K 188 to T 202; E-18 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-19S to G-209- Y 196 to U- 2'O: Ito L 2&l Q-198 to s-2r2; v j99 Q-213, L 200 Io R 4; z 2z o wK 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; H11-210 to L 224; 1 211 to S- 22 5 1- 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 1-233 F-220 to Q-234; G-221 to N- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 16:09 BLAKE DAWUSON UWALDRON LlAWYERS 4 062837999 NO. 19? 164 00 o 429 0 C 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 t 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; 1-233 to S-247- Q-23410 A-248; N-235 to G-249- M-236 to 1- 0250; P 237 to A-251, E-238 to K 252; T 239 to L 253, L 240 to E-254- P 241 toE- 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 m 1-263; 1-250 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- 0-256 to 1-270: 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; Ao 262 to V 276; 1-263 to T 277' P 264 t10 F-278; R 265 to F-279- E-266 to G.280; N-267 00 o to A-281 A-268 toL 282; Q-269 to K 283.1-270 to L 284; and S-271 to L 285 of SSEQ ID NO:2. Preferably these polypeptide fragments have one or more functional Cactivities biological activity anigenimciy and inmmunogenicity) of Neutrokeine alpha and/or Neutrokine-alpha SV polypeptides of the invention and may be used, for example, to generate or screen for antibodies, as described further below The present is invention is also directed to polypeputides comprising, or alcemrnatively consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence described above. The present invention also encompasses the above amino acid sequences fused to a heterologous anmino acid sequence as described herein. Polynucleoides encoding these polypeptides are also encompassed by the invenion.

Additional preferred polypepude fragments of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group consisting of residues: M-1 io C 15; D-2 toL 16; D-3 toK 17 S-4toK 18;T 5toR 19- E-6 toE- 7 o E-21 E-8 toM-22;Q-9 to K 23, S-10 toL 24 R 11 to K 25;L 12 to E-26: T 13 to C 27- S-14 toV28:C 15 to S-29- L 16 to 1-30; K I7 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 to V 39" E-26 to RA40; C 27 to S41 V 28 to S-42; S-29 to K-43 1-30 to D-44 L 31 to G-45, P 32 to K-46; R 33 to LA7- K 34 to L-48, E-35 to A-49" S-36 to A-50; P 'J'20.o-r C I A A20..n o 5 S-3S 39 o 53, R- to tu r%-55; S-42 w L 5u; 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; A- 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 0-68; Ato V-69- L 56 to A-70; L 57 to A-71 S-58 to L 72; C 59 o Q-73 C-60 o10 G0-74; 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 COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 197 00 0130 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- 0-74 to A-88, D-75 to E-89- L 76 to K-90: A-77 to L-91 5-78 to P-92; L 79 to A-93, R-80 to 0-94 A-81 to A-95; E-82 to G-96; L-83 to A-97- Q-84 to P-98; GtoK-99- H-86to A-100: H-87to 0-101 A-88to L. 102;E-89to -103,K-90to Es 104 L-91 to A-105 P-92 to P 106; A-93 to A-107, G-94 ro V 108; A-95 to T 109, G- 96 to A-110; A-97to G-1 I1 P-98 to L 112;K-99 toK 113.A-100to 1-114; 0-101 to F-115; L 102 to E-116; E-103 top 117- E-104 to P I 18: 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 13 to Q-127 I-114 to N-128; F-1I 15s to S-129- E-116 to R 00 iu 130: P 17 to.N-131 P i18 to K 132; A-119 to R 133, P 120 to A-134: G-121 to V o 135 E-122 to Q- 136; G-123 to 137- N-124 to P 138, S-125 to E 139- 5-126 to E ~C 140: Q-127 to T 141 N-128 to 0-142: S-129 to S-143, R 130 to Y 144, N-131 to T 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-I40 to K 154- T 141 to R is 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 s151 to K 165; S-152 to1-166; -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-ISS to G-172; L 159 to Y 173 E- 160 to F-174; E-161 to F- 175; K 162 to 1.176; E 163 toY 177' N-164 to G-178; K 163 to Q-179- -166toV 180: L 167 to L 181 V 168 toY 182:K 169toT 183 E-170 toD-184;T 17 1 to K 185: G-172 to T 186; Y 173 to Y 187- F 174 to A-188, F-175 to M-189 1-176 to G- 190: Y 177 to H-191, G-178 to L 192; Q-179 to 1-193. 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 i-199- T 186 to V 200 Y 187 to F-201 A-88 to G-202: M-89 to D-203, G-190 to E-204; H-191 to L 205; L 192 to 5-206; 1-193 to L 207- Q-194 to V 208; R 195 toT 209- K 196 to L 210; K 197 to F-211 V 198 to R 212; H-199 to C 213 V 200 to1-214; F-201 to Q- 215; G-202 toN-216; D-203 to M-217t 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 2 3 to 22"- o i

G-

230; M-217 to 1-231 P218 toA-232;E 219 to K 233 T 220 to L 234; 1 221 o E 235; P 22 to E236 N-223 to 0-237- 1N-224 to D-238, S-225 to E 239- C-226 to L 240; Y 227 to Q-241 8-228 to L 242: A-229 to A-243 G-230 to 1-244 1-231 toP 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- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.197 D66 00 C237 to 1-251 D-238 to S-252; E-239 to L 253, L 240 to D-254; Q-241 to G-255; L 242 ct to D-256; A-243 to V 257- 1-244 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; 1-251 to L 265; and S-252 to L 266 of SEQ ID NO: 19 Preferably these polypeptide fragments have one or more functional activities biological activity antgenicity and unmunogenicty) of Neutrokmne-alpha and/or Neutrokme-alpha SV polypeptldes of the invention and may be used, for example, to generate or screen for antibodies, as described further below The present invention is also directed to polypeptdes comprising. or 0 alternauvely consisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 00 10o 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence described above.

O The present invention also encompasses the above amino acid sequences fused to a heterologous ammo acid sequence as described herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Additional preferred polypeptde fragments of the invention comprise, or is alternatively consist of, an aminno acid sequence selected from the group consisting of residues: M-1 to F 15; D-2 to C 16; E-3 to S-17- S-4 to E 18: A.5 to K 19" K-6 to G- T 7 to E-21 L-8 to D-22; P-9 to M-23. P 10 to K 24. P I 1 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 -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 0-37- K 24 to A-38; V 25 to W 39- G-26 to F-40; Y 27 to G-41 D-28 to 1-42; P 29 to C-43, I1-30 to R.44- T 31 to D-45; P 32 to G-46; Q-33 to R-47- K 34 lo L-48. E-35 to L-49- E-36 to G-37 to A-51, A-38 to T 52; W 39 to L 53; F-40 to L 54 0-41 to L 55; 1-42 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 52to S-66; L 53 to L-67- L 54 to Y68; L 55 to Q-69- A-56 to L 70 L 57 to A-71 L 58 to A-72; S-59to L 73 8-60 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-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-'9 to n-93. 80 to n.4; Ri-8a to t-95, n-.22 t0 n-96, E-83 u G-7 8 IV n-9; Q-85 to P-99- S-86 to E-OO: Y-87 to L 101 R-88 to T 102: 0-89 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- 96toP l10;G-97 to A-II A-98 to A-112: P-99 to P 113. E 00 to R 114L 101 to P 115-T 102toH-116:A-03 to N-I7-G-.104 oS.118.V 10toS-119-K 106 toR COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWUSON WALDRON LiAWYERS 4 062837999 NO.197 G67 00 0 0

CI

120; L 107 to G-121 L 108 to H-122: T 109 to R 123. P 110 m N-124. A-l 11 to R t 125;A-112 toR 126; P 113 to A-127 R 114 to F-128.P S115 toQ-129- H-1I16 to G- S130; N- 117 toP 131 S-118 to E 132: S-119 to E 133, R 120 toT 134; 0-121 to E- S135; 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 toP 143; G-130 toP 144, P 131 to A- S145;E-132 toP 146;E 133 toC 147 T 134 toL 148: E-135 toP 149- Q-136toG- 150l D.l37 toC 151, V 138 to R 152; D-139 to H-153, L 140 to S-154, S-141 toQi 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- 0 160;C 147toM-161 L 148toN-162:P l49toL 163, G-5iOtoR 164;C151 toNoo 10 165;R 1 5 2 to 1-166; H-153 to 1-167-S-154 to Q-168; Q-155 to D-169- H156 to C S170; fD-157 toL 171 D-158toQ-172; N-159 toL 173, G-160 to I-174; M-161 to A- C 175;N-162 toD-176;L 163 to S-177-R 164 toD-178. N-165 toT 179- 1-166 to P 180 1-167 to A-181 Q-16 to L 182:D-169 toE-183: C 170 to E 184 L 171 to K 185;Q-172toE-186:L 173 toN-187- 1-174 to K 188;A-175 to 1-189- D-I76toV iS 190; S-177 to V 191 D-178 toR 192: T 179 loQ-193,P 10 toT 194-A-181 toG- 195;L 182 to Y 196;E 183 toF-197, E-184toF-198: K ]8 to 1-199-E-186toY 200; N-187 to S-201, K 183 to Q-202; 1-189 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-19. to 1-209- Y 196 toF- 210; F-197 toA-211 F-198 to M-212; 1-I99 to G-213, Y 200 to H-214 S-201 to V 21S;Q-202to 1-216;V203 toQ-217. L 204 toR2 18;y 205 to K 219- T 206toK 220; D-207 to V 221 P 208 to H-222; 1-209 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- 1-216 to L 230;Q-217 toV 231 R 218 toT 232; K 219 to L 233, K 220 to F.234; V 221 to R 3 235; H-222 to C 236; V 223 to 1-237- F-224 to Q-238, 0-225 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 PF-234 to S-248, R 235 to C 249" C 236 to Y 250; 1-237 to S-251 Q-238 to A-252; N-239 to G-253, M-240 to 1-254; 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-7AR in E 262; C 249 to' 263. v 250 to Q-264 S-25. to 265; A-252 to A-266; G-253 to 1-267- 1-254 to P 268: A-255 to R 269- R 256 to E- 270; L 257 to N-271 E-258 io A-272; E-259 to Q-273 G-260 to 1-274; D-261 to S- 275: E-262 to R 276; 1-263 to N-277- Q-264 to G-278; L 265 to D-279- A-266 to D- 280; 1-267 toT 281 P 268 to F-282: R 269 to P 283 E 270 to G-284; N-271 to A- COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO.197 968 00 cN 285; A-272 to L 286; Q-273 to K 287 1-274 to L 288: and S-275 to L 289 of SEQ ID SNO:38. Preferably these polypepude fragments have one or more functional acvities biological actuviy anttgenicty and immunogemcity) of Neutrokne alpha and/or Neuirokine-alpha SV polypeptdes of the nvention and may be used, for example, to generate or screen for antibodies, as described further below The present invention is also directed to polypeptdes comprisng, or alternatively consisting of, an amnno acid sequence at least 80%, 85%, 90%, 92%. 95%. 96%, 97%, 98% or 99% idential to an amino acid sequence described above. The present invention also 0 encompasses the above amino acid sequences fused to a heterologous amino acid 00 10 sequence as described herem. Polynucleoudes encoding these polypeptides are also Sencompassed by the invention.

It will be recognized by one of ordinary skill in the art that some anrno acid sequences of the Neutroklne-alpha and Neutrokine-alphaSV polypeptides can be vared without significant effect of the structure or function of the polypeptide. If such la differences m sequence are contemplated, it should be remembered that there will be cntical areas on the polypeptide which determine activity Thus, the invention further includes vanauons of the Neutrokine-alpha polypeptide which show Neutrokme-alpha polypeptide functional activity biological acuvlty) or which include regions of Neutrokine-alpha polypeptide such as the polypepide fragments described herein. The invention also includes variations of the Neutrokine-alphaSV polypeptide which show Neutrokine-alphaSV polypeputde funcuonal activity biological activity) or which include regions of Neutrokine-alphaSV polypeptide such as the polypeptade fragments described herein.

Such mutants include deleons, insertions, mversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity For example, guidance concerning how to make phenotypically silent amino acid substtutions is provided in Bowie, J. U. ex al., "Deciphenng the Message m Protein Sequences: Tolerance to Amino Acid Substitutions, Science 247 1306-1310 (1990), wherein the authors indicate that there are *wo approaches for study g tc tolerance of an anuno acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.

The second approach uses genetic engmeenng to introduce amino acid changes at COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:09 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 197 P69 o 46', 0 l specific positions of a cloned gene and selections or screens to identify sequences that C maintain functionality SAs the authors state, these studies have revealed that proteins are surprisingly ON tolerant of amino acid substtuuons. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buned armno acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substtutions are described in Bowie, J. U et aL, supra, and the references cited therein.

0 Typically seen as conservatve substitutions are the replacements, one for another, 00 10 among the aliphatic amno acids Ala, Val, Leu and Ile; interchange of the hydroxyl Sresidues Ser and Thr, exchange of the acidic residues Asp and Glu. substitauon C" between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of Figures IA and Il (SEQ ID N0O2), or that encoded by the deposited cDNA plasmid, may be one in which one or more of the amino acid residues are substituted wih a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted arnno acid residue may or may not be one encoded by the genetic code, or (ii) one m which one or more of the amino acid residues includes a substtuent group, or (iii) one in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypepride (for example, polyethylene glycol), or (iv) one in which the additional anuno acids are fused to the extracellular domain of the polypeptide. such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for punfication of the extracellular domain of the polypeptide or a proprotem sequence.

Such fragments, derivatives and analogs are deemed to be withm the scope of those skilled in the art from the teachings herein Furthermore, the fragment, derivative or analog of the polypeptde of Figures and SB (SEQ Tl or that encoded by the deposited cDNo plasmid. ray one in which one or more of the ammo acid residues are substituted with a conserved or non-conserved ammno acid residue (preferably a conserved amno acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a t COMS ID No: ARCS-183615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 19/032009 16:09 BLAKE DAWJSON WJPLDRON LPAWYERS 4 0622337999hU19 P0 NO. 19? PO 00 o 435 SU 'bstuent group, or (iii) one iM wih the eXtraCellular domain of the polypepuide ms fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the extracellular domain of the polypepuide, such as, a soluble s biologically active fragment of another TNF Jigand family member Ligand). an lgGi Fe fusion region peptide or leader or secretory sequence or a sequence which is emiployed for purification of the extracellular domain of the polypepude or a pruprotin sequence. Such fragments, denvataves and analogs are deemed to be withinl O the scope of those skilled in the ant from the teachings herein 00 10 Thus, the NeuzrokmnealphAanMd/of Neurrokrnc-alphaSV polypeptides of the o presiR invention may include one or more amino acid substitutions, deletions or Ci additions. either from natural mutations or human manipulation. As indicated, changes are preferably of a minor narure, such as conserarave amino acid substitutions that do not significantly affect the folding or activity of the protein (set Table 11).

TABLE 11. Conservative Amino Acid Substitutions.

Aromatic Phenylalanzne Tryptophan Tyrosine Hydrophobic Leucane tsoleucine Yalin.

Polar Glumunne Aspa1ragine Basic Afginine

K.

Lysine Histidinc Acidic Aspartic Acid Glutamic Acid Small Alane Scoine Threonine COMS ID No: ARCS-i 83615 Received by IP Australia: Time 16:38 Date 2008-03-19 19/o3/2ooe 19/032008 1:12 81 2 92598999 4 082937999 N.2 0 NO. 726 Pol FAX TRANSMISSION 7 No of pages (including this set) 7 Level 38. Grosvenor Place 225 George Street Sydney NSW 2000 AUSItAi Blake Dawson PATENT ATTORNEYS

TO

The Commissioner of Patents IP Australia F 02 6283 7999 Human Genomne Sciences, Inc New Australian divisional patent application Title: Neutvoklne-alpha and neutrokine-aipha splice variant PART 3 OF 6 T 6128925806000 F 81 2 9258 2999 DX 355 Sydnecy Locked Bag No Grosvenor Place Sydney NSW 2000 Australia www.blakedawson.won 19 March 2008 Our reference DGC SJ 02 1430 4462 Paitra David Clark T 612 925886839 davidoleark Obiakedawson.com Plezae check that you have received this document In fujll, If not, please telephone the sender or call G1 2 9258 6000.

coot idemility Thia document is confidential and may contain legally privleged informnallon. if you are not a namea or euthorised recipient you must not read, copy, distribute or ac In reliance on it It you nave received this document In error.

please telaplhona cur opertor immediately on 61 2 8255 0000 and return tne document by mnal.

Sydney Melbourne Brisbane FI'Ath Canberra 203937378-11 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 D02 00 S-136 In one embodiment of the invention, polypeptide comprises, or alternatively Sconsists of, the amino acid sequence of a Neutroklne-alpha or Neuurolkne-alphaSV polypeptLde having an amino acid sequence which contains at least one conservative amino acid subsumton, but not more than 50 conservative ammno acid substitutions, even more preferably not more than 40 conservative amino acid substitutions, still.

more preferably not more than 30 conservative arnno acid substitutions, and still even more preferably not more than 20 conservative anuno acid substutions. Of course, in order of ever-increasing preference, it is highly preferable for a pepide or polypeptlde (N to have an amnmo acid sequence which comprises the amino acid sequence of a 00 Neutrokme-alpha polypeptide, which contains at least one. but not more than 10, 9 8, 0 7 6, 5, 4, 3, 2 or I conservative amino acid substitutions.

r For example, ste directed changes at the amino acid level of Neutrokme-alpha can be made by replacing a particular amino acid with a conservative substitution.

Preferred conservative subsututon mutations of the Neutrokine-alpha amino acid is sequence provided in SEQ ID NO:2 include: MI replaced with A. G. L L. S. T or V D2 replaced with E; D3 replaced with E: S4 replaced with A, G, I. L, T M. or V TS replaced with A, G, I, L. S, M. or V E6 replaced with D- R7 replaced with H. or K, E8 replaced with D- Q9 replaced with N: S10 replaced with A, G. I. L, T M, or V RI I replaced with H, or K, L12 replaced with A, G, I, S, T M. or V T13 replaced with A, 0.1. L, S. M, or V 14 replaced with G I L, T M, or V L16 replaced with A. G.

I, S, T M, or V K 1 replaced with H. or R; K18 replaced with H, or R; R19 replaced with H, or K, E20 replaced with D- E21 replaced with D- M22 replaced with A, G, 1, L, S, T or V K23 replaced with H. or R. L24 replaced with A, G. I, S. T M, or V replaced with H, or R; E26 replaced with D V28 replaced with A, G. L, S, T or M; 529 replaced with A, G. I, L, T M. or V 130 replaced with A, G, L, S. T M. or V L31 replaced with AG.G S. T M, or V R33 replaced with H, or K, K34 replaced with H, or R; E35 replaced with D- 536 replaced with A. G, 1, L. T M. or V S38 replaced with A, G. 1. L, T M. or V V39 replaced with A. G, 1, L. S. T or M: R40 replaced with H, or K, St i replacen with A, G, i, L, T mw, or v S42 replace wnn A, 1, L. T I. w V 143 replaced with H. or R, D44 replaced with E, G45 replaced with A. I. L, S. T M. or V K46 reDlaced with H. or R. L47 replaced with A. G, I. S. T M, or V L48 replaced with A. G, 1. S. T M, or V A49 replaced with G, L, S, T M. or V replaced with G. I, L. S. T M. or V T5 I replaced wnh A. G. 1. L. S, M, or V L52 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92596999 4 062837999 N0.726 P03 00 o %.A37 C replaced with A, G, I, S. T M, or V L53 replaced with A. G. I. S, T M. or V L54 replaced with A, G, S, T M, or V A55 replaced with G, I, L, S, T M, or V L56 S.replaced with A, G. I, S, T M, or V 1L57 replaced with A, G, I, S, T M, or V S58 replaced with A. G I, L. T M, or V L61 replaced wilh A, G, 1, S, T M, or V T62 replaced with A. G, I, L, S, M, or V V63 replaced with A, G, I, L, S, T or M; V64 replaced withA, G, 1, L. S,T orM;S65replaced withA, 1, L,T M, orV F66 replaced with W or Y Y67 replaced with F or W- Q68 replaced with N; V69 replaced with A. G,1. L S, T orM; A70 replaced with G,I, L, S,T M, orV A71 replaced with o G. 1. L S, T M, or V L72 replaced with A, G, I, S, T M. or V Q73 replaced with N: 0010 G74 replaced with A, I, L. S. T M. or V D75 replaced with E; L76 replaced with A, G, I, S, T M, or V- A77 replaced with G, 1. L, S. T M, or V S78 replaced with A. G, 1.

1L, T M, or V- L79 replaced with A. G, S. T M, or V R80 replaced with H. or K, AS1 replaced with G, 1. L. S. T M, or V E82 replaced with D- L83 replaced with A, G, I, S, T M, or V- Q84 replaced with N; G85 replaced with A. I, T M. or V I H86 replaced with K. or R; H87 replaced with K, or R, A88 replaced with G0, 1, L, S, T M, or V ES9 replaced with D; K90 replaced with H. or R, L91 replaced with A, G, 1, S. T M, or V- A93 replaced with G, I, L. S, T M. or V G94 replaced with A, I. L, S, T M. or V A95 replaced with G. I. L, S, T M. or V G96 replaced with A, I, L, S, T M, or V A97 replaced with G, 1, L, S, T M. orV K99 replaced with H, or R; A100 replaced with G, L, S, T M, orV G0 101 replaced with A, 1. L S, T M. orV Ll 02 replaced with A. G. I. 1.S, T M, or V B103 replaced uwith D- E104 replaced with Dreplaced with G, L S T M or replacedwith,,,.7 replaced with G, I, L, S. T M. or V VI08 replaced with A, G. I, L. S. T or M; T109 replaced with A, G, I. L. S. M, or V Al 0 replaced with G. 1. LS,T M, orV GI 1 replaced withA. I,L,S. T M, orV Ll 12 replaced with A, G, 1, S.T M, or V KI 13 replaced with H, orR. 14 replaced withA.G.L,S,T M.orV Fll 5 replaced with W orY Ell6replaced with DA 119 replaced withG. L, S,T M, orV G121 replaced with A, I,L. S.T M, orV E122 replaced with D- G123 replaced with A. I. L. S,T M, orV N124 replaced with Q S 125 replaed with ,C,IL,'r M, or"I Sl26replaced with G,a. T Zv, v v Q127 replaced with N; N 128 replaced with Q; S129 replaced with A, G. 1, L. T M. or V R130 replaced with H, or K, N131 replaced with Q- K132 replaced with H. or R: R133 replaced with H, or K, A 134 replaced with 0.1. L. S. T M. orV V135 replaced with A. G, 1. L, S,T or M; Qi136 replaced with N; 0 37 replaced with A. 1 L, S. T M, COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062937999 NO.726 004 00 O 433 or V E139 replaced with D- E140 replaced with D; T141 replaced with A, G, 1. L, S, <t M. or V- V 142 replaced with A, G. 1. L, S, T or M; T143 replaced with A, G, I. L, S.

M, or V- Q144 replaced with N; D145 replaced with E; L147 replaced with A. G, I, S, _T M, or V Q148 replaced with N; L149 replaced with A, G. 1. S, T M, or V- 1150 s replaced with A, G,L,S,T M,orV AI51 replaced wth G, L, S.T M.orV D152 replaced with E; S153 replaced with A. 0, I, L.T M. or V E154 replaced wih D' T155 replaced with A. 0, 1, L, S, M, or V T157 replaced with A, G, I, L, S, M, or V 1158 replaced with A, L, S. T M, or V Q159 replaced with N; K160 replaced with SH, or R; 0161 replaced with A, L, S, T M, or V S162 replaced with A, G, I. L. T 0 0 1 M, or V- Y163 replaced with F or W- T164 replaced with A, G. I, L. S, M, or V F165 o replaced with W orY V 66 replaced with A. G, 1. L. S. T or M; WI68 replaced with F or Y L169 replaced with A, S. T M, or V L170 replaced with A. G. 1. S, T M, or V S171 replaced with A, G, I, L, T M. orV F172 replaced with W or Y K173 replaced with H, or R, R174 replaced with H, or K, G175 replaced with A, I. L. S. T is M, orV S176replacedwithA, G,I,L,T M, orV AIT7 replaced wth G, L L, S, T M, or V L178 replaced with A, G. I, T M, orV E179 replaced with D- replaced with D- K181 replaced with H, or R; E182 replaced with D- N 183 replaced with Q; K184 replaced with H, or R, 1185 replaced with A. T M, or V L186 replaced with A, G. I. S, T M, or V V 187 replaced with A. G. L. S. T or M; KI88 replaced with H, or R; E189 replaced with D- T190 replaced with A, G. I. L, S. M, or V 0191 replaced with A, I. L, S. T M. or V Y192 replaced with F or W F193 replaced with W or Y F194 replaced with W or Y 1195 replaced with A, G, L, S. T M.orV Y196 replaced with F orW G197 replacedwithA, I, L. S.T M.orV Q198 replaced with N; V199 replaced with A. G, L. S. T or M; L200 replaced with A. 0. I.

S. T M, or V Y201 replaced with F or W- T202 replaced with A, G, I, L, S. M, or V D203 replaced with E: K204 replaced with H, or R; T205 replaced with A. 0, 1. L, S, M, or V Y206 replaced with F or W A207 replaced with 0, 1, L, S, T M, or V M208 replaced with A. G,1, L, S, T or V 0209 replaced with A, 1, L, S, T M. or V H210 replacu with K, ui R; -2i an replaceu widi A. G, i. S. T tvi. ui v 1212 replaceu wi;h a, G, L, S..T M. or V Q213 replaced with N; R214 replaced with H, or K. K215 replaced with H. or R. K216 replaced wit H, or R. V217 replaced with A. G. I, L, S, T or M; H218 replaced with KL or R, V219 replaced with A. G, I, L. S, T orM. F220 replaced with W or Y G221 replaced with A, I. L, S, T M, or V D222 replaced with E; E223 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 e 062837999 NO.726 905 00 (Cl replaced with D- L224 replaced with A, G, I, S, T M. or V- S225 replaced with A, G, I, ct L, T M, or V L226 replaced with A, G. L S, T M, or V V227 replaced with A. G, I, L, S, T or M; T228 replaced with A. G, I, L, S. M, or V L229 replaced with A. G. I, S, T M. or V F230 replaced with W or Y R231 replaced with H, or K, 1233 replaced with A. G, L, T M, or V Q234 replaced with N; N235 replaced with Q; M236 replaced with A, G. I, L, S, T or V E238 replaced with D- T239 replaced with A. 0, 1.

L, S, M. or V L240 replaced with A, G, I, S, T M, or V- N242 replaced with Q; N243 replaced with Q; S244 replaced with A, G, L, T M. or V- Y246 replaced with F or 0 W- S247 replaced with A 0, I, L, T M, or V A248 replaced with G. L. S. T M. or 0 0 10 V' G249 replaced with A, I. L. S. T M, or V 1250 replaced wnh A, G, L, S, T M. or 0 V A251 replaced with G, L L, S, T M. or V K252 replaced with H, or R. L253 replaced with A, 0, I. S, T M. or V E254 replaced with D' E255 replaced with D- G256 replaced with A, 1. L. S. T M, or V D257 replaced with B; E258 replaced with D- L259 replaced with A. G.1, I, T M. or V Q260 replaced with N; L261 replaced with A, G, S. T M, or V A262 replaced with G, 1, L, S. T M. or V I1263 replaced with A, 0, L, S, T M. or V R265 replaced with H. or K. E266 replaced with D- N267 replaced with Q; A268 replaced with G. I, L. S, T M. or V Q269 replaced with N; I270 replaced with A, G, L, S, T M, or V S271 replaced with A, G, I, L, T M, or V L272 replaced with A, G. I, S, T M, or V D273 replaced with E; G274 replaced with A, I, L, S, T M. or V D275 replaced with E, V276 replaced with A. G. I. L, S, T or M; T277 replaced with A, G. I, L, S, M, or V F278 replaced with W or Y F279 replaced with W or Y G280 replaced with A. I. L. S, T M, or V A281 replaced with 0, L, S. T M. or V L282 replaced with A, G. I. S. T M. or V K283 replaced with H, or R; L284 replaced with A, 0, 1, S. T M, or V- and/or L285 replaced with A. G. I, S, T M. or V Polynuclcoudes encoding these polypepudes are also encompassed by the invention. The resulting Neutrokune-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutroklnc-alphaSV functional activity and/or physical properties (such as, for example. enhanced or reduced stability and/or sulubility). Pic iamy ia resultting piOCirk ouf [Eb nivviIu nIsave an Imcrekiscu and/or a decreased Neutrokine-alpha and/or Neutrokine-alphaSV functional activity More preferably the resulting Neutrokme-alpha and/or Neutrokine-alphaSV proteins of the invention have more than one increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV funcuonal activity and/or physical propeny COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2oo8 16:12 61 2 92586999 4 062837999 N0.726 D06 00 0 0 44o 2_ In another embodiment, site directed changes at the amino acid level of iNeutrokine-alphaSV can be made by replacing a particular armno acid with a conservative substituution. Preferred conservative substitution mutations of the Neutrokuie-alphaSV ammo acid sequence provided in SEQ ID NO- 19 include: M l s replaced with A, G, I, L. S. T or V D2 replaced with E; D3 replaced with E; S4 Sreplaced with A, G. L L, T M. or V T5 replaced with A, G. I, L. S, M, or V E6 replaced with D; R7 replaced with H, or K, E8 replaced with D' Q9 replaced with N; replaced with A. G. 1 L. T M. or V RI I replaced with H. or K. L12 replaced with c0 A.G. T M. or V- T13 replaced with A. G. I. L,S. M.orV S14 replaced with A, G 1L, T M.or V Ll6 replacedwith A, G,1 S. T M. or V Kl 7 replaced with H, or 0' R. K1I replaced with H, or R; R19 replaced with H. or K, E20 replaced with D- -21 replaced with D" M22 replaced with A. G. I. L. S. T or V K23 replaced with H. or R.

L24 replaced with A. G, I. S. T M. or V K25 replaced with H. or R; E26 replaced with D' V28 replaced with A. G, 1. L, S, T or M; S29 replaced with A, G. 1, L, T M, or V is 130 replaced with A. 0, L. S. T M, or V L31 replaced with A. G. I. S. T M. or V R33 replaced with H. or K. K34 replaced with H. or R. E3S5 replaced with D- S36 replaced with A, G.1 L T or V S38 replaced with A. G, 1. L, T or V V39 replaced with A, G, I. L. S, T orM; R40 replaced with H, or K. S41 replaced with A. G. I. L.T M,orV S42 replaced with A. G. I, L. T M,orV K43 replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L. S. T M. or V K46 replaced with H. or R.

L47 replaced with A. G. I. S. T M, or V L48 replaced with A. G, I. S. T M. or V A49 replaced with G. I. L, S. T M, or V A50 replaced with G. 1, L, S, T M. or V T51 I replaced with A, G. 1, L, S. M, or V L52 replaced with A, G, 1, S. T M. or V- L53 replaced with A, G. I. S, T M. or V L54 replaced with A, G, I, S. T or V- replaced with G0, 1, L. S. T M, or V L56 replaced with A. G. 1, S, T M. or V- L57 replaced with A. G, 1. S, T M. or V S58 replaced with A, G, L, T M, or V- L61 replaced with A. G, I, S. T M, or V T62 replaced with A, G. 1, L. S. M, or V V63 replaced with A. G, I. L, S. T or M; V64 replaced with A. G. 1, L, S, T or M; 'epiaccu wiihi G, 1, L, T iv, ur v F6o repoccu wii "W or i 167 rcpaccu wiil F or W- Q68 replaced with N; V69 replaced with A, G, 1, L, S, T or M: A70 replaced with 0. I, L. S. T M. or V A71 replaced with G. 1. L. S. T M. or V L72 replaced with A, G. 1. S. T M. or V Q73 replaced with N; G74 replaced with A, I. L, S, T M, or V replaced with E: L76 replaced with A, G. 1, S. T M. or V A77 replaced with G. I, COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 PO? 00 o ~.44-1 L. S, T M, or V- S78 replaced with A, G, 1, L, T M, or V L79 replaced with A, G, 1, ct S, T M, or V- R80 replaced with H. or K. A8I replaced with G, 1. L, S, T M, or V E82 replaced with D" L83 replaced with A, G. 1. S, T M, or V Q84 replaced with N; replaced with A, 1, L, S. T M, or V- H86 replaced with K, or R, H87 replaced s with K. or R, A88 replaced with G, L, S, T M. or V E89 replaced with D; replaced with H, or R, L91 replaced with A. 0. I, S, T M, or V A93 replaced with G, L. S, T M. or V G94 replaced with A, I. L, S. T M, or V A95 replaced with G. I. L, S, T M, or V G96 replaced with A, I. L. S, T M, or V A97 replaced with G, 1, L. S.

0T M, or V K99 replaed wh H or Aplacedwh orAOeplaced with G, I, L. S, T M, or V 0 0 tO G 101 replaced with A, I. L, S, T M, or V L102 replaced with A, G, I, S,T M, orV 0E103 replaced with D- E104 replaced with D- AI05 replaced with G, I, L, S. T M. or V A 107 replaced with G. L.S, T M.or V V 108 replaced with bA, CI, 1L. S, T or (D M; T109 replaced with A, G, 1, S. M. or V- Al 10 replaced with G, 1. L, S, T M, or V G 111i replaced with A, LL, S.T M.orV LI 12 replaced with A, G. S, T M,or V K113replacedwithH.orR:lI14replacedwithA. G,L,S,T M,orV PF115 replaced with W or Y El 16 replaced with D- At 19 replaced with G. 1. L, S, T M, or V GI121 replaced with A. 1. L.S.T M. or V E122 replaced with D- G123 replaced with A. I L, S. T M, or V N 124 replaced wnith Q- S 125 replaced with A. G, L. T M, or V S126 replaced with A. G, I. L. T M, or V Q127 replaced with N; N128 replaced with Q: S129 replaced with A. G. 1. L, T M, or V R130 replaced with H, or K, N131 replaced with Q: K132 replaced with H, or R; R133 replaced with H, or K, A134 replaced with G, 1, L. S. T M. or V V135 replaced with A, G, 1, L, S, T or M; 0136 replaced with N; Gl137 replaced with A, L L S, T M, or V El 39 replaced with D- E140 replaced with D- T141 replaced with A, G, I. L, S. M. or V G142 replaced with A,I.L.S,T M.orV S143 replaced withA.G, LT M. orV Y144 replaced with F or W- T145 replaced with A, G0. 1. L. S. M, or V F146 replaced with W or Y V147 replaced with A. G,I. L, S, T or M; W 149 replaced with F or Y L 150 replaced with A.G.1,S,T M.orV LIS1 replaced with A.G, 1, S,T M, orV S 152 replaced with A.G .l T M.orV Fs11 replacedw th w orv K154 rpii-ccd replaced with H, or K, G 156 replaced with A, I, L, S. T M, or V S 157 replaced with A, G, 1, L. T M, or V A158 replaced with G0. 1, L. S. T M, or V- L159 replaced with A, 0G, 1, S, T M. or V E160 replaced with D- E161 replaced with D" K162 replaced with H. or R; E163 replaced with D- N 164 replaced with Q: Kl6a replaced with H, or COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208Q 16:12 61 2 92586999 4 062837999 N0.726 P08 00 O0 o A42- R; 1166 replaced with A, G, L, S, T M, or V. LI67 replaced with A, G, 1, S. T M, or V" V168 replaced with A, G0 L, S, T or M; K169 replaced with H. or R; Sreplaced with D- T171 replaced with A, G, I, L, S. M, or V G172 replaced with A, I, L, ST M,orV Y173 rcplaced with F orW F 174replaced withW orY F175 replaced with W orY II76replaced with A. G, L, S,T M, or V Y177replaced with F orW- GT178 replaced with A, I, L. S, T M, or V Q179 replaced with N; V 180 replaced with SA I, L, S. T or M" LIS1 replaced with A, G, 1, S. T M. or V Y182 replaced with ~F or W- T183 replaced with A, G. 1, L, S, M, or V D184 replaced with E; K185 0 replaced with H. or R, T186 replaced with A, G, I. L. S. M, or V Y187 replaced with 00 00 0 F orW- A188 replaced dwith 0G, L. S.T M,orV M189 replaced with A. S, T or V G190 replaced with A. 1, L. S, T M, or V H191 replaced with K, or R L1 92 replaced with A. G,I, S, T M. or V 1193 replaced with A, G, L. S. T M, or V Q194 .replaced with N; R195 replaced with H, or K, K196 replaced with H, or R, K197 replaced with H. or R; V 198 replaced with A, G,1. S, T or M; H199 replaced with Is K. or R, V200 replaced with A, G, 1. L. S, T or M; F201 replaced with W or Y G202 replaced with A, I. L, S, T M, or V D203 replaced with E; E204 replaced with D- L205 replaced with A, G, S, T M, or V S206 replaced with A, G. I, L, T M. or V L207 replaced with A, G, I, S, T M, or V V208 replaced with A, G, 1, L. S. T or M; T209 replaced with A, G, L, S, M. or V L210 replaced wKith A. 1, S, T M. or V P211 replaced with W orY R212 replaced with H. or K, 1214 replaced with A. G, L, S, T M, or V Q215 replaced with N; N216 replaced with Q; M217 replaced with A, G, I, L, S, T or V E219 replaced with D- T7220 replaced with A, G. 1. L. S, M. or V L221 replaced with A, G, 1, S, T M, or V N223 replaced with Q; N224 replaced with Q; Cr 5225 replaced with A, G, I, L, T M, or V Y227 replaced with F or W S228 replaced with A. G. I, L. T M. or V A229 replaced with G, I, L, S, T M, or V G230 replaced with A, 1, L, S, T M, or V 1231 replaced with A, G, L S. T M. or V A232 replaced with G, I. L, S. T M. or V K233 replaced with H, or R, L234 replaced with A, G. 1. S.

T M. or V E235 replaced with D- E236 replaced with D- G237 replaced with A. 1, L.

S T M. or V D238 repaed with E; E230 replaced with D- L24C placcd with n. G.

I, S,T M. or V Q241 replaced with N; L242 replaced with A, S. T M, orV A243 replaced with G. I. L, S, T M, or V 1244 replaced with A, G. L. S. T M, or V R246 replaced with H, or K, E247 replaced with D, N248 replaced with Q: A249 replaced with G, I. L, S. T M, or V Q250 replaced with N; 1251 replaced with A. G, L.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 D09 00 0 N S, T M, or V S252 replaced with A, G, I, L, T M, or V L253 replaced with A, G, I, ct S, T M, or V D254 replaced with E; G255 replaced with A, L, S, T M, or V D256 Sreplaced with E: V257 replaced with A. G, I, L, S, T or M; T258 replaced with A, G, I, O L. S. M. or V F259 replaced with W or Y F260 replaced with W or Y 0261 s replaced with A, L, S, T M. or V. A262 replaced with G 1, L. S, T M. or V- L263 replaced with A, G, 1, S, T M, or V K264 replaced with H, or R, L265 replaced with A, G, T M, orV and/or L266 replaced wih A, G, I. S,T M, orV Polynucleotides encoding these polypeptdes are also encompassed by the invention.

o The resulting Neutrokine-alpha proteins of the invention may be routinely screened for 00 10 Neutrokine-alpha and/or Neutrokne-alphaSV functional activity and/or physical O properties (such as, for example, enhanced or reduced stability and/or solubility).

Preferably the resulting proteins of the invention have an increased and/or a decreased Neutrokmne-apha and/or Neutrokme-alphaSV functional activity More preferably the resulting Neutrokune-alpha and/or Neutroane-alphaSV proteins of the invention have more than one increased and/or decreased Neutroklne-alpha and/or Neutrokine-alpha SV functional acuvlty and/or physical property In another embodiment, site directed changes at the amino acid level of Neutrokine-alpha can be made by replacing a particular amino acid with a conservative substitution. Preferred conservative substitution mutations of the Neutrokine-alpha amino acid sequence provided in SEQ ID NO:23 include: R replaced with H, or K. V2 replaced with A. G. 1. L. S, T or M; V3 replaced with A, G, I, L, S. T or M; D4 replaced with E: LS replaced with A, G, I. S, T M, or V S6 replaced with A. 0, 1, L, T M, or V A7 replaced with G,1, L, S. T M. or V A 0 replaced with G. I L, S, T M, or V L13 replaced with A, G, 1, S, T M, or V G15 replaced with A, I, L, T M. or V- R17 replaced with H, or K. HI8 replaced with K, or R, S19 replaced with A, G, 1, L, T M, or V Q20 replaced with N; H21 replaced with K. or R, D22 replaced with E; D23 replaced with E; N24 replaced with Q; G25 replaced with A, 1, L. S, T M, or V M26 replaced wiih A. G, I, L, S, T or V N27 replaced with Q- L28 replaced with A, C, I, S. or R29 rcplaced wth Il, or K. N30 replaced with Q R3 replaced with H, or K. T32 replaced wih A. G, I. L, S. M. or V Y33 replaced with F or W- T34 replaced with A, G, I, L, S, M, or V F35 replaced with W or Y V36 replaced with A, G, I, L. S, T or M: W38 replaced with F or Y L39 replaced with A, G. I. S. T M, or V- L40 replaced with A, G, 1, S, T M, or V- S41 replaced with A, G, I. L, T M, or V COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 00 o 44 0

C

F42 replaced with W or Y K43 replaced with H. or R; R44 replaced with H. or K.

C G45 replaced with A, I. L, S. T M. orV N46 replaced with Q; A47 replaced with G. 2 S, T M, or V L48 replaced with A. 0,1, S, T M, or V E49replaced with D- Sreplaced wuith D- K,51 replaced with H. or R; E52 replaced with D- N53 replaced with Q; K54 replaced with H, or R. 155 replaced with A, G. S, T M, or V V56 replaced with A, G, I, L, S. T or M; V57 replaced with A, G, I, L. S. T or M; R58 replaced with H, or K, Q59 replaced with N; T60 replaced with A, G. 1, L, S, M, or V G61 replaced Swith A, I. L. S. T M. or V Y62 replaced with F or W F63 replaced with W or Y F64 Sreplaced with W or Y 165 replaced with A, 0. L, S. T M, or V Y66 replaced with F 0 0 to or W- S67 replaced with A. G, 1, L. T M, or V Q68 replaced with N; V69 replaced o with A. G, 1, L, S, T or M; L70 replaced with A, G, 1. S, T M. or V Y71 replaced with C F or W- T72 replaced with A, G, I. L, S, M, or V D73 replaced with E, 175 replaced with A, G, L, S, T M, or V F76 replaced with W or Y A77 replaced with G, 1, L, S, T M. or V M78 replaced with A, G, I. L. S. T or V G79 replaced with A, 1, L, S. T M, or V H0 replaced with K, or R, V81 replaced with A, G, I, L, S, T or M; 182 replaced with A. G. S, T M, or V Q83 replaced with N; R84 replaced with H, or K, replaced with H. or R; 86 replaced with H. or R. VS7 replaced with A, G0, I, L, S.

T or M; H88 replaced with K, or R; V89 replaced with A, G, I, L, S. T or M; replaced with W or Y G91 replaced with A, I, L, S. T M, or V 1D92 replaced with E; E93 replaced with D- L94 replaced with A, G, I. S. T M. or V S95 replaced with A, G.

1, L. T M. or V L96 replaced with A, G. 1, S. T M, or V V97 replaced with A, G, 1, L, S, T or M; T98 replaced with A, G0, 1. L, S, M. or V L99 replaced with A. G. I, S.

T M. or V F100 replaced with W or Y RI11 replaced with H. or K, 1103 replaced with A. G, L, S, T M, or V Q104 replaced with N; N105 replaced with Q; M106 replaced with A. G. L, S, T or V KIOS replaced with H, or R; T109 replaced with A, G,I, L, S,M, orV Ll 10 replacedwithA, G, 1, S,T M, orV N 112replaced with Q; N113 replaced with Q- S114 replaced with A, G. 1. 1, T or V YR 16 replaced with F or W- SI 17 replaced with A. G, 1, L, T M, or V A 118 replaced with G, 1, L, S.

I fu-.

T vi, Or" G 9 Yrepimcu Willh a, n. S, T iv, ui v 120 rcpiaccu wiIII G, S, T M. orV A121 replaced with G, I. L, S. T M, orV R122 replaced with H, or K.

L123 replaced with A, G. 1, S, T M, or V E124 replaced with D' E 25 replaced with -D G126 replaced with A, I. L. S, T M. or V D127 replaced with E; E128 replaced with D-1129 replaced with A, G. L, S.T M,orV Q130 replaced with N: L131 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 Dll o 00 N replaced with A, G, I, S, T M, or V A132 replaced with G, I, L,S, T M, or V 1133 C replaced with A, G. L S. T M, or V- R135 replaced with H, or K, E136 replaced with SD- N137 replaced with Q; A138 replaced with G, L. S, T M, orV Q139 replaced with N; 1140 replaced with A, G, L, S, T M, or V S41 replaced with A. G, 1, L. T M, s or V R 142 replaced with H. or K. N143 replaced with Q-G 144 replaced with A, I, L, S, T M. or V D145 replaced with E; 0146 replaced with E: T147 replaced with A, G, I, L, S, M, or V F148 replaced with W or Y F149 replaced with W orY G150 replaced with A, 1, L, S, T M, or V A151 replaced with G, I. L, S. T M, or V- L152 o replaced with A G, G S, T M, or V K153 replaced wth H. or R. LI 54 replaced with 00 to A, G, 1S, T M, or V and/or L55 replaced with A, G, I,S, T M, or V SPolynucleotides encoding these polypeptides are also encompassed by the invention.

The resulting Neutrokine-alpha protins of the invention may be routinely screened for Neutrolune-alpha and/or Neutrokne-alphaSV functional activity and/or physical properties (such as, for example, enhanced or reduced stability and/or solubility).

is Preferably the resulting proteins of the invention have an increased and/or a decreased Neutrolune-alpha and/or Neutrokime.alphaSV functuonal activity More preferably the resulting Neutrokine-alpha and/or Neutrolkne-alphaSV proteins of the invention have more than one increased and/or decreased Neutrokme-alpha and/or Neutrokme-alpha SV functonal activity and/or physical property In another embodiment, site directed changes at the amino acid level of Neutrokine-alpha can be made by replacing a particular amino acid with a conservative substitution. Preferred conservative substitution mutations of the Neutrokne-alpha amino acid sequence provided in SEQ ID NO:38 include: M1 replaced with A. G, I, L, S, T or V D2 replaced with E; E3 replaced with D- 54 replaced with A. 0, 1. L, T M, or V A5 replaced with G, I, L. S. T M. or V K6 replaced with H, or R; T7 replaced with A, G, 1, L, M, or V LB replaced with A, T M. or V L 13 replaced with A, G, 1. S. T M, or V F15 replaced with W or Y S17 replaced with A, G. I. L, T M.

or V E18 replaced with D- K19 replaced wih H, or R. G20 replaced with A, I, L, S, T M, or V E21 replaced with D- D22 replaced with E; 23 replace, with G, I, S.

T or V,.K24 replaced with H, or R; V25 replaced with A. G. 1, L. S, T or M: G26 replaced with A, I. L, S, T M, or V Y27 replaced with F or W- D28 replaced with E; 130 replaced with A, G. L. S, T M, or V- T31 replaced with A, G, 1. L, S, M, or V Q33 replaced with N; K34 replaced with H, or R; E35 replaced with D; E36 replaced with COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 i9/o3/2oe8 916:12 61 2 92596999 4 862837999. NO,1.72G P12 00 o 0 D- G37 replaced with A, I, L, S. T M, or V A38 replaced with G, L, S. T M, or V Ct W39 replaced with F or Y F40 replaced with W or Y G41 replaced with A. 1. L, S.

T or V 142 replaced with A. G. L. S, T M, or V R44 replaced with H, or K, replaced with E; G46 replaced with A, L L. S. T M. or V- R47 replaced with H, or K.

s 148 replaced with A, G, S. T M, or V L49 replaced with A. G, I, S, T M, or V replaced with G, IL, S, T M. or V AS1 replaced with G,I, L, S. T M. or V T52 replaced with A. G, S, M or V L53 replaced with A, 0, 1, S, T M, or V L54 replaced with A. G. 1, S. T M, or V L55 replaced with AG, 1, S, T M, or V A56 0 replaced with G. I, L. S. T M. or V L57 replaced with A. 0. 1. S. T M, or V 58 0010 replacedwthA, G..S,T M, or V S59replacedwithA.G.1,L.T M.orV replaced with A. 0. 1. L. T Mor V S61 replaced with A, G, 1. L, T M. or V F62 replaced with W or Y T63 replaced with A, G, 1. L, S. M, or V A64 replaced with G, I. L, S. T M, or V M65 replaced with A, G. 1. L, S, T or V S66 replaced with A. G, L, T M, or V" L67 replaced with A. 0, 1. S. T M. or V Y68 replaced with F or W IS Q69 replaced with N; L70 replaced with A, G,1.S.T M, or V A71 replaced with G,1.

L. S, T M, or V A72 rplaced wth G, I, L, S, T M. or V L73 replaced with A,0. L S. T M, or V Q74 replaced with N; A75 replaced with L. S, T M, or V D76 replaced with E; L77 replaced with A, G, 1, S, T M, or V M7 replaced with A, G, 1, L. S, T or V N79 replaced with Q:L 80 replaced with A, G, 1. S. T M, or V RSI replaced with H. or K, M82 replaced with A 1 L. S. T or V E83 replaced with D" L24 replaced with A, G, 1, S, T M. or V Q85 replaced with N; S86 replaced with A.

G 1, L. T M. or V- Y87 replaced with F or W- R88 replaced with H. or K, G89 replaced with A. 1. L, S, T M, or V S90 replaced with A. G, 1, L, T M, or V A91 replaced with G,1, L, S. T M, or V 792 replaced with A, or V- A94 replaced with G, 1, L, S. T M. or V A95 replaced with G, 1, L, S. T M, or V" A96 replaced wth G. 1, L, S, T M, or V G97 replaced with A, I, L, S, T M, or V- A98 replaced with 0, I. L. S, T M, or V El{0 replaced with D- LI0I replaced with A. 0. 1.

S, T M, or V T102 replaced with A, G,1. L, S M. or V A103 replaced with G, I, L, S.T M.orV C IndrcplaccdwIh 4. 1, 1 T pw., c L.S.T orM: K106 replaced with H, orR; LI07replacedwith A. iS, T M, orV LIDS replaced with A, G, 1. S. T M, or V T109 replaced with A. 0. 1, L. S, M, orV AII I replacd with 1. L, S, T M, or V A112 replaced with G, 1, L, S, T M, or V R 114 replaced with H, or K. HI 16 replaced with K, or R. N 117 replaced with Q, S I 18 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 iq/o3/2oo8 16:12 61 2 92596999 4 062837999 N0.726 P13 00 0 replaced withA, G, L,T M.orV Sll 9 replacedwithA, G. I, L,T M. orV R120 it replaced with H, or K, G 121 replaced with A, 1, L, S, T M, or V H 122 replaced with SK, or R; R123 replaced with H. or K, N124 replaced with Q; R125 replaced with H, or C" K, R126 replaced with H, or K. A127 replaced with G, I. L, S, T M, or V F128 replacedwithW orY Q129 replaced with N: G130 replaced with L, S,T M.or V E132 replaced with D- E 133 replaced with D- T 134 replaced with A. G. I, L. S, M.

SorV E135 replaced with D- Q136 replaced with N; D137 replaced with E; V138 Sreplaced with A, G, 1, L, S, T or M; D139 replaced with E; L140 replaced with A, G. 1, 0 S. T M, or V- S 141 replaced with A. G, L. T M. or V A 142 replaced with G. I, L, 0 0 10 S,T M, or V- A 145replacedwithG, I. L,S,T M.orV L48 replacedwith A. G, 1.

SS, T M. or V G 150 replaced with A, I, L. S, T M. or V Rl 52 replaced with H, or K, H 153 replaced with K. or R; S 154 replaced with A, G, I, L. T M, or V Q155 replaced with N: H156 replaced with K. or R, D157 replaced with E; D158 replaced with E; N159 replaced with Q; G160 replaced with A, I. L, S, T M, or V MI61 replaced with A, 0, I, L. S, T orV N162 replaced with Q: L163 replaced with A, G, I, S,T M. or V R164 replaced with Hl, or K. N 165 replaced with Q- 1166 replaced with A. G, L. S, T M, or V 1167 replaced with A, G. L. S.T M, or V- Q168 replaced with N; D169 replaced with E; LI71 replaced with A, G. I. S.T M, or V Qi72 replaced with N: L173 replaced with A, G, I, S, T M. or V 1174 replaced with A. G, L, S, T M, or V A175 replaced withG, L, S, T M, or V D 176 replaced with E; S 177 replaced with A. G, 1, L, T M. or V D 178 replaced with E; T179 replaced with A, G, I, L, S. M, or V AIS I replaced with G. I, L,S,T M,orV L182replaced withA.G.l,S.T M, or V E183 replaced with D" E184 replaced with D- K85 replaced with H, orR, E186 replaced with D; N 187 replaced with Q K188 replaced with H, or R; 1189 replaced with A, G,L, S,T M, orV Vl 90replacedwith A, G, I. L,S.T orM; V 191 replaced with A, G. L, L, S, T or M; R192 replaced with H, or K. Q193 replaced with N; T194 replaced with A. G. 1, L, S, M. or V G 195 replaced with A, I. L. S, T M. or V Y1 96 replaced withF or W F197 replacedwith W orY F198 replaced with W or Y 1199 reDlaced with A- T M, or V- V20 replaced w;:th 1 r W" S2 replaced with A. G, 1, L, T M, or V Q202 replaced with N; V203 replaced with A. L, S, T or M; 1L204 replaced with A, G, I. S. T M, or V- Y205 replaced with F or W T206 replaced with A. 1. L, S. M. or V D207 replaced with E; 1209 replaced with A, G, L, S. T M, or V- F210 replaced wth W or Y A2ll I replaced with G. I, L. S. T M. or V COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2e 16:12 61 2 92586999 4 062837999 N0.726 D14 00

CI

M212 replaced with A, G, L. S, T or V G213 replaced wtnb A. I. L, S, T M, or V t H214 replaced with K, or R; V215 replaced with A. 1. LL, S, T or M; 1216 replaced i with A, 0, L, S, T M, or V Q217 replaced with N; R218 replaced with H, or K, K219 replaced with H. or ftR, K220 replaced with H, or R; V221 replaced with A, 0,1. L. S.

T or M; H222 replaced with K, or R: V223 replaced with A. G, I1, L. S. T or M; F224 replaced with W or Y G225 replaced with A I. L, S, T M. or V D226 replaced with E; E227 replaced with D L228 replaced with A, G, S, T M, or V S229 replaced Swith A. G, 1I, L, T M. or V 1L230 replaced with A, G, I, S, T M, or V V231 replaced Switbh A, 0, IL, S. T orM; T232replaced withA.G, I,L, S,M,orV L233 replaced 00 o to with A, I. T M, or V F234 replaced with W or Y R235 replaced with H, or K, 0 1237 replaced with A, G, L, S. T M, or V 0238 replaced with N- N239 replaced with C Q; M240 replaced with A, G0, 1, L, S, T or V K242 replaced with H. or R.T243 replaced with A, G, 1, L, S, M, or V L244 replaced with A, 1, S, T M, or V N246 replaced with Q; N247 replaced with Q; S248 replaced with A,G, 1, L. T M, or V is Y250 replaced with F or W S251 replaced with A, G, I, L, T M, or V A252 replaced with G, 1. L.S,T M. or V G253 replaced wth A, 1.L. S, T M,orV 1254 replaced with A. 0, L. S, T M, or V A255 replaced with G, 1. L, S, T M. or V R256 replaced with H, or K, L257 replaced with A. G 1, S, T M. or V E258 replaced with D- E259 replaced with D- G260 replaced with A. 1. L. S, T M. or V D261 replaced with E; E262 replaced with D 1263 replaced with A, 0. L, S, T M, or V Q264 replaced with N; L265 replaced with A, G, I, S, T M, or V A266 replaced with G, 1, L, S. T M, or V- 1267 replaced with A, G, L, S, T M, or V R269 replaced with H. or K. E270 replaced with D N271 replaced with Q; A272 replaced with G, 1, L, S. T M. or V Q273 replaced with N; 1274 replaced with A. G, L, S, T M, or V S275 replaced with A. G, I, L, T M, or V R276 replaced with H. or K, N277 replaced with Q- G278 replaced with A. I, L. S, T M, or V D279 replaced with E; D280 replaced with E; T291 replaced with A, G. 1, L, S. M, or V P282 replaced with W or Y F283 replaced with W or Y G284 replaced with A, I, L, S, T M, or V A285 replaced with G. I, L, S, T M, or V L286 reolaced with A. r 1. S T M, or v K28 7 replaced with or R; 1.288 replaced with A, G, I, S. T M, or V- and/or L289 replaced with A, G. I, S, T M, or V Polynucleoides encoding these polypeptides are also encompassed by the invention. The resulting Neutrokine-alpha proteins of ihe invention may be routinely screened for Ncutrokine-alpha and/or Neutrokine-alphaSV functional activity and/or COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92586999 4 062837999 NO.726 00

C

N physical properties (such as, for example, enhanced or reduced stability and/or c solubility). Preferably the resulting proteins of the invention have an increased and/or C a decreased Neutrokmne-alpha and/or Neutrokme-alphaSV functional acuvity More Spreferably the resulting Neutrokone-alpha and/or Ncutrokioe-alphaSV proteins of the invention have more than one increased and/or decreased Neutrokine-alpha and/or Ncutroknlu-alpha SV functional activity and/or physical property Amino acids n the Neutrokne-alpha and/or Neutrokine-alphaSV polypeptides of the present invention that are essential for function can be identified by methods 0 known m the art. such as site-directed mutagenesis or alanine-scanning mutagenesis 00 10 (Cutingham and Wells, Science 244-1081 1085 (1989)). The latter procedure Smtroduces single atanne mutations at every residue in the molecule. The resulting mutant molecules are then tested for functional activity such ligand binding and the ability to stmulate lymphocyte B cell) as, for example, proliferation.

differentuauon, and/or activaton.

s1 Of special interest are substitutions of charged ammno acids with other charged or neutral amino acids which may produce proteins with highly desirable improved charactensucs, such as less aggregation. Aggregation may not only reduce activity but also be problematic when prepanng pharmaceutical formulations, because aggregates can be immunogenic (Pinckard et aL. Clin. Exp. Inununl. 2:331-340 (1967); Robbms et al., Diabetes 36: 838.845 (1987); Cleland et aL, Crn. Rev. Therapeutic Drug Carer Systems 10-307-377 (1993).

In another embodiment, the mventon provides for polypepudes having amino acid sequences containing non-conservative substiutons of the amino acd sequence provided m SEQ ID NO:2. For example, non-conservatve substitutions of the Neutrokne-alpha protein sequence provided in SEQ ID NO:2 include: M replaced with D, E, H, K, R, N, Q, F W Y P or C,.D2 replaced with H, K. R, A. G, 1, L, S, T M,V NQ.F W Y P or C;D3 replaced with HK,R. A. G, 1. L,S, T M,V N, Q.

F W Y P or C, S4 replaced with D E, H, K, R, N, Q, F W Y P or C, T5 replaced with H. K. R, N. Q F w v P or C, E6 replaced :i H.K. C, ST M, V N,Q,F W Y P or C, R7 replaced with D E, A, G, I L. S, T M.V N,Q, F W Y P orC, E8 replaced with H, KR, A. G, LS, T MV N.Q,F W Y P orC, Q9replaced with D E H. K, R A, G,I,L.S,T M.V F W Y P or C,S10 replaced with D, E, H, K, R, N, Q, F W Y P or C, RI1 replaced with D, E. A. G, 1. L S. T M.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92596999 4 062837999 NO.726 P16 00 0 49o c ^V N,Q.F W Y P orC. Ll2replacedwithD,E,HK,RN,Q,F W Y P orC, t T13 replaced with D, E. H, K, R, N, Q, F W Y P or C, S 14 rplaed with D, E, H, K.

i R.N. Q,F W Y P orC. C15 replacedwith D. E,H, K, R,A. G.I, L. S. T M, V N, SQ,F W Y or PL6replaced withD, F H,K,R,N,Q.F W Y P orC,K17 s replaced with DE. A. 0.1. L. S,T M,V N.Q,F W Y P orC: KI 8 replacedwith SED,.,A,G,I.L.S.T M.V N.Q,F W Y P orC, R19 replaced with D, EA. G IL, S.T M,V N.Q.F W Y P or C, E20 replaced with H. K, R. A, G.I. L. S,T M, V NQ,F W Y P orC, E21 replaced with H .KR. A,G,1,L,S,T M.V N.QF W CY P or C; M22 replaced with D, E, H, K, R, N, Q, F W Y P or C. K23 replaced Sio witihD A, L,S,T M.V N,Q,F W Y P orC,L24 replaced with D, E. H, K.

SR,N,Q,F W Y P or C, K25 replaced with D E,A,G, I. L, S,T MV N.QF W Y P or C, E26 replaced with H. K, R. A. G. 1, L.S, T M.V N, Q F W Y P orC.

C27 replaced with D E, Hl, K,R,A. G. L,S.T M. V N,Q,F W Y or P V28 replaced with D E, H, K, R N. Q. F W Y P or C. S29 replaced with D E, H, K. R.

is N. Q. F W Y P orC, 130 replaced with D. E, H, K, R. N, Q. F W Y P orC. L31 replaced with D, E, H, K, R. N. Q F W Y P or C. P32 replaced with D E, H, K, R, A,G, 1, L. S,T M, V N,Q, F W Y orC, R33 replaccdwith D E,A,G, ,IL,ST M,V N.Q.F W Y P orC, K34replaced withDE,A. G. I, L. S.T M.V N.Q,F W Y P orC, E35replacedwthH.K, R,A.G, 1, L, S,T M,V N,Q,F W Y P or C, S36 replaced with D, E, H, K, R, N. Q, F W Y P or C, P37 rcplaced with D E, H.

K,R,A,G. I,L,S,T M.V N, Q, F W Y orC, S38 replaced with D E.H, K,R,N.

Q,F W Y P or C,V39 replaced with D E, H, K, R, N. Q, F W Y P orC, replaced wnith D, E, A.G, I, L.S, T M.V N.Q. F W Y P orC, S41 replaced with D E,H,K,R, N,Q,F W Y P orC, S42 replaced wnith D E. H. K,R.N.Q,F W Y P orC. K43replaced with D.E G,,L,S,T M,V NQ.F W Y P orC,D44 replaced with H. K, R, A, G. 1, L. S, T M. V N, Q. F W Y P or C, G45 replaced with D E,H,K. R,N,Q, F W Y P orC. K46 replaced withD E, A,G, L, S, S.T M, V N,Q.F W Y P or C, L47 replaced withD, EH.K,R,N.Q,F W Y P orC, L-4 replaceu w.I D E, H, K R, Q, F v V P us- C, t49 repiauu wh ,D F, n. K, R.N,Q.F W Y P orC. A50 replacedwith D E.H,KR.&N,Q.F W Y P orC.

TSI replaced wuth D H. K, R, N, Q, F W Y P or C, L52 replaced with D E. H, K.

R.NQ.F W Y P orC,L53 replacedwithD E, H,K. R,N,Q, F W Y P orC.

L54 replaced with D, E. H, K, R. N, Q, F W Y P or C, A55 replaced wth D E. H. K.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 16:12 61 2 92586999 4 062837999 N0.726 P17 00 0.

R,N.,Q.F W Y P orC. L56 replaced wthD, E.H.K, R, N, Q. F W Y P orC; t L57 replaced with D, E. H, K. R, N, Q, F W Y P or C, S58 replaced with D. E. H. K.

i R,NQ.F W Y P orC;C59replacedwith D, E,H,KR. A. G. I.L,S,T M,V N.

Q,F W Y orP C60 replaced wtth D E,H. K,R,A, G, I. L,S,T M,V NQF W S Y or P- L61 replaced with D. E. H. K, R. N, Q, F W Y P or C. T62 replaced with D, E,H,K,R,N.Q,F W Y P orC. V63replaced withD, E,H, K. R. N, Q, F W Y P ~or C, V64 replaced wnh D. E, H. K, R, N, Q, F W Y P or C, S65 replaced with D, E.

SHK,RN,QF W Y P or C, F66replaced with D. E.H.K. R,N,Q,A, G, I. L, S, T M,V P or C, Y67 replaced with D E. HK,R,N,Q,A,G,I.L S,T M.V P or 00 i o C, Q68 replaced withD,E,HK,R, A,GI,L.S.T M.V F W Y P orC. V69 o replaced with D E, I, K, R, N, Q F W Y P or C, A70 replaced with D, F. H. K. R, N.Q, F W Y P orC. Al7 replaced with D E.H. K.R,N,Q.F W Y P or C. L72 I replaced with D E, H. K. R.N, Q F W Y P or C.Q73 replaced with D, E. H, K,R.

A, G,I. L.S.T MV F W Y P orC, G74 replaced withD E,H.KRN,Q,F W is Y P orC.D75 rcplacedwithH. K R. A, G, I, L,S.T M, V N, Q, F W Y P orC.

L76 replaced with D. E. H. K. R, N, Q, F W Y P or C, A77 replaced with D E. H, K, R,N,Q,F W Y P orC.S78 replaced withD E,HK,R,N,Q,F W Y P orC.

L79 replaced with D, E, K, R, N. Q, F W Y P or C. R80 replaced with D, E, A, G.

IL,S,T M,V N.Q,F W Y P orC, ASl replaced with D E,H, K. R.N,Q,F W Y P or C. E82 replaced with A, G, I. L.S.T M.V N.Q. F W Y P orC.

L83 replaced with D. E. H. K. R. N, Q,F W Y P or C, Q84 replaced with D E, H.K.

R,A,G,IL,S,T M.V F W Y P orC, G85replacedwuhD E.H. K, R. N,Q,F W Y P orC.H86replaced with D, E,A.G0.l.L.S.T M.V N.Q.F W Y P orC.

H¥7 replaced wnth D E, A, G, I, L S. T M, V N, Q F W Y P or C A88 replaced with D, E. H, K, R, N. Q, F W Y P or C; E89 replaced with H, K. R. A. G. I. L, S. T MV N.Q.F W Y P or C.K90 replacedwith D E.AG. L, S.T M,V N.Q.F W Y P or C, L91 replaced with D, E, H, K, R. N, Q, F W Y P or C, P92 replaced wih D. E. H, K, R, A. G, 1, L, S, T M, V N, Q, F W Y or C. A93 replaced with D E. H. K.R.N.Q.F W Y P or C, G repaced wth D E.HK.R,N,Q,F W z P or C. A95 replaced with D E. H, K. R, N, Q, F W Y P or C, 096 replaced with D, E, H, K, R, N, Q, F W Y P or C, A97 replaced with D, E, H, K, R, N, Q. F W Y P or C, P98 replaced with D E, H, K.R, A, G,I.L.S. T M. V N, Q, F W Y orC: K99 replaced with D. E. A. 1. L. S, T M, V N, Q. F W Y P orC. AIOO replaced with COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:12 61 2 92586999 4 262837999 N0.726 D18 00 0 o

W

SD,E,H.KRN,Q,F W Y P orC, GlOl replacedwth D, E.HK,R.N.Q.F W i Y P orC, L02 replaced withD E, H, K. R,N,Q,F W Y P orC, E103 replaced withH. K,RAG,ILL,S,T MV N,Q,F W Y P orC, El 04replaced withH.K.

A RA,G,I,L.ST M,V NQ,FW Y P or C, AI105 replaced with D E.H.KR,N, SQ.F W Y P or C, P106replaced withD.E.H,K,R, A, G. 1L.ST M.V N,Q,F SiW Y orC. A 107replacedwith D, E K, RN,Q,F W Y P orC, VlO 108 replaced S- witthD,E,H,K,R,N.QF FW Y P orC,TI 09replaccdwithD, F.H.K,RN,Q,F SW Y P orTC.AlO1 rcplcd with D, E,H,K,R.N,Q,FW YP orCGlll] C, replaced wuh D E, H. K, R, N, Q. F W Y P or C, LI 12 replaced with D, E, H, K, R, 0 1 N,Q,F W Y P orC,Kll3replaccdwthDEA,G.I,L.S.T MV N,Q,F W Y 0 P or C. I 114 replaced wnth D E, H, K, R, N, Q. F W Y P or C. Fl15 replaced with D, E. H, K,R,N.Q, A, G, 1 L. S.T M. V P orC, El 16 replaced with H. K. R, A. G, LL,S,T M.V N.Q F W Y P orC; PI 17 lreplaced with D E, H, K.R, A,G,I.L.

S.T MV N,QFW Y or C, P 18 replaccdwithD E, H, K, R, A, L.S, T M, V N.Q, FW Y orC, Al 19replaced with D. E, HK, R,N,Q,F W Y P orC, Pl2Orcplaced withD E, H. KR, A.G, I, L, S. T M,V N,Q,F W Y or C: G 21 replaced with D E. H. K, R, N, Q, F W Y P or C, E 122 replaced with H. K, R, A. G.

1,L,S,T M.V NQ,F W Y P orC. G123replacedwith D E. H. K.R,N.Q.F W Y P orC.N24replaccdwithD EHK,R,A,G.I,L,S,T M,V F W Y P orC, S 125 replaced with D, E,H,K,R.N, Q,F W Y P orC. S 126replacedwithD E,H, K,R,N,Q,F W Y P orC, Q 127 replacedwithD E.H,K,R, A, G.1,L.S.T M,V F W Y P orC, Nl28 replacedwith DE,-K, R, A G,LL,S,T M,V FW Y P or C, S129 replaced with D, E, H. K, R, N. Q. F W Y P or C. R130 replaced with D EAG,1,L.ST M.V NQ.F W Y P orC, N 131 replaced wth D E.HK,R,A.

G, IL,S,T M. V F W Y P orC. K132replaced with D E. A. G. IL,S,T M,V NQ.F W Y P orC,R33replaced with D E,A,G,I,L,S,T M,V N,Q F W Y P or C, A 134 replaced with D, E. H, K, R, N, Q. F W Y P or C, V 135 replaced with D E, HK,R,N,Q, F W Y P orC, Q136replacedwith D,E,H. K.R. A.G,I.L.S.

T

r M. J' V Y P or C, G 3Y replcu waiih D K. R. i, Q. F 'V Y F uc C.

P138 replaced with D. E, H. K. RA, GI, S.T M,V N, Q, F W Y orC, E139 replaced with H. K,R.A.G.I, L,S,T M,V N. Q.F W Y P orC. E140 replaced with H, K,R,A,GI,L, S,T M.V N. Q,F W Y P orC, T141 replaced with D E.

H,K.R.N,Q,F W Y P orC, Vl42replaced with D E,H.KR.N,Q,F W Y P COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 D19 00 0 AS 0 (-45 orC; T143 replaced with D, E, H, K, R, N. Q, F W Y P orC, Q 144 replaced with D, Ct E, H,K. R. A, G.I. L, S, T MV F W Y P orC;D145replaced with H, K. R, A. G, I.L,S.T M,V N.Q.F W Y P orC. Cl46replaced with D.E, H. K, R, G. L, 2~ST MV N.Q.F W Y or P-L47replacedwithD E, H, K,R,N,Q,F W Y Por C, Q148 replaced withD, E, H, K, R, A, 0,1, S,T M. V F W Y P orC, L 149 Sreplaced with E. H. K, R, N, Q, F W Y P or C; 1150 replaced with D, E, H. K, R, N,Q, FWY P orC.A151 replaced withD,EH,K.R,N.QF W YP orC.

SDI52 replaced with H, K, R.A, GI, L. S.T M. V N, Q, F W Y P orC, S153 (N replaced with D, E, H. K, R, N, Q. F W Y P or C. E154 replaced with H, K, R. A. G, 00 o 10 ILS,T M,V N.Q.F W Y P orC,T155 replaced withD F. H.1 K. R.N. Q.F W SY P orC: P156replacedwith D E, H, K, R, A, G. I. L, S,T M,V N, Q,F W Y or C, T157replacedwithD. E. H. K, R, N, Q, F W Y P orC. 1158 replacedwith D, H, K, R,N,Q,F W Y P orC, Q159 replaced wh D,E, H, K, R,A.GI. M, V F W Y P orC, Kl6OreplacedwithD E.A,G,I.LS,T M.V NQF W Y P orC, G0161 replaced with D E, H, K. R. N. Q, F W Y P orC. S162 replaccd with D.

E, H. K, R, N. NQ,F W Y P orC, Y163replaced withD, E,H,K,R.N.Q,A.G, L.

ST M,V P orC.Tl64 replaced withD E, H. K.R,N,Q,F W Y P orC,F163 replaced with D, E. H.K.R.N,Q,A,G 1. L, S,T M, V P or C, V 166replaced wnh DE, H,KR. N, Q,F W Y P or C, P167 replaced with D, E. H. K, R. A. G. 1. L. S.

T M, V N, Q, F W Y orC. Wl68 replaced with D E, H, K, R, N, Q.A. G, I, L, S.

T M.V P arC. L169replaced withD E, H, K. R,N,Q,F W Y P orC, 1170 replaced with D. E. H, K, R. N, Q, F W Y P or C, S171 replaced with D E, H. K, R.

N,Q,F W Y P orC,F 172replaced withD E, H,K, R,N, Q, A, G0 1, ,L,S,T M.V P orC. K173 replaced wnth D E, A. G, I, L. S, T M.V N,Q,F W Y P orC. R174 replacedwith D. E,A,G.I L.S.T M.V N,Q, F W Y P orC, G 175 replaced with D.E,H,KR.NQ,F W Y P or C;S176 replaced with D, E,H.K.R.N, Q, F W Y P or C, A177 replaced with D, E. H. K, R, N, Q, F W Y P or C. L17S replaced wnh D E, H.K. R, N. Q, F W Y P orC.E179 replacedwith H. K.R. A, G, 1. L. S.

T M.V N.Q F w V P orC. E180 replaced w.h H, K, R, A, I, L, S r

N,

Q.F W Y P orC,K81 SlrcplacedwithD E. A, G, I, L, S, T M.V N.Q.F W Y P orC, E182 replaced withH. K, R, A, 0G.I. L, S. T M, V N.Q,.F W Y P orC. N183 replaced with D E. H. K. R. A, G. 1, L. S, T M, V F W Y P or K 1 4 rplaced withD. E.A.G I. L S.T M. V N. Q, F W Y P orC. 1185 replacedwih D. E.H.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 862837999 NO.?26 00 K,R,N,Q,F W Y P orC,.LIB6replaced with D E,H.K.RN,Q,F W Y P orC.

c VI87 replaced with D, E.H. K.R, N,Q. F W Y P orC. K188 replaced with D E,A, S I. LL,S,T M.V N. Q,F W Y P orC, E189replacedwithH, K. R,A,G, I.LS, T MV N, Q,F W Y P orC, T190replaced with D. E, H, K,R,N.Q,F W Y P s orC, G 191 replaced with D. E H, K.R.N, Q.F W Y P orC. Y 192 replacedwith D.

E, H,K,R. A, G, I. L,S,T M, V P or C. Fi93replaced with D E,H,K.R, N, S;Q, A. G, I, L, S, T M, V P or C, F194 replaced wlth D E, H, K, R, N, Q, A, G, 1, L, S,T M.V P or C, 1195 replaced withD, E,H,K, R,N, Q,F W Y P orCYl96 0 replaced with D, E. H, K, R, N, Q. A, G0. I, L, S. T M, V P or C, G197 replaced with 0 0 10 D,EH.K,RN,QF W Y P orC, Q198 replaced with D E,H, K,R G,1,L,S, o T M,V F W Y P orC,V199replaced withD E,H,K,R,N,Q,F W Y P orC.

L L200 replaced with D E, H, K, R, N, Q. F W Y P or C, Y201 replaced with D E, H, K, R, N.Q, AG. I. L,S,T M, V P orC. T202replaced withD E. H,K, R.N. Q,F W Y P or C, D203 replaced with H, K, R, A. G, I, L. S, T M, V N. Q. F W Y P or C, K204replaced withD E.A, G, I. L,S,T M.V N,Q.F W Y P orC, T205 replaced with D E. H, K, R, N Q, F W Y P or C, Y206 replaced with D. E, H, K, R, N,Q,A, L,S.T M,V P or C. A207 replaced withD. E.H,K,R.N. Q,F W Y P or C, M208 replaced with D, E, H, K, R, N. Q, F W Y P or C. G209 replaced with D E. H. K, R. N, Q, F. W Y P orC.H210Oreplaced with D E, A, G 1, L, S,T M, V 20 N,Q,F W Y P orC. L2llreplacedwithD F, H, KR,N, Q F W Y P orC.1212 replaced with D. E, H, K, R, N, Q. F W Y P or C, Q213 replaced with D. E, H, K. R, A.G.1L, S,T M,V F W Y P orC. R214 replaced wth D. E,A,G, 1. LS,T M, V N,Q,F W Y P orC,K215replacedwithD,E,A,G,1,L,S.T M.V N.Q,F W Y P orC,K216 replaced with D. E, A, G, l, L, S. T M,V N,Q,F W Y P orC, V217replaced with D, E, H,K.R, N,Q,F W Y P orC, H218 replaced with D E.A.

G, 1. L,S,T M,V N,Q,F W Y P or C; V219 replaced with D, E,H, K. R,N,Q,F W Y P orC, F220 replaced withD E. H,K. R.N.Q,A. G,1,L, S, T M,V P orC.

G221 replaced with D E. H. K, t, N, Q,F W Y P orC, D222 replaced with H, K. R- A, C, I, S.T N. Q, F W v D cr E 223 rplacd H. K, 5 L, S, T M,V N,Q, F W Y P or C, L224 replaced with D, E, H, K, R. N. Q, F W Y P or C. S225 replaced with D. E, H. K, ft, N. Q F W Y P or C, L226 replaced with D, E, H, K,R,N. Q,F W Y P or C. V227 replaced with D, E. H, K.R.N, Q. F W Y P or C, T228 replaced with D E. H, K, R, N, Q. F W Y P or C; L229 replaced COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92586999 4 062837999 NO. 726 P21 00 0 withD,E,H,K,R.N.Q,F W Y P orC, F230replacedwth D, E, H. K.RN. Q, A.

Ct 0. G,,L,S.T MV P orC. R231 replacedwithD. E.AG,ILS,T MV N.Q,F W Y P orC, C232replacdwithD, E,H,K.R,A.G.lL.S.T MV N,Q,F W Y or F- 1233 replaced wth D E, H. K. R. N, Q, F W Y P or C, Q234 replaced with D s EH,KR,AYG,.1.LS,T MV F W Y P orC, N235 replacedwtth E, H. K.R, A,GI,L,.S,T MV F W Y P orC, M236replacedwithD, E.H,K,R.N.Q,F W Y P orC, P237replaedwthD, E, H, K, R, A, G, I. S. T M,V N,Q,F W Y or C, E238 replaced with H, K, R, A. G. 1, L, S, T M. V N, Q, F W Y P or C. T239 0replaced with D, fE, H, K, R, N, Q, F W Y P or C. L240 replaced with D E, i, K, R, o io N.Q.F W Y P orC, P241 replacedwthfD EH,K,R, A,GI,1, S,T M.,V N,Q, oF W Y or C, N242 replaced with D E H, K R, A. G. I. L, S. T M, V F W Y P or C.N243 replaccd wsrhD E,H,K,R.A,G,1.L,S,T MV F W Y P orC,S244 replaced with D E. H. K. R, N. Q. F W Y P or C. C245 replaced with D, E, H. K, R, A,G.,L,S,T MV NQ,F W Y orP- Y246 replaced with D E. H. K. R. N. Q. A, G,IL.S.T M.V P orC, S247 replaced withI0 E,H,K,R.N,Q,F W Y P orC.

A248 replaced with D, E, H, K, R, N, Q, F W Y P or C. G249 replaced with D E. H, K,R,N,Q,F W Y P orC, 125DreplacedwithD, E,H,K.R,N,Q,F W Y P orC, A251 replaced with D, E, H, K, R, N, Q. F W Y P or C, K252 replaced with D E, A, G,1I.LS, T M,V N,Q,F W Y P orC, L253 replacedwithD EH,K.RN.Q F W Y P orC, E254replacedwtH, K,R, A,G, I,L,S,T M,V N,Q,F W Y P or C. E255 replaced with H, K. R, A. 0, 1. L. S. T M, V NQ, F W Y P or C, 0256 replaced with D. E, H. K. R. N, Q, F W Y P or C, D257 replaced with H. K, R, A. G.

L S, T M, V N.PQ. F W Y P or C. E258 replaced with H, K, R, A, G. I. L. S, T V N.Q. W Y P or C: L259 replaced wth D E. H, K, R, N, Q. F W Y Pr C, Q260 replaced with D, E, H, K. R. A. G 1. S. T M, V F W Y P or C. L261 replaced with D E H. K R. N. Q, F W Y P or C. A262 replaced with D E. H. K. R.

N,Q,.F W Y P orC, I263replacedwith D E. H. K.R,N,Q,F W Y P orC, P264 replaced with D, EH K, R, A, G. 1, L, S, T M, V N, Q F W Y or C, R265 replaced withD F A, S.1,L,S,T N,Q, F W v D orc,E26repla,,, cwihH, K.R.

3a A,G,I.L,S.T M.V N.Q,F W Y P orC. N267replaccdwithfD E,H, K. R.A,G.

LLST MV F W Y P orC. A268 replaced with D H. K, R. N,QF W Y P or C, Q269 replaced with D, E. H. K, R, k G.1, L, S. T M, V F W Y P or C. 1270 rcplarcdwith D E.HK.R,N,Q.F W Y P orC. S271 replacedwithD EH.KR.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 P22 00 0 0 N. Q, F W Y P or C; L272 replaced with D E. K K, R, N. Q. F W Y P or C.

^C D273 replaced with H, K, R. A, G, 1. L. S. T M. V N, Q, F W Y P or C. G274 replaced with D E, H. K, R. N. Q, F W Y P or C. D275 replaced with H. K, R, A, G, I, L, S. T M,V N,Q,F W Y P or C, V276 replaced with D, E, H, K, R, N, Q, F W Y P or C, T277 replaced wh D. E. H. K. R. N. Q, F W Y P or C; F278 replaced Swith D, E, H, K, R. N, Q, A. G, 1. L. S. T M, V P or C; F279 replaced with D. E, H.

K. R, N, Q. A, G. I, L. S. T M, V P or C, G280 replaced wilh D. E, H. K. R, N. Q. F W Y P or C. A281 replaced with D E. H. K, R N. Q. F W Y P orC, L2 8 2 0 replaced with D E. H, K, R, N, Q, F W Y P or C. K283 replaced with D E, A. G, 1.

0 0 L, S, T M V N,Q,F W Y P or C. L284 replaced with D.E.H. K, R, N,QF W oY P or C. and/or L285 replaced with D, E. H. K, R. N, Q, F W Y P or C.

Polynucleondes encoding these polypeptides are also encompassed by the invention.

The resulting Neutrokme-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokme-alphaSV functional actvities and/or physical properties (such as. for example, enhanced or reduced stability and/or solubility) described throughout the specification and known in the art. Preferably the resulting proteins of the invention have an increased and/or a decreased Neutrokine-alpha and/or Neutrokine-alphaSV functional activity More preferably the resulting Neutrokmealpha and/or Neutroinke-alphaSV proteins of the invention have more than one increased and/or decreased Neurokine-alpha and/or Neutrokine-alphaSV functional activity and/or physical property In an additional embodiment, Neutrokine-alpha polypeptides of the invention comprise, or alternatively consist of, more than one anuno acid 2, 3,4. 5. 6.7 8, 9 10, 15,20, 30 and 50) replaced with the substituted amino acids as described above (either conservative or nonconservauve).

In another embodiment of the invention, non-conservative substitutions of the Neuirokine-alphaSV protein sequence provided in SEQ ID NO- 19 include: Ml replaced with D E. H. K. R, N, Q, F W Y P or C, D2 replaced with H. K, R, A. G. 1, S,T w k, N, Q. r W Y P or C. D3 repicu with H. K, R. n, G, S, T ivi. v N,Q,F W Y P or C:S4 replaced with D E,H,K.R,N,Q, F W Y P replaced with D E, H, K. R, N. Q F W Y P or C, E6 replaced with H. K. R. A. G. I.

L, S. T M. V N. Q, F W Y P or C, R7 replaced with D E. A. G,I. L, S, T M. V N, Q.F W Y P orC.ES replacedwith H,K.R,A. 0, I,L,S,T M.V N.Q.F W Y P COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92596999 4 062937999 N0.726 U23 00 0 orC, Q9 replacedwihD, E.H,K,R,A,G,IL,S,T MV F. W Y P orC, S0 :t replaced with D E, H, K. R, N, Q, F W Y P or C, RI I replaced with D, E, A, G, I. L.

S.T M,V N,Q,F W Y P orC, L12replacedwithD E,H,KIR,N,QF W Y P orC, Tl3 replacedwithD EH,K,R,N,Q,F W Y P orC, Sl4replacedwithD E, SH,K.R,N.Q,F W Y P orC,.CdreplacedwithD, E,H,K, R.A.0.LS, T M, V N,Q,F W Y orP' Ll6replacedwi hD E,H,K,R,N,Q,F W Y P orC, K17 rcplacedwithD, E, A, G, I, L, S, T MV N,Q,F W Y P orC, Ki8replaced with D.E,A,G,I.L.S.T MV N.Q,F W Y P orC, Rl9replacedwith D, E, A, G,,L, S.T MV N,QF W Y P orC, E2OreplacedwithH AG,I,L.ST MV o io N.Q..F W. Y P orC, E.21 rcplaced wih H, K, R. A, G, 1, L. S, T M,V N,Q,F W oY P or C, M22 replaced wih D. E Ii, RN. Q, F W Y P or C. K23 replaced with D EA. L, S, T M, V N Q. F W Y P orC, 24 replaced withD. E. H. K.

R,N.Q.F W Y P orC, K25replacedwithD. FAG,I.L.S,T M,V NQ.F W Y P orC, E26replacedwithH, K,R.A, G.LL.S.T M,V NQ.F W Y P orC.

13 C27 replaced with D, E, H, K, R. A, G, 1. L. S. T M, V N, Q, F W Y or P- V28 replaced with D E, H, R, N, Q, F W Y P or c; S29 replaced with D, E, H, K, R, N,Q.F W Y P or C.130 replaced with D E,H,K,R,N,Q,F W Y P orC L31 replaced with DE. H, K, R. N, Q, F W Y P or C. P32 replaced with D, E, H, K, R, A,G,I.LS,T M,V NQ,F W Y orC, R33replaced with D E,A,G.I.LS.T M.V NQ,F W Y P orC, K34replacedwithD, E.AG,IL,S.T MV N,Q.F W Y P orC. E35replacedwithH, K,RA.G,I.L.S.T M.V NQF W Y P or C; S36 replaced with D. E. H, K, R, N, Q. F W Y P or C. P37 replaced with D, E. H, K.R.AtGIL,S,T MV NQ.IP W Y orC;S38replacedwithD F,H,K.R.N.

QF W Y P orC. V39replaced withD E,H,K,R,N,Q.F W Y P orC. R40 replaeedwth D,E.A.G.tL.S,T M.V NQ,F W Y P orC, S41 replaced with D E.H.K,R.N.Q.F W Y P orC,S42 rcplaccdwithD, E.H.K.R,N.Q.F W Y P orC. K43 replaced wthD, E,A, GI.L,S. T M.V N,Q,F W Y P orC. D44 replaced with H, K, R. A. G, 1, S, T M, V N. Q, F W Y P or C, G45 replaced with D E, H, K. R, m, Q, F Vv Y P or C, Ko rcpiacea wirn D E, A, G,I L, S, T V N, Q, F W Y P or C, L47 replaced wth D. E, H, K. R, N. Q, F W Y P or C.

L4 replaced with D. E. H. K. R, N- Q. F W Y P or C. A49 replaced with D E. H. K.

R,N,Q,F W Y P orC, A50replaced withD E,H.K,R,N,Q,F W Y P or C.

T51 replaced with D. E. H, K. R. N, Q. F W Y P or C. L52 replaced with D E, H, K, COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92586999 4 062837999 NO.'726 124 00 O0 R,NQF W Y P orC. L53replaced with D.E,H. K. RNQ.F W Y P or C.

L54 replaced with D, E. H. K, R, N, Q, F W Y P or C. A55 replaced wth D, E, H. K, R. N,Q,F W Y P orC;L56replacedwithD, E,H. K.R,N,Q,F W Y P orC.

L57 replaced with D, E. H. R. N. Q, F W Y P or C, S58 replaced with D E, H, K, R, N,Q.F W Y P orC, C59replacedwithD E.H,K,R,A,G,1,L,S,T M.V N.

Q,F W Y orP-C6OrcplwdwthD, E,H,R.A.G.i,L,.ST M.v N,Q.F W Y or PL 61 replacedwith D EH.K,R,N.Q,F W Y P orC.T62replaccd with D E HK.R.N.Q.F W Y P orC, V63 replaced with D. E,H,K,R.N.Q.F W Y P 00 or C,V.64 replaced with D, F, H, K. P. N. Q. F W Y Por C, S65 replaced with D E.

oo H K, R.N.Q,F W Y P orC. F66 replaced with D, E, H. K, R,NQ AG. LL, S.

T M,V P orC. Y67 replacodwithD, E,H, K, R, G.1, L. S.T MV P or C, Q68replacedwithD E.H.K,R.A.GI1,L.S.T M,V F W Y P orC.V69 replaced with D. E. H, K, R. N. Q. F W Y P or C. A70 replaced with P E, E. K R.

N,Q.F W Y P orC, A7l replacedwth D.E,.H,K.R,N.QF W Y P orC. L72 is replaced with D, E,H, K,R,N,Q,F W Y P orC, Q73 replaced with D F, H, K, R.

A, G. I. L. S, T M, V F W Y P or C, G74 replaced with D. E, H, K. R, N, Q, F W Y P orC, D75replaced with H, K, R,A,G,1, L,S,T M.V N.Q,F W Y P orC.

L76 replaced with D, F, H, K. R, N, Q, F W Y P or C. A77 replaced with D E, H. K, RN,Q,F W Y P orC. S78replacedwithD, K. R.N,Q,F W Y P orC, L79 replaced with D, E, H, K I, N. Q. F W Y P or C. R80 replaced with D. E. A, G.

1,L, S,T M,V N.Q.F W Y P orC, A81 replacedwithD E,H,K,R,N,Q,F W Y P orC, E82replacedwithH, K,R,A,G, LL. S,T M,V N,Q,F W Y P orC.

L83 replaced with D, E, Ii K. R, N, Q, F W Y P or C, Q84 replaced with D EH,K.

R,A.G.,LS,T MV F W Y P orC, G85replacedwithD FH, K, R, N, Q, F 2S W Y P orC, H6replaced withD E,A.G,1.L,S.T MV N,Q,F W Y P orC.

H87 replaced with D E, A, 0, I, L, S, T M. V N, Q, F W Y P or C. A88 replaced with D, E, H. K, R, N, Q, F W Y P or C. E89 replaced with H, K. R. A. G. I. L, S. T M.V NQ F W Y P orC. K9OreplaedwithD EIA.GI.,S.T M,V NQ,F W r P Or C. L91 replaceo with D E, H. K, R, r, Q, F W Y P wj C. r92 ieplaccu withD. EH,K,R,A,G,,L.S,T MV NQF W Y orC. A93replacedwith D, BHK,R.NQ.F W Y P orC. G94replacedwithD.E.H.K.R.N.Q.F W Y P or C, A95 replaced with D E. H. K. R. N, Q. F W Y P or C. G96 replaced with D. E, H,K,R,N,Q F W Y P orC.A97replac~dwith D E.H.K.R,NQ,F W Y P or COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200 16:12 61 2 92586999 4 062837999 N0.726 025 00 O0 C, P98 replaced with D E. H, K, R. A. G, I, L, S, T M,V N.Q,F W Y orC, K99 replaced with D E. A. G, I, L, S, T M, V N. Q, F W Y P or C, At00 replaced with D,F ,H.K,R,N,Q,F W Y P or C, Gl01 replaced with D E,H,K,R. N,Q,F W Y P orC. L102 replaced with D E, H, K, R. N, Q, F W Y P or C; El03 replaced wih H. K. R, A. G, I. L, S. T M, V N, Q. F W Y P or C. E104 replaced with H. K, R, A, G, I. L S. T MV N,Q.F W Y P orC, Ai05 replaced wsth D. E.H.KR. N, Q.F W Y P or C.Pl06 replaced with D E, H. K, R. A, 0.I, L, S. T M.V N.Q.F W Y or C, A 107 replaced wth D E, H. K, R, N, Q. F W Y P or C. V 108 replaced Swith D, E, H, K, R, N, Q, F W Y P or C, TI09 replaced with D, E, H, K, R, N.Q. F 0 10 W Y P or C, AllOreplaced wth D E, H.K,RN,Q,F W Y P orC,G111 0replaced with D E. H. K, R, N, Q. F W Y P or C. L 112 replaced with D, E, H. K, R.

N,Q,F W Y P orC. K113 replacedwithD E,AG,I,L,S.,T M.V N,Q,F W Y P or C. 114 replaced wth D E.H, K, R. N, Q,F W Y P or C. F 115 replaced with D. E. H. K. R. N.Q. A, G, 1, L. S.T M,V P or C, El 16 rplaced wth H, K, R, A, G, IS LL, S,T M,V N,Q,F W Y P orC, Pll17replacedwithD. E,H.K.R.A.G, 1. L, S, T M. V N, Q, F W Y or C. Pl 18 replaccd with D E. H. K. R. A. G. 1, L. S, T M.

V N.Q.F W Y orC, AllreplacedwithD E.H,K,R,N,Q.F W Y P orC.

replaced wth H,K, R, A. 0, 1, L, S.T M,V N,Q,F W Y orC. G121 replaced with D E, H, K, R, N, Q F W Y P or C, El22 replaced with H. K, R, A, G.

1,L,S,T M.V N,QF W Y P or C. G123 replaced with D E. H, K, R, N, Q, F W Y P orC, N124 replaced with D E, H, K, R, A, G,I, L, S, T M, V F W Y P or C, S125 replaced with D E. H, K. R. N, Q, F W Y P or C; S126 replaced with D E, H.

K,R,N.Q,F W Y P or C, Ql27 replaced with D E, H. R.A,G,I.L.S,T M. V F W Y P orC, N128 rplaced wahith D. E,H.K,R, A,G, I,L,S,T M,V F W Y P or C, S129 replaced with D. E, H. K, R, N. Q, F W Y P or C, R!30 replaced with D E, A, 0, L, S. T M, V N, Q,F W Y P or C, N 131 replaced with D. E, H, K, R, A, G.I,L,S,T M.V F W Y P or C, K132 replaced with D. E. A G, I, L. S, T M,V N,Q,F W Y P orC, R133 replaced with D E, A. G, I L. S, T M.V N,Q.F W Y Tor rC. -134 replnced with D. E, H, K, R. N, Q, F V Y P or C, v 35 reptacez with D E. H, K, R, N, Q, F W Y P or C, Q136 replaced with D E, H, K, R, A,G, I. L, S, T M,V F W Y P orC, Gl37replacedwithD E.H. K, R,N. Q.F W Y P orC.

Pl38 replaced with D E, H, K. R,A,G, I, L,S.T M,V N,Q,F W Y orC, E139 replaced with H, K,R. A, G. 1,L.S,T M.V N.Q.F W Y P or C. E140 replaced COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:12 61 2 92596999 4 062937999 N0.726 U26 00 0 with H, K, R, A, G, L, S. T M, V N. Q, F W Y P or C. T141 replaced with D, E, C H. K,RNQ.F W Y P or C, G142replacedwithD EHK,R,.NQ,F W Y P or C. S 143 replaced with D E, H, K, R, N. Q, F W Y P or C, YI44 replaced with D E. H.K, RN, K Q,AGI.L.S.T M.V P orC, T145replacedwithD. E.H.K.R.N, Q,F W Y P orC, F146replacedwithD. E,H, KR, N,QA,G.I,L,S.T MV P or C; V 147 replaced with D L H, K. N, Q, F W Y P or C, P148 replaced with D E,H,K. IA, G,.L,S.T MV NQ,F W Y orC, W149replacedwith D EH, If, I, A, G, 1, L. S, T M,V P orC. LlSOreplacedwihD E, H, K. RN. 0,F W Y P or C. LI51 replaced with D E,H.KKR. N,.F W Y P orC. Sl52replaced o 10 withD, EHK.R,N,Q,F W Y P orC. Fl 53 replaced with D, EH,K.R,N.Q,A.

o 0,IL. S.T MV P or C, K154rcplaccd with D. E.A.G.I.LS.T M.V N.Q.F C W Y P orC. Rl55replacedwithfD E. A,G.1, L,ST M.V N,Q.F W Y P orC, G136 replaced with D, E, H, K, t N, Q, F W Y P or C, S157 replaced with D, E H, K,RN,Q,F W Y P orC. AI5BrcplacedwnhD.E.H,K,R.N,Q,F W Y P or C. L159 replaced wih D, E, H, K R, N, Q, F W Y P or C; Ei60 replaced with H. K, R.A G,1,L,S,T MV N,Q F W Y P orC, El61 replacedwith H.K, P.A.G.I, L,S,T M,V N,Q,F W Y P orC, K162replacedwithDE.A,G.1.L,S.T M,V NQ.F W Y P orC, El63replacedwithH, K,R,A,G,,LS.T M,V NQ,F W Y P orC, N164rcplaccwithD E.H,K,RA.G,1,L,S,T M,V F W Y P orC.

Kl65replacedwithD, E.A,0.1,LS,T M,V NQ.F W Y P orC, Il66replaccd with D,E,H,K,R,NQ,F W Y P orC, Ll67replacedwith D E,H.K.R,N,Q,F W Y P orC. V168replacedwth D,E.H.K.R.N,Q.F W Y P orC, K169 replacedwth D E.A.G,1,L,S,T M.V NQ.F W Y P orC. H, K, R, A, G, 1. L, S,T MV N,Q,F W Y P orC, TI71 replaced with D. E. H. K, R.NQ.F W Y P orC.GI72replaCedwithD.E,H,.R,N,Q,F W Y P orC.

Y173 replaced with D, E, H, K. R, N. Q, A, G. 1. L, S. T M, V P or C. F174 replaced wthD, E,H K. RN,Q.A,GIL,S,T MV P orC, Fl75replacedwithD E.H.

K.RIN.Q.A,G.I,L, S,T MV P orC, 1176replacedwith DE.H,K,R,N,Q F W V n orC, 7 replacuwith D E.H. F4 L. Q. A,Ci, j. 1- 3,7 wi, v P orC.

178 replaced wnh D. E. H, K R, N, Q, F W Y P or C. Q179 replaced with D E, H, KR.A.G, IL, ST M.V F W Y P orC, Vi80 replaced withD E. H. K. l. N. Q.

F W Y P orC. Lll rcplaced with D E.H,K, RLN, Q,F W Y P orC. Y82 replaced with D, E, H. K, Rt N, Q A, G. 1. L, S, T M. V P or C, T193 replaced with COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P27 00 0 (C D,E.H.K.R.N,Q,F W Y P orC, Dl84replaced withH, K, R,A, 0,I.L.S,T M, MC V N,Q.F W Y P or C. K185replaced with D.E. A, G,I, L,S,T M,V N.Q,F W Y P or C, T186 replaced wth D E, H, K, R. N. Q. F W Y P or C. Y187 replaced wsthD, E,H,K,R,N,Q.A,G,I.L,S.T M,V P orC.Al88 replaced with D,E.H.

S K,R. N,Q,F W Y P or C, M19 replaced with D E,H,K,R, N, Q.F W Y P or C, 0l190 replaced with D E,H.K. R,N,Q.F W Y P orC, Hl91 replaced wh D E, A.G.I.L.S.T M.V N.Q,F W Y P or C. L92 replaced wth D, E. H. K, R. N, Q, SF W Y P or C. 11l93 replaced with D, E, H, K. R, N. Q. F W Y P orC. Q194 0 replaced with D. E. H. K, R, A, G. I, L, S. T M. V F W Y P or C, R195 replaced 00 1o with D.E,A.G,I,L,S,T M.V N,Q,F W Y P orC, Kl96replaced wiuhD E, A.

0 G.1. I, T M, V N, Q, F W Y P or C; K197 replaced with D, E, A, G, I, L. S, T M, V N. Q. F W Y P orC, V198 replaced with D E, H, K, R, N, Q, F W Y P or C; H199 replaced with D E, A. G, I, L, S, T M. V N, Q, F W Y P or C, V200 replaced with D. E, H, K. R. N, Q, F W Y P or C. F201 replaced with D, E, H, K, R, 1> N,Q,A,G,I,L,S,T M,V P or C, G202 replaced with DE, K,R.N.Q F W Y P orC. D203 replaced with H, K, R, A. G. I L. S, T M. V N, Q, F W Y P or C.

E204 replaced wth H, K, R. A, G.I.L, S. T M. V N, Q. F W Y P orC, L205 replaced with D, K, R, N, Q, F W Y P or C, S206 replaced with D, E. H. K. R N, Q. F W Y P orC. L207 rplaced wih D E. H, K, R, N, Q, F W Y P orC, V208 replaced with D, E, H, K, R, N, Q, F W Y P or C; T209 replaced with D E, H, K R, N. Q,F W Y P orC, L210 replaced with D E, H, K, R, N, Q.F W Y P or C, F211 replaced with D E, H. K, R, N. Q. A. G, I, L. S. T M, V P or C, R212 replaced wth D E, A, G, I, L. S, T M, V N, Q, F W Y P or C, C213 replaced with D. E. H, K, R, A, G. I, L. S. T M, V N. Q, F W Y or P-1214 replaced with D, E, H. K R, N, Q.F W Y P or C, Q215 replaced with D,E.H,K, A, G, 1. L, S.T M.V F W Y P orC. N216replaced withD E, H.K,R.A.G,I.L,S.T M,V F W Y P orC, M217 replaced with D. E. H, K, R. N. Q, F W Y P or C, P218 replaced wnh D E, H.

K. R, A.G, I. L. S, T M. V N, Q, F W Y or C, E219 replaced with H, K, R, A, G, 1 L. S,'r M, I N, Q. v or C, T220 icpauecu wih D E. H, K. R. I. Q, F W Y P or C, L221 replaced wnh D EH, K. R, N. Q, F W Y P or C, P222 replaced with D, E, H. K, R, A, G, I, L, S, T M. V N. Q. F W Y or C. N223 replaced with D E, H, K, R, A, G. I, L. S. T M. V F W Y P or C. N224 replaced wth D E, H. K, R.

S,T M,V F W Y P or C, S225 replaced with D E. H. K. R.N. Q.F W COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 D28 00 0 N Y P orC, C226 replaced with D.E.H K,R, A. .LL, S.T M.V N.QF W Y or t P- Y227 replacedwithD E,H. K,R, N,Q.A,G, I,L,S,T M,V P orC.8228 2replaced with D E, H, K, R, N, Q F W Y P or C, A229 replaced wuth D, E, H, K, R, N, Q. F W Y P orC, O230 replaced with D E H. K, R, N. Q,F W Y P orC, 1231 replaced with D E. H, K, R, N, Q, F W Y P or C, A232 replaced wilh D, E, H, K, R.

N,Q.F W Y P orC; K233 replaced with D.E. A.Gl L.S.T M,V N,Q,F W Y SP or C. L234 replaced with D, E, H. K, R, N, Q, F W Y P or C, £235 replaced with SH, K, R, A, G. I. L S, T M.V NQ F W Y P or C. E236replaced withH, K. R.A, 0 GI.L S,T M,V N,Q,F W Y P orC, 0G237 replacedwith D,E.H,K, R.N,Q,F 0 0 10 W Y P orC, D238 replaced withHK. R,A,G I, L,S,T M,V N,Q,F W Y P or SC. E239 replaced withH, K. R, A, G. L, S,T M.V N,Q. P W Y P or C, L240 replaced with D E. H, K. R. N, Q, F W Y P or C; Q241 replaced with D E, H, K, R.

AGI,L.S.T M.V F W Y P orC. L242 replaced wathD E, H, K. R,N,Q.F W Y P or C, A243 replaced with D. E, H. K, R. N, Q, F W Y P or C. 1244 replaced is with D, E, H, K, R, N, Q, F W Y P or C" P245 replaced with D E, H, K, R, A, G, I, L,S,T M, V N, Q, F W Y orC; R246rcplaced with D E,A, G,I. L,S,T M, V N, Q,F W Y P orC.E247replaced with H. K,R,A. GI. L,S,T MV N,Q.F W Y P orC, N248 replaced withD. FE.H, K,R, A,G, I. L.S,T MV F W Y P orC, A249 replaced with D E. H. K. R, N, Q. F W Y P or C, Q250 replaced with D E, H, K.R.A G. I, L.S, T M. V F W Y P orC.I 1251 replaced with D E,H,K.R, N.Q, F W Y P or C"S252replaceddwithD, E, 1iK.R,N, Q, F W Y P orC, L253 replaced with D, E. H, K, R, N, Q, F W Y P or C, D254 replaced with H, K. R, A, G, I,L,S,T M.V N.QF W Y P orC. G255 replaced withD E, H, K, R, N,Q,F W Y P orC, D256replacedwithH, K, R, A. G. I, L. S, T M. V N,Q,F W Y P orC.

V257 replaced with D E. H. K. R. N. Q, F W Y P or C, T258 replaced with D E, H, K, R, N,Q,F W Y P orC, F259 replaced with D, E. H. K, R. N, Q, A.G. I, L, S.T M,V P orC. F260 replaced dwith D E. H,K. R. N, Q.A, G, I,L,S,T M, V P orC.

G261 replaced with D E, H. K. R. N, Q, F W Y P or C. A262 replaced with D E. H, K. R, N. Q, C W v a r C. L253 rplac "vd wl D, E, H, K, R. Q, F W Y P u C, K264 replaced with D E. A. G, I, L, S, T M, V N, Q, F W Y P or C, L265 replaced with D. H. K, R, N, Q. F W Y P or C, and/or L266 replaced with D E, H, K. R.

N. Q. F W Y P or C Polynucleotides encoding these polypeptidcs are also encompassed by the invention. The resulting Neutrokinc-alpha proteins of the COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO. 726 U29 00 Cl Inventrion may be routinely screened for Neutroklne-alpha and/or Neutrokine-alphaSV functonal activities and/or physical properties (such as, for example, enhanced or reduced stability and/or solubility) described throughout the specification and known in the art. Preferably the resUlting proteins of the mnvention have an increased and/or a decreased Neutrolune-alpha and/or Neutrokne-alphaSV funcutional activity More preferably the resulting Neutrokwealpha and/or Neutrokine-alphaSV prormns of the invention have more than one increased and/or decreased Neutrolunc-alpha and/or Neutrokme-alphaSV functional activity and/or physical property In an additional embodiment, Neutrolone-alpha polypepudes of the invention 00 10 comprise. or alternatively consist of, mom than one ammo acid 2, 3, 4. 5, 6, 7 8, 9 10, 15. 20, 30 and 50) replaced with the substituted amino acid. as described above cl (either conservative or nonconservauve).

For example, preferred non-conservacive substtutions of the Neutrokme-alpha protein sequence provided in SEQ ID NO:23 include: RI replaced with D E, A, G, 1.

L S. T M, V N, Q. F W Y P or C. V2 replaced wlh D, E, H. K, R, N, Q, F W Y P or C, V3 replaced with D E. H, K, R, N, Q, F W Y P or C, D4 replaced with H.

K,R,A,G.1,L,sT MV N,Q,F W Y P orC. L5 replaced with D, E. H, K. R. N, Q, F W Y P or C, S6 replaced with D E. H, KR, N, Q, F W Y P or C, A7 replaced with D E, H, K, R. N, Q. F W Y P or C, P8 replaced with D E, l K. R. A.

G, L. S. T M, V N, Q F W Y or C. P9 replaced with D E, H. K. R, A. G, L S.

T M, V N, Q,F W Y or C, Al0 replaced with D, H. K, R, N, Q F W Y P or C.

Pl I replaced with D,E. H. KR. A.G. I,L, S, T M,V N, Q,F W Y or C. C12 replaced with D E. H, R, A. G. L, S. T M, V N. Q, F W y or P, L13 replaced with D. E. H. R, N. Q, F W Y P orC, Pl4 mplaced with D E, E. K. R. A, L S, T M,-V N, Q F W Y or C. GIS replaced with D E, H. K, R. N, Q. F W Y P or C, C16 replaced with D E. H. K. RA, G, I. L, S, T M, V N, Q F W Y or P- R17 replaced with D. E. A, G,1. T M, V N.Q, F W Y P or C. H18 replaced with D E,A, G.IL, S,T M, V N.Q,P F W Y P orC. Sl9replaced wth D, E. H. K, R, N, QF W Or C, Q20 replaced w::h D E. H, K R, A. C, i, S' W Y P or C, H21 replaced wnith D E, A, G. I L. S, T M. V N. Q, F W Y P or C, D22 replaced with H, K, R, A. 1. L S, T M, V N, Q. F W Y P or C, D23 replaced with H, K. R. A. G. I, L, S. T M. V N, Q. F W Y P or C. N24 replaced wnith D E.

H, K, R. A, G, L. T M. V F W Y P or C, 025 replaced with D E. H, K, R, N.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:12 61 2 92586999 4 062837999 NO.726 130 00 0 Q. F W Y P or C. M26 replaced with D. E, H, K. R. N, Q, F W Y P or C, N27 rplaceCd with .D E. H. K, R, A, G, L L, S, T M. V F W Y P or C, L28 replaced with DIE,H.KRN,QF W Y P orC. R29replacedwthD E,A.G,I.LS.T M,V N.Q..F W Y P orC: N3OreplacedwithD E,H,K,R,A,O,.LL,S,T MV F W Y P or C; R31 replaced with 1, E. A, G, 1L. S, T M, V N, Q. F W Y P or C, T32 replaced with 1 E. IH, K R, N, Q, F W Y P or C, Y33 replaced wth D, E, H, K, R, N,Q,A,0, 1 L,S,T M.V P orC, T34replacedwith D E, H, K, R, N, Q, F W Y P orC, F35 replaced with D EH, K. R, N,Q,A,0, I, LS,T M.V P orC,V36 replaced with D, E, H, K. R, N. Q. F W Y P or C. P37 replaced with D EI. &R.

o 10 A,G. 1, L,S,T MV NQ,F W Y orC. W38replacedwithD E H.K.R.N.Q.

A,

G. LLS.T MV P orC. L39replacedwth D.E.H, K.R,N,Q.F w Y P orC, replaced with D, E. H. K. R. N, Q, F W Y P or C. $41 replaced with D, S H. K, R.N.Q.F W Y P orC. P42replacedwithD.E.H.K.RN,QA.uG..L.S ,T M.

V P or C. K43 replaced with E A. G. I, L. S. T M, V N. Q, F W Y P or C, R44 rplaced with D, E, A. G. I, L, S,T M, V N, Q, F W Y P orC. G45 replaced with D,E,HK.R,N,Q.F W Y P orC. N46replaced withD E,H,K.R,A,0,1,L,S, T M, V F W Y P or C. A47replaced wtth D, E, H, K, R, N. Q, F W Y P or C, L48 replaced with D, E. H, K. R. N. Q. F W Y P or C, E49 replaced wifh H, K, R, A.

G.I.L,S.T M.V N,Q.F W Y P orC.E5OreplaedwithH. K.R,A,0,.tS.T M,V N,Q F W Y P orC. K51 replacedwithD D A,G,1,L,S, T M.VN, Q,F W Y P orC, E52replacedwithH. K,R,A,G,L,S.T MV N.Q.F W Y P or C. N53 replacedwJthID, E,H,KR.A.G.I,LS.T M.V F W Y P orC, K54 replaced with D E, A, G, 1 L, S, T M, V N, Q, F w Y P or C. 155 replaced wl D, EH.KRN,Q.F W Y P orC. V56replacedwithD E,H,K,R.N,Q.F W Y P 2s or C, V57 replaced with D F H, K, R. N. Q, F W Y P or C. R5S replaced with D. E, A,G,I.L.S.T M.V N.Q,F W Y P orC. 059replacedwnlhD. E.H.K.R.A.G.

1.L,S.T MV F W Y P orC, T6OreplacedwithD EH,K,R,N,Q,F W Y P or C, 061 replaced with D E. H, K, R, N. Q, F W Y P or C, Y62 replaced with D. E.

,.KK,N.Q,A,G.1,L.S,T M.v P orC, F63rcplacea winD, EiH,KR. riQ, 3o A.G,1,L,S.T MV P orC. F64 replaced with D, E,H K,R.NQ, AG.IL,S.T M. V P or C, 165 replac d with D E.H.K.R, N Q. F W Y P or C, Y66 replaced wthD. EH,K,R.NQ,A.GIL.S.T MV P orC. S67replaced withD EH.K, RN,Q.F W Y P orC. Q68 replacedwthD. E.HK.R.A.G..L.LS.T M.V F COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 16:12 61 2 92596999 4 062937999. NO. 726 IP31 00 0 0 W Y P or C, V69 replaced with D, H, K, R, N, Q, F W Y P or C. L70 replaced Swith D.E.H,K,R.N,Q,F W Y P orC, Y71 mplacdwithD, E,H,KR,N,Q,A, G,.L,JS,T MV P orC.T72replacedwithD, E,H,K,R, NQ,F W Y P orC, C*N D73 replaced with H. K, R, A, G, L L, S, T M, V N, Q, F W Y P or C, P74 replaced s with D.E.HK.R,A.G.I,L S,T MV NQ,F W Y orC, 175 replaced withD, E, H,K,R,N,QF W Y P orC, F76nrplacedwithD E,H ,K,RN.Q.AG,I.L S, T MV P orC, A77replacedwthD, ,H,KR,N,Q,F W Y P orCM7S replaced with D, E, H, K, R, N, Q, F W Y P or C, G79 replaced with D. F, Hi K R, N, Q, F W Y P at Q H80 mplacdwithD. EA, G.1. L,S, T M, V N, Q.IF W Y P or C. V81 replaced with D E, H.K R, N,Q. F W Y P or C, 182 replaced wth D, SE.H.K,R,N,Q,F W Y P orC, Q83rplacedwthD E.H,K,RA,G.I.L,S,T M.V F W Y P orC. R84replacedwithD, E,A.0IL.S. T MV N,Q.F W Y K) P or C, K85 replaced with D E, A, S, T M, V N, Q, F W Y P or C; K86 replaced with D. E, A, G, 1, L, S, T M. V N, Q. F W Y P or C, V7 replaced with D.E.H.,K.R.N,Q,F W Y P orC.H88replacedwithfD E,A,G,1,L.S.T MV N.Q,F W Y P orC. V89replacedwithD. E.H,K,R,N.QF W Y P orC: replaced with D E, H. K. R. N. Q. A. G, 1. S.T M. V P or C. G91 replaced with D.

E,H,K,R,N,Q,F W Y P orC, D92replaced with H, K. R. A,G,I.L,S,T MV N,Q.F W Y P orC, E93 replacedwithH KR,A,G,TL,S,T MV N.Q.F W Y P or C. L94 rcplaced with D E, H, K, R. N, Q F W Y P or C; 95 replaced with D,E,H.K,R,N,Q,F W Y P orC, L96replacedwithD E,H,K,R,N,Q,F W Y P or C, V97 replaced with D. E, H. K. It, N. Q. F W Y P or C, T98 replaced with D, F. H. K,R. N.Q.F W Y P orC. L99replacedwithD E, H, K, R, N, Q, F W Y P orC. FI00replacedwithD,)E,, K.R,N,QA,G,IL,S,T MV P orC;R0I replacedwthD. E.A..LLS.T MV N.QF W Y P orC Cl2 replaced with D.E,H.K.R.A.G,I,LS,T MV NQ,F W Y orP" l03replacedwithD. EH.

K.R.N,Q.F W Y P orC, Q104rcplaced withD EHIK.R.A.G,IL,S.T

M.V

F W Y P orC, NO5replacedwithD, FH.K, R,AG,,L,S.T M.V F W Y P orC, Ml06replacedwith E.H,K,R,N.Q,F W y P orC. Pi07rcpaceu wtD 3o E.H, K,R,A.G.1.L.S,T MV N.Q,F W Y orC, KO8 replacedwith D F,A.G, I,L,S,T MV N,Q,F W Y P orC TlM replaced with D. E.H.K.R.N.Q.PF W Y P orC, LllOreplacedwithD E.lH.,K,R,N,Q,F W Y P orC. Plll replaced wthD, EH,K,R,A.G,1,1.,S,T MV NQ,F W Y orC. Nll2replacedwtth D.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/20e 16:12 61 2 92586999 4 062837999 N0.726 P32 00 0 C E H. KR.A, G, I, L, S, T M, V F W Y P orC. N113replaced withD.E. H, K, R, ct A.GI.L,S.T M.V FW Y P orC. Sl l4replaced wih D. E. H, K, R. N.Q.F W SY P orC.C 15 replaced withD E, H, K, R, A, G, 1, L, S, T M.V N,Q,F W Y or P- Y116 replaced with D, E. H. K, R, N, Q. A, G. I. L, S, T M, V P orC, S117 replaced with D. E.H. K.R. N. Q, F W Y P orC. Al 18 replacedwithD. E. H, K, R, N.Q.F W Y P orCG119 replaced with DE.H,KR.N,Q.F W Y P orC. 1120 Sreplaced with D, E. H, K. R. N, Q, F W Y P or C, A121 replaced with D, E. H. K. R.

SN,Q,F W Y P orC, Rl22 placed with D.E. A, G.I. L.S.T M.V N.Q.F W Y o P or C. L123 replaced wih D, E. H. K. R. N, Q. F W Y P or C. E124replaced with ci 00 ic H.K.R. A.G. I.L.S.T M, V N.Q.F W Y P or C; E125 replaced with H, K, R, A.

SG.I.L.S.T M.V N.Q.F W Y P orC. G 126replaced wnhD E, H. K, R. N. Q, F W Y P or C. D127rcplaced with H,K R.A. G,L L.S.T M.V N.Q,F W Y P or C.E128 replaced withH. K.R.A, G, I, L. S, T M. V N, Q F W Y P orC, 1129 replaced with D, E, H. K. R. N, Q, F W Y P or C. Q 130 replaced with D. E. H. K, R, IS A,G, I. L, L.S. T M. V F W Y P orC: Ll31 replaced with D, E, H. K. R, N. Q. F W Y P or C, A 132 replaced with D E. H. K. R. N, Q, F W Y P or C; 1133 replaced withD. E, 1H. K, R. N. Q, F W Y P orC, P134replacedwith D E.H, K. R.A. G.1, L, S. T M. V N. Q, P W Y orC; RI35 replaced with D, E. A, G, 1. L, S.T M. V N.

Q.F W Y P orC. E 136 replaced with H,K.R, A.GI, I, L.S.T M.V N.Q.F W Y P orC,NJ 37 replaced withD.E.H.K.R. A. G,LLST M.V F W Y P orC, A138 replaced with D E, H. K, R. N. Q. F W Y P or C, Q 39 replaced with D, E,1 H.

K.R.A.G.1,L.S.T M.V F W Y P or C, 1140 replaced withD E1.H-,K.R.N.Q.

F W Y P orC. S141 replaced wh D. E, H, K. R, N.Q. F W Y P or C. R142 replacedwth D, E.A. G, I.L.S. T M, V N.Q, F W Y P orC, N 143replaced with D.E.IH.K.R.A.G.I.L.S.T M.V F W Y P orC. G144replaced wih D E.H.K, R.N, Q, F W Y P orC. D145replaced with H, K. R. A, G, 1. L, S, T M. V N.Q, F W Y P or C. D146 replaced with H. K.R, AG.GL L.S.T M.V N.Q.F W Y P or C;T147 replaced with D E.H. K.R. N, Q. F W Y P or C.F 148 replaced with D E, H, K. R, N, Q, A, C.I, L, S.

T M, orC, l49 rpaccu wthD IIK. R.N. Q, A.G..L.S.T M.V P orC.Gl150OreplacedwizhD E,H.KRNQ.F W Y P or C. A 151 replaced with D E, K, R, N, Q, F W Y P or C. L 152 replaced with D, E.

H. K. R.N, Q, F W Y P or C;K153replacedw D.E. A. G. I. L. S. T M, V N, Q.

F W Y P or C. L54 replaced with D F. H. K, R. N, Q, F W Y P orC. and/or COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2808 16:12 61 2 92586999 4 062837999 N0.726 D33 00 0 cs L155 replaced with D, E, H, K. R, N, Q, F W Y P orC. Polynucleotides encoding these polypeptides are also encompassed by the invention. The resulting Ncutrokine- Salpha proteins of the invention may be routinely screened for Neutrokme-alpha and/or Neutrokme-alphaSV functional activiues and/or physical properties (such as, for example, enhanced or reduced stability and/or solubility) described throughout the specification and known n the art. Preferably the resulting proteins of the invention have an increased and/or a decreased Neutrokmne-alpha and/or Neutrokne-alphaSV functional activity More preferably the resultng Neutrokme-alpha and/orNeutrokne O alphaSV proteins of the iventuon have more than one increased and/or decreased 00 io Neutrokme-alpha and/or Neutrokme-alphaSV functional activity and/or physical Sproperty cl In an additional embodiment, Neutrokme-alpha polypeptides of the invention comprise, or alternatively consist of, more than one anuno acid 2, 3, 4, 5, 6, 7 8, 9 10, 15, 20, 30 and 50) replaced with the subsuitued amino acids as described above (tither conservative or nonconservative).

For example, preferred non-conservative substitutions of the Neutrokine-alpha proemn sequence provided in SEQ ID NO:38 include: MI replaced with D. E. H. K, R.

N, Q,F W Y P or C, D2 replaced withH K,R. A. G. L, S.T M, V N, Q. F W Y P or C. E3 replaced with H, K, A, G. I, L,S. T M, V N.Q,F W Y P orC, S4 replaced with D, E, H. K, R, N, Q, F W Y P or C; AS replaced with D, E, H. K. R, N, Q,F W Y P or C: K6 replaced with D, A. G, I, L,S, T M, V N.Q,F W Y P or C; T7 replaced with D, E, H, K, R, N, Q. F W Y P or C, L8 replaced with D E. H, K,R, N, Q,F W Y P or C P9 replaced wth D E. H. K,R, A, G, I, L, S, T M, V N, Q. F W Y or C. PO replaced with D. E, H. K. R. A, G 1, L, S, T M, V N, Q, F W Y or C, PI replaced wthD E,H, K, R, A.G.I.L.S.T M,V N,Q.F W Y orC.

C12 replaced with D EH. K.R. A G, L. S. T M,V N, Q,F W Y or P-Ll3 replaced with D. E, H, K. R. N. Q. F W Y P or C. C14 replaced with D, E, H. K, R.

A,G,I,L,S,T M,V N.Q.F W Y or P Fl5replacedwithD E, H,K, R,N,Q,.A, L. T M,V or C, C'6 replaced w:hD E, K,R. C, L N.

Q, F W Y or P Sl7 replaced wth D, E, H. K R, N. Q, F W Y P or C, E18 replaced with H.K, R, A, G. I, L. S, T M. V N, Q, F W Y P or C; K19 replaced with D. E, A, G, I, L, S. T M, V N, Q. F W Y P or C, G20 replaced with D E, H.

K. R. N, Q. F W Y P or C. E21 replaced wth H. K. R. A, G,1. L, S. T M, V N. Q COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2088 16:12 61 2 92586999 4 062837999. N0.72G 934 00 O0 IF W Y P orC:D22replacedwith H, K. R. A, GIJ L, S, T MV N,Q,F W Y P Ct or C. M23t eplaced with D, E. H, K, R. N, Q, F W Y P or C, K24 replaced with D E, A. G. I, L, S, T M, V N, Q, F W Y P or C. V25 replaced with D, E. H, K, R. No Q, F W Y P or C, 026 replaced with D E. H. K. R, N, Q, F W Y P or C, Y27 S replaced with D, E, H, K. R, N. Q. A, L, S, T M V P or C. D28 replaced with H, K, R, A, Go 1. 1, S, T MV NQ,F W Y P orC.P29replacedwith D,E,H. K,R.

A,G.I,L,ST MV NQ,F W Y orC, 13Oreplaced with D E.H. K. R, N. F W Y P orC, T31 replaced with D, E. ,HK, R.N,Q,F W Y P or C; P32 replaced ci wlthD, E,HK,R.A,G,I,L,S,T MV N.Q.F W Y orC, Q33 replaccd wthD.

00 oo E .H,.KRA.G,1,L.S.T M.V F W Y P orC. K34 replaced with D. SIT MV N.Q,F W Y P orC. E3nreplacCdwithH. RAG. LS.T M.V N,Q.F W Y P orC, E36replacedwith-H. K. R.A,G, i. L,S.T MV N.Q,F W Y P or C, G37 replaced with D E. H, K, R, N, Q. F W Y P or C: A38 replaced with D.E,H,K,R,N,Q,F W Y P orC, W39replaced withD E. H. K, R. N,Q.A. G1.

*1 L,S,T M,V P orC, F4OreplacedwithD, E,H,K.R,N,Q,A.G,,L,SJT M.V P or C; G41 replaced with D E, H, K, R. N, Q F W Y P or C, 142 replaced wtlh D, E.

H, K,R,N,Q,F W Y P orC; C43replacedwithD E.H,KR.AG..LL.S.T M, V N.Q.F W Y orP R44replacedwith D E,A,G,1,L,S,T MV N,Q F W Y P orC. D45 replacd with H, KR,A,GI.L,S,T MV NQ,F W Y P orC G46 20 replaced with D, H. K, R, N, Q. F W Y P or C, R47 replaced with D, E. A, G. I, L, S,T MV N.Q,F W Y P orC. L48replaced withD.E.H, K, R.N,Q,F W Y P or C, 1A9 replaced with D E. H, K, R. N, Q, F W Y P or C, A50 replaced with D, E.

H,K.R.N,Q, F W Y P orC. A51 replacedwthD. E.H,K.RN. Q. F W Y P or C, T52 replaced with D E, H, K, R, N, Q. F W Y P or C. L53 replaced with D. F H.

KRN,Q, FW Y P orC. L54replacedwithD E.H. K. R, N. Q, F WY P rC, replaced with D E, H, K, R N. Q, F W Y P or C, AS6 replaced with D E, H. K, R,N,Q.F W Y P orC. L57replaccdwithD E.H.K,R.NIQ F W Y P orC, L.58 replaced with D E, H, K, K. N, Q. F W Y P or C. S59 replaced with D F H. K, RM.,Q, V v 13 -r C, AA %I. IAI P aCS E, H, K, R,MH, WX v n arC S6 replaced wih D. E, H. IL R. N, Q. F W Y P or C, F62 replaced with D, E. H, K R, N,Q.A,G.I, LS.T M,V P orC. T63 replacedwithD EH,K,R.N.Q,F W Y P or C, A64 replaced with D E, H. K, R. N. Q. F W Y P or C. M65 replaced with P. H, K, R, N,Q,F W Y P or C. S66replacedwth D, E, H, K, R, NQF W Y COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 925896999 4 062937999 NO.726 00 O0

CI

P or C,.L67 replaced with D, E, H, K, R, N. Q, F W Y P orC, Y68 replaced with D t E,H.KR,N,Q.A,G,I.LST MV P orC, Q69 replaced with D, E, H, K, R. A, 2GI L,ST M,V F W Y P orC, L70 replaced withD, E. H, K. R. N, Q.F W Y P or C, A71 replaced with D, E. H. K. R. N. Q. F W Y P or C. A72 replaced with D.

EH,KR,N,Q,F W Y P or C. L73replacedwith D, E, K, R.N, Q, F W Y P orC; Q74 replaced withD. E.H.K.R, A, G.I.LS.T MV F W Y P orC. Sreplaced with D, E. H, K. R. N. Q, F W Y P or C, D76 replaced with H. K, R, A, 0G, 1I.LST M,V N,Q.F W Y P orC, L77 replaced with D. E, H. K, R, N. Q. F W SY P or C, M78 replaced with D, E. H. K. R, N, Q, F W Y P or C; N79 replaced 0 0 io with D,E, H. K,R,A.G,I,L.S,T M. V F W Y P or C, LSO rcplaced with D E, H, SK.R. N.Q.F W Y P orC. R8I replacedwith D. E.AG, I, L.S.T M.V N,Q.F W Y P or C. M82 replaced with D E, H, K R, N. Q, F W Y P or C. E83 replaced with H. K, R. A, GI, L S. T M. V N. Q, F W Y P orC. L84 replacedwith D, E. H, K.R.N,Q,F W Y P orC. QS5 replaced wth D,E, H. K.R, A, G, IL, S,T MV is F W Y P or C.S86 replaced with D.E, H,K,R. N,Q, F W Y P or C,Y87 replaced with D. E, H, K, R. N, Q. A. G, I, L, S. T M. V P or C, R89 replaced with D, E.A.G.I.L.S,T M.V N,Q,F W Y P orC. GS9 replaced withD E, H.K.,R.N, Q.F W Y P orC:S90 replaced withD.E.H.K,R.N,Q,F W Y P orC;A91 replaced with D. E, H. K, R. N. Q. F W Y P or C. T92 replaced with D. E. H. K. R, N. Q. F W Y P orC. P93replaced withD, E, H. K. R, A.G, I, L, S, T M, V N, Q, F W Y or C; A94 replaced with D, E. H. K, R. N. Q. F W Y P or C; A95 replaced with DE. EH. K, R N. Q, F W Y P orC, A96replacedwth D E. H, K. R, N.Q, F W Y P orC, G97 replaced with D E. H. K, R, N, Q, F W Y P or C. A98 replaccd with D. E, H.K. R, N, Q, F W Y P orC.P99replaced with D E, H, K. RA. GI.L. S.T M.V N.Q.F W Y orC.ElOO replaced with H. KR. A.G.,l .L.S.T M.V N.

Q,.F W Y P or C,.L1OI replacedwithD. E. H. K, R, N, Q.F W Y P orC, T102 replaced with D, E, H, K, IR, N, Q, F W Y P or C, A103 replaced with D E. H, K R, NQ,F W Y P orC;G104replacedwith Q.F W Y P orC; VOS renlard with D E. H. K, R,N. Q. W v D o C, .106 replaced wih l 3D G,J,LS.T M,V N,Q,F W Y P orC. Ll 07replacedwithD.E.H.K.R, N.Q.F W Y P orC.LI08 replaced with DE.H.K.R, N.Q.F W Y P orC,T109 replaced with D, E, H, K, R, N, Q, F W Y P or C. Pi 10 replaced with D. E K. KR.

A.G,1,L.S,T M.V NQF W Y orC, A 11 replaced with D E.H, K, R, N, Q. F COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92586999 4 062937999 N0.726 336 00

N

W Y P orC. A12replaced with D B,H,K,R.N,Q,FW Y PorCP113 Ct replaced with D, A G, I,L,S,T M.V NQ,F W Y orC, Rll4replaced withD G, I, L, S,T M,V N,Q,F W Y P orC, Pll5 replaced withD EH, SK,.R,A,G,I.L, S,T M.V N,Q,F W Y orC; H1ll6replacedwnhD E A.G.I,L.

ST M.V N.Q.F W Y P orC;N117 replacedwithD, E,H.K,R. A, G,I. L,S,T SMV F W Y P or C;Sl replaced with D. E. H, K, RN, Q, F W Y P orC,S)199 Sreplaced with D.E,H,KR, N, Q,F W Y P or C, R 120 replaccd withD E. A,G,1.

L,ST M. V N,Q, F W Y P orC, Gl21 replacedwith D, E, H, K,R,N,Q.F W SY P orC.Hl 122 replaced withD E, A, S, T M,V N,Q,F W Y P orC, 0 0 10 Rl23 replaced with D E, A,G, I, L, S,T M, V N,Q,F W Y P orC.N 124replaced Swith D.E, H. K. R. A, G, I,L, S,T M,V F W Y P orC. Rl25 replaced withD E.

A,G,I,L.S,T M,V NQ.F W Y P orC.Rl26rplaced with D EA,GI,L,S, T M, V N.Q, F W Y P orC, A127 replaced with D E, H, K, R. N, Q,F W Y P or C, F128 replaced with D. E, H. K. R. N, Q. A, G. I, L, S, T M, V P or C. Q 129 replaed with D, E, H, K, R, A, G, I, L, S, T M, V F W Y P orC; G130 replaced with D E.H. K, R,.N,Q.F W Y P orC, Pl31 replaced with D E,H. K. R,A,G, I.

L,S,T M.V N.Q,F W Y or C: El32 replaced with H,K,R,A,G.I,L,S,T M,V N. Q,F W Y P orC, E133 replaced with H. K.R. A,G, 1,L,S.T M. V N,Q,F W Y P orC. Tl34 replaced with D E. H, K. R, N. Q, F W Y P or C. E]3: replaced withH, K. R. A, G I,L. S,T M, V N, Q, F W Y P orC. Ql36replacedwithD E, H. K. R,A. G.I,L. S,T M,V F W Y P orC: D137 replaced withH, K,R,A, G, I, L.S.T M,V N,Q,F W Y P orC. Vl38 replaced withD,E. H, K, R, N, Q, F W Y P orC. D139replaced with H,KR, A,G,I,L,S,T M.V N.Q,F W Y P orC, rcplaced with D. E. H. K, R. N. Q, F W Y P or C. S 141 replaced wath D E. H, K.R.N,Q,F W Y P or C, A142replaced with D E, H,K.R.N, Q.F W Y P or C, P143replaced with D. E,H, K, R, A, G, I, L. S, T M, V N, Q. F W Y or C. P144 replaced with D, E, H, K, R, A. L. S,T M. V N, Q, F W Y or C, A14S replaced with D, E, H, K, R. N, Q, F W Y P or C, P146 replaced with D E, H, K, R. A, G, I, L. S,r tN. Q, c W v or C, C14' replaccd with D, E, H, K. R. G, 1, 3, T M, V N,Q,F W Y or P, Ll4 replaced wuhD E, H. K,R,N,Q, F W Y P orC.

P149replacedwithD. E R. A, G, 1. L, S,T M. V N, Q. F W Y orC, G150 replaced with D, E, H. K, R. N. Q, F W Y P orC, C151 replaced with D E, H, K, R, A. G, 1. L, S,T M,V N,Q.F W Y or P- R152 replaced with D E.A, GI, 1.L,S. T COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 P37 00 O 414- M.V N.Q.F W Y P orC.H 153 replaced with D A. G, I, L. S.T M.V N,Q,F ct W Y P orC. S154replacedwith D. E. H. KRN, Q,F W Y P Sreplaced with D, E, H, K. R, A 1. L. S. T M, V F W Y P or C, H156 replaced with D. E. A. G. L, S, T M. V N, Q. F W Y P orC, Dl57 replacedwithH, K,R.

A.G,IL.S.T M,V N.Q,F W Y P or C, Dl 5 replaced with H. K.R,A. G, l,L, S,T M,V N,Q,F W Y P orC. Nl59 replacedwith D E. H,KR,A,G.I, L,LST SMV F W Y P or C, 160 replaced with D, E H. K,R,N,Q.F W Y P orC, M161 replaced with D, E. H, K R. N, Q. F W Y P or C, N162 replaced with D, E.

0 H,K,R,A,G,I,L,S.T MV F W Y P orC, L163 replacedwith D E, H, K, R, N, 00 10 QP W Y P orC, R164 replaced withD EA, G.I,L.ST M,V N.Q,F W Y P SorC,N 165 replaced wilh D E, H, K,R, A. IL,S,T MV F W Y P orC, 1166 replaced with D. E, H, K. R. N. Q, F W Y P or C. 1167 replaced with D E H, K. R, N.QF W Y P orC,Q168rplacedwithD, S.T M,V F W Y P orC, D169 replaced with H, K,R, A. G, I.L,S.T M.V N,QF W Y P orC, is C170 replaced withD E, H. K. R, A. GI 1 L,S T M, V N Q. F W Y or P L171 replaced wzith D, E, H. K, N, Q, F W Y P or C, Q172 replaced wih D E, K, R.

A, 1, L, S,T M,V F W Y P or C, L173 replaced wthD E,H, K.R.N.Q.F W Y P or C, 1174replaced wiLh D,E, H, KR, N,Q, F W Y P or C, A175 replaced withD. E, H. K. R. N, Q, F W Y P or C, 0D176 replaced with H, K. R, A, L. S, T M,V N.Q,F W Y P orC. S177 replacedwnhD EFH, K.R.N, Q, F W Y P orC, D178 replaced with H. K. R, A, G. 1I,L, S,T M, V N,Q,F W Y P orC, T179 replaced with D, E. H. K, R. N. Q. F W Y P or C, PI8O replaced with D, E. H. K, R, A, G, I, L, S, T M. V N, Q.F W Y orC, A18I replaced wnh D E. H, K, R. N. Q.F W Y P orC. L182rplaced with D E.H, K,R. N,Q,F W Y P orC. E183 2S replaced with H, K, R, A, G. I S, T M, V N, Q. F W Y P or C, El184 replaced withH, K, R. A.G. I, L, S. T M, V N. Q, F W Y P orC, K185 replaced withD E, A. G. IL, S,T M,V N. Q,F W Y P or C, E186 replaced withH. K, R, A. G, I, L.

ST M,V NQF W Y P orC. N187replacedwIIhD,E,H.K,R,A, 0G,I, L,S,T M, V= W v or C, KISSreplaced withD, E,G. T V, v N, Q. F W Y P or C, 1189 replaced with D E. H. K, N. Q, F W Y P orC, Vl90 replaced with D. E. H, K R, N. Q F W Y P orC. VI 91 replaced with D E,H, K, R,N. Q. F W Y P or C. Rl92replaced with D E,A,G,I,L,S,T M.V NQ,F W Y P orC.

Q193 replaced with D. E.H.K. R. A,G,I, L. S,T M,V F W Y P orC. TI94 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 16:12 61 2 92586999 4 062837999 N0.726 938 00 S 4;Q- N replaced with D. F. H. K, R, N, Q, F W Y P or C; G 195 replaced with D. E. H, K, R.

t N,Q,F W Y P or C. YI96replaCd with D, E.H.KR,NQ. A. G,0I. L.S.T M,V S*P or C, F197 replaced with D, E, H, K. R, N, Q, A, G, I, L, S, T M. V P or C; F198 Sreplaced with D. E. H.K, R.N, Q. A. G, I. L, S. T M,V P orC, I 99 replaced with D. E. H, K. RN, Q, F W Y P orC, Y200 replaced with D E, K, R, N, Q, A G. I, SL,S,T M,V P or C;S201 replaced withD, E,H,K,R,N,Q,F W Y P orC, Q202 Sreplaced with D, E. H, K, R, A, G, I. L, S. T M, V P W Y P or C; V203 replaced SwithD E. H. K. R, N. Q. F W Y P orC, L204 replaced with D E, H, K,R.N, QrF SW Y P or C, Y205 replaced with D, E, H. K, R, N, Q, A, G, I. L, S. T M. V P or C.

0 0 10 T206 replaced with D, E, H, K. R, N, Q, F W Y P or C, D207 replaced with H. K. R.

SG A.G,1, L.S,T MV NQ.F W Y P orC, P208 replaced with D E.H.K,R. A,G.

1 LS.T M,V N,Q,F W Y or C, 1209 replaced with D E,H, K,R.N. Q, F W Y P orC, F210 replaced with D, E, H. KR. N, Q. A. L. S.T M,V P orC. A2II replaced with D. E, H. K R. N, Q, F w Y P or C, M212 replaced with D, E, H, K, R, is N,Q,F W Y P orC, G213 replaced withD E. H. K. R,N, Q. F W Y P orC, H214 replaced wilh D. E, A, G, I, L. S, T MIVL V N. Q. F W Y P or C, V215 replaced with D, E.H,K,R. N,Q, F W Y P orC. 1216 replaced wzth D E,H,K, R,N,Q,F W Y P orC, Q217 replacedwith D F. H, K,R.A,G, LL, S,T M.V F W Y P or C. R218 replaced with D, E, A, G, I,L, S. T M,V N,Q, F W Y P orC. K219 replaced with D E. A, G, I, L. S. T M, V N, Q, F W Y P or C, K220 replaced with D. E,AG. I, L.S. T M, V N, Q,F W Y P orC. V221 replaced withD. E,H, K, R.

NQ,F W Y P orC, H222 replaced with D. E, A. G. I, L.S, T M,V N,Q,F W Y P or C, V223 replaced with D E. H, K, R, N, Q, F W Y P or C. F224 replaced with D, E. H. K. R.N.Q,A, G. 1.L. S. T M. V P or C. G225 replaced with D. E,H, K, R, N,Q,F W Y P orC, D226replaced with H.K.R A, G. I,L.S.T M,V N,Q.F W Y P or C, E227 replaced with H, K.R. A. G.1.L,S,T M.V N,Q,F W Y P orC, L228 replaced with D, E, H. K, R, N. Q, F W Y P or C, $229 replaced with D E, H, K.R,N,Q,F W Y P or C.L230 replaced with D,E,HK,R,N,Q,F W Y P orC.

V231 repnl-sced 0, H. K, R, Q, W v or C, T232 rtepla.u wilh D E, H.

K, R,N,Q,.F W Y P or C, L233 replaced with D, E, H, K. R, N, Q, F W Y P orC.

F234 replaced with D, E, H. K, R, N, Q, A, G, 1, L, S. T M. V P or C; R,235 replaced with D. E, A, G, I, L,S,T M, V N. Q.F W Y P orC. C236 replaced withD, E. H.

K,R,A,G.I, LS.T M.V N,Q.F W Y or P- 1237replaced withD. E, H.K,R, N.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 I39 00 o l (N Q,FW Y P or C. Q238 replaced with D.E,H, K, R,At,I, LS.T M,V F W Y ct P or C. N239 replaced with D. E, H. K. R. A, G. I1, L S, T M. V F W Y P or C, S M240 replaced with D, E, H, K, R. N. Q, F W Y P or C, P241 replaced with D, E, H, KR.A.GI.LST M.V N.Q.F W Y orC, K242 replaced with D.E. A, G. I.L.

S ,T M.V N.Q,F W Y P or C, T243 replacedwith D E H, K, R.N. Q, F W Y P or C.L244 replaced with D. E, H. K, R. N, Q. F W Y P or C. P245 replaced with ID. E.H.K.R.A. G. I.L.S. T M,V N,Q,F W Y or C, N246replaced withD,E,H., K.R.A.G.I.L.S.T M.V F W Y P or C. N247 replaced withD E.H.K.R.A.G.

I. L. S.T M. V F W Y P or C, S248 replaced with D, E, H. K. R.N, Q, F W Y P 00 or C. C249 replaced with D, E.HKR, A. G. I. LST MV N,Q,F W Y or P SY250 replaced with D, E, H, K, R, N, Q, A, G, 1, L, S, T M, V P or C, S251 replaced with D E. H, K. R, N, Q. F W Y P orC, A252replaced withD E, H, K, R, N, Q,F W Y P or C. G253 replaced with D. E. H, K. R. N. Q, F W Y P or C, 1254 replaced with D. E. H, K. R. N. Q. F W Y P or C. A255 replaced with D, E, H, K, R. N, Q; F W Y P orC.,R256replaced w:thD E,A,G,I.L.S.T M,V NQF W Y P orC, L257 replaced with D. E. H. K. R. N. Q, F W Y P or C; E258 replaced with H, K, R, A, G.I, LS.T M. V N, Q, F W Y P or C. E259replaced with H. K. R, A. G, 1, L, S,T MV N.Q.F W Y P or C, G260 replaced withD, EH, N. Q. F W Y P or C D261 replaced with H. K. R, A, G, I, L, S. T M, V N.Q. F W Y P orC, E262 replaced with H, K, R, A, G, 1, L, S, T M, V N, Q, F W Y P or C. 1263 replaced with D, E, H. K, R, N. Q. F W Y P or C; 0264 replaced with D. E, H, K. R, A,G.I.L.S,T M.V F W Y P orC. L265 replaced with D, E. H. K. P, N. Q, F W Y P or C.,A266 replaced with D, E. H. K, R, N, Q, F W Y P or C, 1267 replaced with E. H. K. R. N, Q.F W Y P orC. P268replacedwnhD, E, H, A. G.l I, L, S. T M, V N, Q, F W Y or C; R269 replaced with D, E, A, G. I.L, S. T M. V N.

Q.F W Y p or C; E270 replaced with H. K A. G. I. L. S. T M. V N.Q.F W Y P orC. N271 replaced with D F. H. K. R. A. G, I.L, S.T M.V F W Y P orC.

A272 replaced with D. E. H, K. R. N, Q. F W Y P or C, Q273 replaced with D. E. H.

K. R. A, C. 1. L. S.r M, W V orC. I274 rplaced wi.th D E. HK, R,.Q, F W Y P or C. S275 replaced with D. E, H, K. R. N. Q. F W Y P or C. R276 replaced with D. E. A, 0G. 1, L. S, T M. V N. Q. F W Y P or C. N277 replaced with DEH.K.R.A.G.1,L.S.T M.V F W Y P orC. G278 replaced with D E.HK.

R. N, Q. F W Y P or C, D279replaced withH. K.R.A. G. I.L. S, T M, V N, Q.F COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/200e 19/032008 1:12 G1 2 92586999 4 2237999 N.2 4 NO.726 P40 00 WY yp or C;fD280replaced withH, K.R A, G, LLS. T M.V N, Q, FW Y P or Ct ~~~CT28l replaced witD, E.IH.K.F, NQ.F W Y P or C,PF282 replaced with DB, HI, R, G, .L.ST M, V P or C: F283 replaced with D, E, H, K. R. N. Q, A,G0,1, L, S,T M. P or C.G02S replacedwithfl K,R, N,Q, F W Y P or s C: A2t5replaced withD. E. H.KR, N, Q.F W Y P or C, L296replaced with D E, N. Q.F W Y P or C, K287replAed withD, E,A, G,1.t" S. T M.,V N, Q.

F W Y P or C,L288 replacd wihD, EH. KR, NQ. F W Y P or C, and/or L289 replaced with D. E, H, IC, R, N, Q, F W Y P or C. Polynucleotides encoding Cl rhese polypeptides are also cUcODmpassed by the invention The resulting Neutrokine- 00 O o0 alpha proteins of the invention may he routinely screened for Neutrokine-aipha and/or 0 Neutrokine-alphaSV functional activities andor physical properties (such as, for C example, enhanced or reduced stability anidlor solubility) described throughout the specification and known in t art Preferably the resulting proteins of the invention have an increased and/or a decreased Neuran-aipha and/or is functional activity More preferably the resulting Newrokine-alpha and/or Neutrokane alpbaSV proteins of the invention have more than ooe increased and/or decased Neurrokine-aipha and/or Neutrokmne-alphaSV functional activity and/or physical property In an additional embodiment, Neutrokine-aiha polypeptides of the invention comprise, or alternatively consist of. more than one arwno acid 2. 3,4,5. 6,7 8.

9 10. 15.20, 30 and 50) replaced with the substituted amumo acids as described above (either conservative or nonconservatiye).

Replacement of amino acids can also change the selectivity of the hind ing of a C ligand to cell surface rccepors. For example, Ostade et aL. Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of 'rNF-alpha to only one of the two known types of TNF receptors. Since Neutrolone-aipha and Neutrokwe-alpbaSV is members of the TNF polypeptide family mutations similar to chose in TNF-alpha are likely to have similar eftets ink Neuirolune-alpha and/or Sites that are critical for ligand-rccepior binding can also be determined by structural analysts such as crystallization, nuclear magnetic re-sonance or photoaffmniity labeling (Smith es alt. I. Mo. Bint 224:899-904 (1992) and de Vos nr aL Science 255-306-312 (1992)).

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 P41 00 Since Neutrokine-alpha is a member of the TNF-related protein family to c modulate rather than completely eliminate functional activities biological Sactivities) of Neutrokme-alpha, mutations may be made in sequences encoding amino acids in the TNF conserved domain, m positions Gly-191 through Leu-284 of Figures IA and IB (SEQ ID NO:2). more preferably in residues within this region which are not conserved in all, most or several members of the TNF family TNFalpha. TNF-beta, LT-beta, and Fas Ligand) (see Figures 2A-B). By making a specific mutation in Neutrokme-alpha m the position where such a conserved amino C acid is typically found in related TNFs, the Neutrokane-alpha mutem will act as an 00 antagomst, thus possessing activity for example, which inhibits lymphocyte B Scell) proliferation, differentiation. and/or activation. Accordingly polypeptides of the present nvention include Neutrokin-alpha mutants. Such Neutrokine-alpha mutants comprise, or altermauvely consist of, fragments, variants or derivatives of the full-length or preferably the extracellular domain of the Neurrokme-alpha amino acid is sequence shown m Figures IA and IB (SEQ ID NO:2). Polynucleoudes encoding the above Neutrokine-alpha mutants are also encompassed by the invention.

Since Neutroklne-alphaSV is a member of the TNF-related protein family to modulate rather than completely eliminate functional activities biological acuvtties) of Neutrokme-alphaSV mutations may be made in sequences encoding amino acids in the TNF conserved domain, m positions Gly-172 through Leu-265 of Figures 5A and 5B (SEQ ID NO' 19), more preferably in residues within this region which are not conserved in all, most or several members of the TNF family TNFalpha. TNF*beta, LT-beta, and Fas Ligand) (see Figures 2A-B). By making a specific mutation in Neutrolne-alphaSV in the posiion where such a conserved amino 32 acid is typically found in related TNFs, the Neutrokme-alphaSV mutein will act as an antagonist, thus possessing activity for example, which inhibits lymphocyte B cell) proliferation, differentiation, and/or activation. Accordingly polypeptudes of the present invention include Neutrokme-alphaSV mutants. Such Ncutrokne-alphaSV muwtants comip is, or alkieratvey sOI:,.ay M u, jragments, variants or uenvaives or me full-length or preferably the extracllular domain of the Neutrokine-alphaSV amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19 Polvnucleotldes encoding the above Neutrokmne-alpha SV mutants are also encompassed by the invention.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 D42 00

O

In addition, it will be recognized by one of ordinary skill in the art that c mutations targeted to regions of a Neutrokine-alpha polypeptide of the mvention which Sencompass the nineteen amnno acid residue insertion which is not found m the SNeutrokine-alphaSV polypepude sequence amino acid residues Val-142 through Lys-160 of the sequence presented in Figures lA and IB and in SEQ ID NO:2) may affect the observed functional activities biological activity) of the Neurokinc-alpha polypeptide. More specifically a partial, non-limiting and non-exclusive list of such residues of the Neutrokme-alpha polypepude sequence which may be targeted for mutation includes the following anuno acid residues of the Neutrokune-alpha polypeptide sequence as shown in SEQ ID NO:2: V 142; T 143 SQ-144. D-145- C 146: L 147" Q-148: L 149 1-150: A-151 D-152: S-153. E-154.

C T 155; P 156; T 157 -158, Q-159- and K 160.

Recombinant DNA technology known to those skilled in the art (see, for instance, DNA shuffling supra) can be used to create novel mutant proteins or mutens including single or multiple anmno acid substtutions, deletions, additions or fusion proteins. Such modified polypeptdes can show enhanced aciuvy or increased stability In addition, they may be purified i higher yields and show better solubility than the corresponding natural polypepude, at least under certai punfication and storage conditions.

Thus, the invenuon also encompasses Neutrokme-alpha and/or Neurokine-alphaSV derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate Neutrokine-alpha and/or Neutroklme-alphaSV polypeptides that are better suited for expression, scale up. etc., in the host cells chosen.

SFor example, cysteine residues can be deleted or substiuted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or hbth nf the first or third amino acid pos:lons on any one or r.nrc of the glycosylaton recognitions sequences in the Neutrokine-alpha and/or Neurokine-alphaSV polypeptides of the invention, and/or an armno acid deletion at the second position of any one or more such recognition sequences will prevent COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/20013 16:12 61 2 92586999 4 062837999 N0.726 943 00 glycosylation of the Neutrokne-alpha and/or Ncutrokine-alphaSV at the modified tnpepude sequence (see, Miyajimo et al., EMBO J 1193-1197).

SAdditionally one or more of the amino acid residues of the polypepudes of the minvention argimne and lysne residues) may be deleted or substituted with another S residue to elaunate undesired processing by proteases such as, for example, funns or Skenms. One possible result of such a mutation is that Neutrokine-alpha polypepude of the invention as not cleaved and released from the cell surface.

In a specific embodiment, Lys-132 and/or Arg-133 of the Netrokine-alpha C-I sequence shown in SEQ ID NO:2 is mutated to another amino acid residue, or deleted 0o Io altogether, to prevent or dimnish release of the soluble form of Neutrokme-alpha from O cells expressing Neutrolne-alpha. In a more specific embodiment. Lys-132 of the Neutrokme-alpha sequence shown in SEQ ID NO:2 is mutated to Ala-132. In another, nonexclusive specific embodiment, Arg-133 of the Ncutrokine-alpha sequence shown m SEQ ID NO:2 is mutated to Ala-133. These muratled proteins, and/or polynucleotides encoding these proteins have uses such as, for example, in ex vivo therapy or gene therapy to engineer cells expressing a Neutrokine-alpha polypepltde that is retained on the surface of the engineered cells.

In a specific embodiment, Cys-146 of the Neutrokme-alpha sequence shown in SEQ ID NO:2 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomernzaton of the mutant Neutrokine alpha polypeptide when expressed n a expression system (essentially as described in Example In a specific embodiment, Cys-146 is replaced with a Scnne ammno acid residue. Polypeptudes encoding these polypeptides are also encompassed by the invention.( In another specific embodiment, Cys-232 of the Neutroke-alpha sequence shown in SEQ ID NO:2 is mutated to another ammo acid residue, or deleted altogether.

for example, to aid preventng or diminishing oligomenzaton ofthe mutant Neutrokme-alpha polypcptide when expressed in a expression system (essentially as dectribd in Example In. specfic embodinicrnt, Cys-232 ittepicu wilh a Senne amino acid residue. Polypeptides encoding these polypept2des are also encompassed by the invention.

In yet another specific embodiment, Cys-245 of the Neutrokine-alpha sequence shown in SEQ ID NO;2 is mutated to another amno acid residue, or deleted altoaether, COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 062837999 NO.726 P44 00 for example, to aid preventing or dimimushmSg oligomerzzation of the mutant Neutrokine-alpha polypeptide when expressed in a expression system (essentally as described in Example In a specific embodiment, Cys-245 as replaced with a Senne anuno acid residue. Polypeptides encoding these polypeptides are also encompassed by the rnventuon.

The polypeptides of the present minvention are preferably provided min an isalated form, and preferably are substantially punfied. A recombmanly produced version of the Neurokmnc-alpha and/or Neuutrokine-alphaSV polypepudes can be substantially 0 purified by the one-step method described m Smith and Johnson Gene 67:31-40 00 to (1988).

O The polypeptds of the present invention include the complete polypepude encoded by the deposited cDNA (ATCC Deposit No. 97768) including the intracellular, transmemrnbrane and extracellular domains of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptde encoded by the deposited cDNA, is the extracellular domain minus the utracellular and transmembrane domains of the protein., the complete polypeptude of Figures IA and IB (amino acid residues 1 285 of SEQ ID NO:2). the mature soluble polypeptide of Figures IA and IB (amino acids 134-285 of SEQ ID NO:2), the exiracellular domain of Figures IA and I B (amno acid residues 73-285 of SEQ ID NO:2) ummus the wiracellular and transmembnne domains, 2o as well as polypeptdes which have at least 80%, 85%, 90% similanrty more preferably at least 95% similanty and still more preferably at eIcast 96%, 97%, 98% or 99% similarity to those described above. Polynueleotides encoding these polypeptides are also encompassed by the invention.

The polypeptides of the present nmventon also include the complete polypeptide encoded by the deposted cDNA including the mintracellular, utransmcmbrane arid extracellular domains of the polypeptide encoded by the deposited cDNA (ATCC Deposit No. 203518), the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the srwraccIllular and transmembrane domains of the proiein, the complete polypaptda of rigures 5 n anu 53 %amnu aCiu residues 266 of SEQ ID NO- 19), the extracellular domain of Figures SA and 5B3 (ammno acid residues 73-266 of SEQ ID NO: 19) minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%. 90% similanty more preferably at least similarity and still more preferably at least 96%.97%, 98% or 99% similanrity to COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 945 00 0 those described above. Polynucleoudes encoding these polypeptides are also C3 encompassed by the invention.

SFurther polypeptdes of the present mvention include polypeptides at least or at least 85% identical, more preferably at least 90% or 95% Identical, still more preferably at least 96%, 97%. 98% or 99% idenucal to the polypeptide encoded by the deposited cDNA (ATCC Deposit No. 97768) or to the polypepude of Figures IA and IB (SEQ ID NO:2), and also include portions of such polypeptdes with at least amnuo acids and more preferably at least 50 amino acids. Polynucleotides encoding- C these polypeptdes are also encompassed by the invention.

00 10 Further polypeptides of the present invention include polypepudes at least Oor at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC Deposit No. 203518) or to the polypepude of Figures 5A and (SEQ ID NO:19), and also include portions of such polypepudes with at least s1 amno acids and more preferably at least 50 amino acids. Polynucleoudes encoding these polypeptdes are also encompassed by the invention.

By similarity" for two polypeptde s sintended a similarity score produced by companng the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix. Genetics Computer Group, University Research Park, 575 Science Drve. Madison, WI 53711) and the default settings for determining similanty Bestfit uses the local homology algorithm of Smith and Waterman (Advances m Applied Mathematics 2:482-489 1981) to find the best segment of similarity between two sequences.

By a polypeptude having an arnno acid sequence at least, for example, 95% "identical" to a reference ammno acid sequence of a Neutrokine-alpha and/or Neutrokmne-alphaSV polypeptde is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amno acids of the reference amino acid of the Neutrokine-alpha and/or Neirfokinc-apiraS'v puiypepouc-. r uiWcr words, to obtain a polypepulde having an amno acid sequence at least 95% identcal to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substtuted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues n the reference sequence may he COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 D46 00 l inserted into the reference sequence. These alterations of the reference sequence may c occur at the amino or carboxy terminal positions of the reference amnno acid sequence G or anywhere between those terminal positions, interspersed either individually among ON residues in the reference sequence or in one or more contiguous groups within the s reference sequence.

As a practical matter, whether any particular polypepude is at least 80%, 95%, 96%, 97%. 98% or 99% identical to, for instance, the amno acid sequence shown m Figures IA and IB (SEQ ID NO2), the anmno acid sequence encoded by the o deposited cDNA clone HNEDU15 (ATCC Accession No. 97768), or fragments 00 10 thereof, or, for instance, to the anuno acid sequence shown in Figures SA and 5B (SEQ SID NO: 19), the amino acid sequence encoded by the deposited cDNA clone HDPMC52 (ATCC Accession No. 203518), or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package. Version 8 for Unix, Genetics Computer Group, University IS Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present mvention, the parameters are set, of course, such that the percentage of identity as calculated over the full length of the reference amino acid sequence and that gaps in homology of up to of the total number of amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query) sequence Ca sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosc. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matnx=PAM 0, k-tuple=2.

Mismatch Penalty= I Joining Penalty=20. Randomization Group Length=0, Cutoff Score=l Window Sizeasequence length, Gap Penalty=5, Gap Size Penalty0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is sooner. Afording tu tLlu embudiihrent. thiie subject :clqueinc is shorter than the 3o query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-ternmmal truncations of the subject sequence when calculating global percent identity For subject sequences truncated at COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 D47 00 cN the N- and C-termuni, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-termnal of Sthe subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determmation of whether a residue is matched/aligned is determuned by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this O embodiment. Only residues to the N- and C-termum of the subject sequence, which are 00 o1 not matched/aligned with the query sequence, are considered for the purposes of Smanually adjusting the percent identity score. That is, only query residue posmons C outside the farthest N- and C-ermunal residues of the subject sequence. For example, a ammo acid residue subject sequence is aligned with a 100 residue query sequence to determne percent idenmy The deletion occurs at the N-tcrminus of the subject Is sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-termnus. The 10 unpaired residues represent 10% of the sequence (number of residues ai the N- and C-termni not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the PASTDB program. If the rimaaing 90 residues were perfectly matched the final percent identity would be 90%, In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This ume the deletions are internal deletions so there are no residues at the N- or C-termni of the subject sequence which are not matched/aligned with the query In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment, The puiypxcplauc of the prefcint inveiiiil nave u.is that incluuc, but are not limited to, as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those skilled m the art. Additionally as described m detail below the polypeptides of the present invention have uses that include, but are not limited to, to raise polyclonal and monoclonal antibodies, which are COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 348 00 c useful m assays for detecting Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide expresion as described below or as agonmss and antagonists capable of enhancing or Sinhibiting Neutrokne-alpha and/or Neutrokine-alphaSV fmcton. The polypepudes of the inventon also have therapeutic uses as described herein. Further, such polypepudes can be used m the yeast two-hybrid system to capture Neutrokne-alpha and/or Neurokine-alphaSV binding proteins which are also candidate agomsts and antagonists according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989).

O

C Thnasgews and "unock-wots" 00 The polypepudes of the mvention can also be expressed in transgenic animals.

c-i Animals of any species, including, but not limited to, mice. rats, rabbts, hamsters, guinea pigs, pigs, nncro-pigs, goats, sheep, cows and non-human primates, e.g..

baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as pan of a gene therapy protocol.

Any technique known m the an may be used to introduce the transgene polynucleoudcs of the invention) into animals to produce the founder lines of transgenc animals. Such techniques include, but are not limned to, pronuclear microinjection (Paterson, er aL. Appl. Microbol. 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.. US. Pat. No. 4,873,191 (1989)); retrovurus mediated gene transfer into germ lines (Van der Putten et al, Proc. Natl. Acad. Sci..

USA 82:6148-6152 (1985)), blastocysts or embryos: gene targeting in embryonic stem cells (Thompson at al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo. 1983, Mol Cell. Biol. 3.1803-1814 (1983)); itroduction of the polynucleotdes of the invenuon using a gene gun (see, Ulmer el al., Science 259-1745 (1993): introducing nucleic acid constructs into embryonic pleunpotent stem cells and transferring the stem cells back Into the blastocyst; and sperm-mediated gene transfer (Lavtrano et al., Cell 57-717 723 (1989); etc. For a review of such techniques, see Gordon. "Transgenic Animals, Intl. Rev Cytol. I l::171 229 (1989), which is incorporated by reference herein n its entirety See also,.U.S Patent No. 5,464.764 COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 P49 00 0 (Capecchi, et aL, Positive-Negative Selection Methods and Vectors); U.S. Patent No.

i 5,631.153 (Capeccht. et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modificanons and Positive-Negauve Selection Methods and Vectors for Making Same); U.S. Patent No. 4,736866 (Leder. et aL, Transgenic Nons Human Animals); and U.S. Patent No. 4,873,191 (Wagner, et Genetic Transformation of Zygotes): each of which is hereby incorporated by reference in its entirety Any technique known in the art may be used to produce transgenmc clones 00 containing polynucleotides of the inventon, for example, nuclear transfer into Sto enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et aL, Nature 380:64-66 (1996): Wilmut et al., Nature 385:810- 813 (1997)) The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their IS cells, mosaic or chnmeric animals. The transgene may be integrated as a single transgene or as multiple copies such as m concatamers, head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a partcular cell type by following, for example, the teaching of Lasko et al.

(Lasko et al., Proc. Natl. Acad. Set. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the ar. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly when such a technique s to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleoude sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivaung the endogenous gene in only that cell type, by following, for example, the reachins of Gu et al. (Gu ct al., Sc;cnce 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactvation will depend upon the particular cell type of interest, and will be apparent to those of skill iD the art. In addition to expressing the polypeptide of the present invention in a ubiquitous or tissue specific manner m transgenmc animals, it would also COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 00 be routine for one skilled m the art to generate constructs which regulate expression of the polypeptide by a variety of other means (for example, developmentally or Schemically regulated expression).

Once transgenic animals have been generated, the expression of the s recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southem blot analysis or PCR techmques to analyze animal tssues to verify that integranon of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgemc animals may also be Ci ;assessed using techniques which include, but are not limited to, Northern blot analysis o in of tussue samples obtained from the animal, mi sit hybridizaton analysis, reverse o transcnptase-PCR (rt-PCR); and'TaqMan PCR. Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or i1 crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgemc animals to produce animals homozygous for a given integrauon site in order to both augment expression and eliminate the need for screening of animals by DNA analysts; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest; and breeding of transgenic animals to other animals bearing a distinct transgene or knockout mutation.

Transgenc and "knock-out" animals of the invention have uses which include.

but are not limited to, animal model systems useful in elaborating the biological function of N.mnrokine-alpha and/or Neuirck:ne-alphaS"' polypeptldes, sudying conditions and/or disorders associated with aberrant Neutrokine-alpha and/or Neuirokme-alphaSV expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 951 00 In further embodiments of the invention, cells that are genetically engieered to t express the polypepudes of the nvention, or altematively that are genetically engineered not to express the polypepudes of the mvention knockouts) are administered to a patient n vwwo. Such cells may be obtained from the patient animal, including human) or an MHC compatible donor and can include, but are not linuted to fibroblasts, bone marrow cells, blood cells lymphocytes), adipocytes.

muscle cells, endothelial cells etc. The cells are genetically engineered an vitro using Srecombinant DNA techmques to introduce the coding sequence of polypeptides of the Sinvention into the cells, or alternatively to disrupt the coding sequence and/or S1to endogenous regulatory sequence associated with the polypeptides of the invention, e.g., 0 by transduction (using viral vectors, and preferably vectors that integrate the transgenc mio the cell genome) or transfection procedures. including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation. liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a is strong consttutve or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the mvention can be introduced into the patient systemically in the circulation, or intrapentoneally Alternatively the cells can be incorporated into a matrix and implanted in the body genetically engmeered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349- and Mulligan Wilson. U.S. Patent No. 5,460,959 each of which is incorporated by reference herein in ts entirety).

When the cells to he administered are non-autologous or non-MHC compatible cells, they can be adrminstered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of .omponents wn:h th munmdia cxtraceilul, cjivrumnilunt., aoes not allow ume introduced cells to be recognized by the hosit mmune system.

Antibodies COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 e 062837999 N0.726 952 00 Further polypepttdes of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment.

Sor varant of SEQ ID NOC2 and/or SEQID NO 19 and/or an eptope, of the present Sinvention (as determined by immunoassays well known in the an for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments. F(ab') fragments, fragments produced by a Fab expression library anti-adiotypic (ant-Id) antibodies (including, ann-sd antibodies CN to antibodies of the invention), and eptope-bnding fragments of any of the above.

00 0 10 The term antibody as used herein, refers to nmmunoglobulin molecules and immunologically active portions of immunoglobulin molecules, molecules that contain an antigen binding sue that tmmunospecifieally binds an anngen. The immunoglobulin molecules of the invention can be of any type IgG, IgE, IgM.

IgD IgA and IgY). class IgG I lgG2, IgG3, lgG4, IgA 1 and IgA2) or subclass of 1is mmunoglobulin molecule. Immunoglobulins may have both a heavy and light chain.

An array of IgG. IgE, IgM, IgD, IgA. and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Anttgen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region. CHI, CH2, and CH3 domains. Also included in the mvention are antigen-binding fragments also comprising any combmation of variable region(s) with a hmge region. CH 1 CH2, and CH3 domains. The antibodies of the invention may be from any animal origm including birds and mammals. Preferably the antibodies are human, murne mouse and rat), donkey ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "humarn antibodies inciluu antibodies navmg tne amino acia sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5.939.598 by Kucherlapani ct at.

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 953 00 c The antibodies of the present invention may be monospecific, btspectiic, mtnspecific or of greater multispecficity Multispecific antibodies may be specific for Sdifferent epitopes of a polypepude of the present nvention or may be specific for both a polypepude of the present invention as well as for a heterologous epltope. such as a heterologous polypeptide or solid support material. See, PCT publications WO 93/17715- WO 92108802; W09 100360; WO 92/05793- Tutt, et al., J. Immunol.

147:60-69 (1991); U.S. Patent Nos. 4,474,893- 4,714,681 4,925,648; 5,573.920; 5,601,819- Kostelny et al., J. Immuno. 148:1547-1553 (1992).

Cl Antibodies of the present Invenuon may be described or specified in terms of o to the epitope(s) or portion(s) of a polypepude of dhe present invention which they Srecognize or specifically bind. The epitope(s) or polypeptade porton(s) may be specified as described hercin, by N-tenmnal and C-terminal positions, by size inm contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypepude of the present nvention may also be is excluded. Therefore, the present mventon includes antibodies that specifically bind polypeptides of the present inventon, and allows for the exclusion of the same.

In specific embodiments, antibodies of the invenuon bind to polypepttdes comprising Phe 115 to Leu-147 Ile 150 to Tyr 163. Ser-171 to Phe-194, Glu-223 to Tyr-246, and Ser 271 to Phe-278 of the amino acid sequence of SEQ ID NO:2. In another specific embodiment, antibodies of the invention bind to polypeptides consisung of Phe-115 to Leu-147 Ue-50 to Tyr-163, Ser-171 to Phe-194, Glu-223 to Tyr-246, and Ser-27 to Phe-278 of the auino acid sequence of SEQ ID NO:2. In a preferred embodiment, antibodies of the invenuon bind to a polypeptde comprising Glu-223 to Tyr-246 of SEQ ID NO;2. In another preferred embodiment, antibodies of s the invention bind to a polypepude consisting of Glu-223 to Tyr 246 of SEQ ID NO:2.

In a more preferred embodiment, antibodies of the invention bind to a polypeptide consisting of Phe-230 to Asn-242 of SEQ ID NO:2. In further preferred, nonexclusive embodiments, the antibodies of the inventon inhibit one or more biological activities of Neu:trokmc-alpha and/o, Ncatwrutic-ipuaS'v poiypepuaes ot te Invention through specific binding. In more preferred embodiments, the antibody of the nvention inhibits Neutrokine-alpha- and/or Neutrokne-alphaSV-mediated B cell proliferation.

Antibodies of the present invention may also be described or spectfied in terms of their cross-reactivity Antibodies that do not bind any other analog, ortholog, or COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 I54 00

O

C

N homolog of a polypeptide of the present mivention are included. Antibodies that bind t polypeptides with 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%, and at least 50% identity (as O calculated using methods known in the art and described herein) to a polypepude of the s present invention ae also included in the presentinvention. In specific embodiments, antibodies of the present invention cross-react with maine, rat and/or rabbit homologs of human protens and the corresponding epttopes thereof. Antibodies that do not brnd polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 0 75%. less than 70%, less than 65%, less than 60%. less than 55%. and less than 0 0 0t identity (as calculated using methods known in the an and described herein) to a o polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific anugemc or immunogenic polypeptide, or combination(s) of 2, 3.4,5, or more of the specific anugemc and/or immunogenw polypeptides disclosed herein. Further is included in the present mventon are antibodies which bind polypeptides encoded by polynucleotides which hybndize to a polynucleottde of the present invention under hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified i terms of their binding affinity to a polypeptlde of the invention. Preferred binding affinities include those with a dssocianton constant or Kd less than 5 X 10' M. 10 M, 5 X 10" M, 10M, 5 X 10" M, 1 0 M. 5 X 10- M, 1

M.

IO'PM, 10 M. 5 X 10" M. lO'" M, 5 X 10'" M, 10 M,5 X lO"' M. 10" M, 5 X M, 10'" M, 5 X 10"" M, 10 1 4 M, 5 X 10"' M, or 10 M.

The inventon also provides antibodies that competively inhibit binding of an antibody to an epilope of the invention as determined by any method known in the an for determining competitive binding, for example, the imunoassays described herein.

In preferred embodiments, the antibody competitively inhibits binding to the epitope 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 Athibodies of the prrst ;ivcnton may act aa agoi-Si ui miwuhumsts or the polypeptides of the present inventon. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polvpepudes of the invention either partially or fully Preferrably antibodies of the present invention bind an antgenic epitope disclosed herein, or a portion thereof The invention features both COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062937999 N0.726 955 00 0 N receptor-specific antibodies and ligand-spccific antibodies. The inventon also features <c receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activaion signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be Sdetermined by detecting the phosphorylaton tyrosine or senrnethreonine) of the receptor or its substrate by immunoprecipnauon followed by western blot analysts (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at 0 least 80%, at least 75%. at least 70%. at least 60%, or at least 50% of the activity in 00 to absence of the antibody SThe invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor ligand complex, and, preferably do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies I which bind the ligand and prevent binding of the ligand to the receptor as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor These antibodies may act as receptor agonists, potentate or activate either all or a subset of the biological activities of the ligandmediated receptor activaton, for example, by inducing dimenzation of the receptor The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprsing the specific biological activities of the peptdes of the invention disclosed herein. The above antibody agonsts can be made using methods known in the an. See. PCT publication WO 96/40281 U.S. Paent No. 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. Set. 11 (Pt2):237-247 (1998); Pitard ct al.. J. Immunol. Methods 205(2): .90 1997); ziautard et CytuLnir 9(4):233-24 t 9 97); Canson ca ai., J.

Biol. Chem. 272(17) 11295-11301 (1997); Taryman et al.. Neuron 14(4):755-762 (1995); Muller et al., Structure 1153-1167 (19981: Bartunck et al.. Cvtokme 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 056 00 -1 So C I Antibodies of the present invention may be used, for example, but not limted to, to purify detect, and target the polypeptides of the present invenion, including both m viro and a vivo diagnostic and therapeutic methods. For example, the antibodies O have use in immunoassays for qualitatively and quantitatively masuring levels of the s polypeptides of the present invention in biological samples. See, Harlow et al..

Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2nd ed.

1988) (incorporated by reference herein n its entirety).

As discussed m more detail below the antibodies of the present invention may C be used either alone or in combination with other compositions. The antibodies may to further be rccombmantly fused to a heterologous polypcpuide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention Smay be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeputdes, drugs, radionuclides, or toxins. See, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No, 5,314,995; and EP 396,387 The antibodies of the invention include denvanves that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.

For example, but not by way of limitanon, the antibody derivatives include antibodies that have been modified, by glycosylanon, acetylation, pegylation, phosphylation, anudauon, denvanzation by known protecung/blockng groups. proteolyti cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carned out by known techniques, including, but not limied to specific chemical cleavage, acetylaton, formylation, metabolic synthesis of tunicamycn, etc. Additionally the derivatve may contain one or more nonclassical amino acids.

The antibodies of the present invention may be generated by any suitable memnoa Known in te art. Polycional antibodies to an antigen-ot-interesi can oe produced by various procedures well known in the art. For example, a polypeptide of the invention can he administered to various host animals including, but not limited to, rabbits, mice. rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to ncrease the COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P57 00 c immunological response, depending on the host species, and include but are not limited c to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolccilun, pluronic polyols, polyantons, pepudes, oil emulsions, keyhole limpet hemocyanms, dinirophenol, and potenually useful s human adjuvants such as BCG (bacille Calmette-Guein) and corynebactenum parvum.

Such adjuvants are also well known m the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybndoma, recombnant, and phage display C technologies, or a combimaton thereof. For example, monoclonal antibodies can be 00 1i produced using hybndoma techniques including those known in the art and taught, for o example, 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 Hybrndomas 563-681 (Elsevier, 1981) (sid references incorporated by reference m their entueties). The term monoclonal antibody as used herein is not limited to antibodies produced through hybndoma technology The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic. prokaryotic, or phage clone, and not the method by which it is produced.

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

Methods for producing and screening for specific antibodies using hybrdoma technology are routine and well known in the art and are discussed in detail in the Examples Example In a non-limrting example, mice can be immunized nwth a polypepude of the mvention or a cell expressing such peptide. Once an immune response is detected, antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybndomas are selected and cloned by liumted dilution. The hybndoma clones are then assayed by methods known in the art for cells that s=ecrete aibodies capable of bindins puypcpuut; u ii v veiunuun.

Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybndoma clones.

Accordingly the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising cultrinng a COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 D58 00 O

A

hybndoma cell secretng an antibody of the invention wherein, preferably the c hybndoma as generated by fusing splenocytes isolated from a mouse immunized with San antigen of the invention with mycloma cells and then screening the hybndomas resultng from the fusion for hybndoma clones that secrete an antibody able to bind a polypepide of the ivention.

Antibody fragments which recognize specific epiropes may be generated by known techmques. For example, Fab and F(ab')2 fragments of the inventon may be produced by proteolyc cleavage of immunoglobulin molecules, using enzymes such ci as papan (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

0 0 10 F(ab)2 fragments contain the vanable region, the light chain constant region and the SCH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known m the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the is polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display anugen-binding domains expressed from a repertoire or combinatorial antibody library human or munne). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombmantly fused to cither the phage gne 111 or gene VIII protein.

Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Bnnkman et al., J. Immuniol. 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); Persac et al., Gene 187 9-18 (1997): Burton et al., Advances in Immunology 57'191 280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90102809- WO 91/10737- WO 92/01047* WO 12l8619- WO 93' 1236; WO 95/15982; WO 95/2P040 ana u.S. Patent Nos.

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; each of which is incorporated herein by reference in us entirety- COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P59 00 c As described in the above references, after phage selection, the antibody coding c regions from the phage can be isolated and used to generate whole antibodies.

including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bactena, as described in detail below For example, techniques to recombinantly produce Fab, Fab and F(ab')2 fragments can also be employed using Smethods known i the an such as those disclosed i PCT publication WO 92/22324; Mollinax et al., BtoTechniques 12(6):864-869 (1992); and Sawal et at., AJRI 34:26-34 (1995); and Better t al., Science 240:1041.103 (1988) (said references incorporated 00 10 by reference m their entireties).

Examples of techniques which can be used to produce single-chain Fvs and C antibodies include those described in U.S. Patents 4,946,778 and 5.258,498; Hustonet al., Methods in Enzymology 203'46-88 (1991): Shu et al., PNAS 90:7995-7999 (1993): and Skerra e aL. Science 240:1038-1040(1988). For some uses, including m vivo use is of antibodies in humans and in vitro detection assays, it may be preferable to use chmnenc, humanized, or human antibodies. A chimenc antibody is a molecule m which different pontons of the antibody are derived from different animal species, such as antibodies having a vanable region derived from a munne monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimenc antibodies are known in the art. See Momson, Science 229-1202 (1985); 01 et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191 202; U.S. Patent Nos. 5,807 715; 4,816,567' and 4,816397 which are ncorporated herein by reference m their entirety Humanized antibodies are antibody molecules from nonhuman species antibody that binds the desired antigen having one or more complementaity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antgen binding.

These framework substtutions are identified av methods well know. :n the at. by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, Queen et al., U.S. Patent No.

5,585,089- Riechmann et al., Nature 332:323 (1988). which are incorporated herein by COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 062837999 N0.726 00 0 c reference in their entireties.) Antibodies can be humanized using a variety of ct technques known mn the at including, for example, CDR-grafung (EP 239 400; PCT Spublication WO 91/09967- U.S. Patent Nos. 5,225.539 5,530,101 and 5.585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studncka et al., Protem Engineering 7(6):805-814 (1994); Roguska. at al., PNAS 91:969-973 (1994)). and chain shuffling Patent No.

5,565.332).

Completely human antibodies are paricularly desirable for therapeutic C treatment of human patients. Human antibodies can be made by a variety of methods o1to known m the ar including phage display methods described above using antibody C librares derived from human immunoglobulin sequences. See also, U.S. Patent Nos.

4,444,887 and 4,716.111 and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096. WO 96/33735. and WO 91/10741 each of which is incorporated herein by reference in is entirety Human antibodies can also be produced using transgentc mice which are incapable of expressing functional endogenous immunoglobulins. but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination nto mouse embryonic stem cells. Altematively the human variable o2 region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain mmunnoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected nto blastocysts to produce chimeric mice. The chimenic nuce are then bred to produce homozygous offspnng which express human antibodies. The transgerc mice are immunized in the normal fashion with a selected antgen, all or a porton of polypepude of the invention. Mon.con-al antibodies directed aga;st the antigen can be obtained from the immunized, uansgenic mice using conventional hybndoma technology The human immunoglobulin transgenes harbored by the transgenic rtce rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, It is possible to produce COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 D61 00 0 Cl therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this Stechnology for producing human antibodies, see Lonberg and Huszar, ant. Rev Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing O human antibodies and human monoclonal antibodies and protocols for producing such s antibodies, see, PCT publications WO 98/24893; WO 92/01047- WO 96/34096; WO 96/33735; European Patent No. 0 598 877- U.S. Patent Nos. 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, which are incorporated by reference herein u their entirety In addiuon.

0 companies such as Abgemx, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be 0 0 to engaged to provide human antibodies directed against a selected antigen using o technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as guided selection. In this approach a selected non-human monoclonal antibody a mouse antibody is used to guide the selection of a completely human antibody recognizing the same epitope. (Jcspers et al., Bio/echnology 12:899-903 (1988)).

Further. antibodies to the polypeptides of the invention can, in turn, be utilized to generate antu-diotype antibodies that "rimic polypeptides of the mvention using techniques well known to those skilled in the art. (See, Greenspan Bona, FASEB J. 7(5):437-444; (1989) and Nissmoff, J. Immunol. 147(8):2429-2438 (1991)).

For example, antibodies which bind to and compeutively inhibit polypeptide mulumenzation and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic the polypeptlde mulumcnzatnon and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing ana-idiotypes or Fab fragments of such ant-idiolypes can be used m therapeutic regimens to neutralize polypeptde ligand. For example, such antiidiotypic antibodies can be used to bind a polypepude of the invention and/or to bind its ligands/receptors, and thereby block us biological acuvity Polynucleotides Encoding Antibodies The invention further provides polynucleotides comprising a nucleottde sequence encoding an antibody of the inventon and fragments thereof. The invention also encompasses polynucleotudes that hybridize under stnngent or lower stringency COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 D62 00 0 hybndization conditions, as defined supra, to polynucleotides that encode an Ct antibody preferably that specifically binds to a polypcpude of the invention, preferably an antibody that binds to a polypeptde having the amino acid sequence of O SEQ ID NO:2. In another preferred embodiment, the antibody binds specifically to a s polypeptde having the anuno acid sequence of SEQ ID NO: 19 In another preferred embodiment, the antibody binds specifically to a polypeptide having the ammno acid sequence of SEQ ID NO:23. In another preferred embodiment, the antibody binds specifically to a polypeptde having the amino acid sequence of SEQ ID NO:28. In 0 another preferred embodiment, the antibody binds specifically to a polypeptide having 0 0 0o the armno acid sequence of SEQ ID SThe polynucleoides may be obtained, and the nucleotide sequence of the polynucleotdes determined, by any method known in the an. For example, if the nucleotUde sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleoudes as described in Kutmreer et al., BioTechnmques 17:242 (1994)), wich, briefly involves the synthesis of overlapping oligonucleondes containing portions of the sequence encoding the antibody annealing and ligating of those oligonucleotides. and then amplification of the ligated oligonucleoudes by PCR.

Alteratively a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a partcular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chenucally synthesized or obtained from a suitable source an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody such as hybndoma cells selected to express an antibody of the mvention) by PCR amplification using synthetic prmers hybndizable to the 3' and 5' ends of the sequence or by clonng using an oligonucleoude probe specific for the particular gene sequence to identify a cDNA clone from a cDNA library that enrndr the antibody .dmpdired uclsC acds generaed by PCR nmay thcu uc utou into replicable cloning vectors using any method well known in the art.

Once the nucleoude sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P63 00 o 47X 0 C recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, c the techniques described in Sambrook et 1990, Molecular Cloning, A Laboratory SManual, 2d Ed., Cold Spnng Harbor Laboratory Cold Spnng Harbor, NY and Ausubel Set al., ds., 1998, Current Protocols in Molecular Biology John Wiley Sons. NY which are both incorporated by reference herein in their entireties), to generate antibodies having a different amano acid sequence, for example to create anuno acid substitutions, deletions, and/or mnsemrons.

In a specific embodiment, the annno acid sequence of the heavy and/or light 0' chain variable domams may be inspected to identify the sequences of the 00 10 complementarty determining regions (CDRs) by methods that are well known in the O art. by comparison to known amino acid sequences of other heavy and light chain variable regions to deirneune te regions of sequence hypervanability Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, into human framework regions to humanize a non-human antibody as described supra. The framework regions may be naturally occumng or consensus framework regions, and preferably human framework regions (see. e.g., Chothia et al., J. Mol. Biol. 278. 457-479 (1998) for a lisung of human framework regions). Preferably the polynucleotde generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invenuon. Preferably as discussed supra. one or more ammno acid substitutions may be made within the framework regions, and, preferably the amino acid substitutions improve binding of the antibody to its antigen. Additionally such methods may be used to make anuno acid substitutions or deletions of one or more variable region cysteme residues particapaung in an intracham disulfide bond to generate antibody molecules lacking one or more intracham disulfide bonds. Other alterations to the polynucleotide are encompassed by the present ivention and within the skill of the art.

In addition, techniques developed for the production of chimenc antibodies (Morrison et al., roc. Nail. '.cad. Sc.. 8':85 -855 t 984); 4eubcrg.- et ai., Nrture 312:604608 (1984); Takeda et al., Nature 314:452454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropnate biological activity can be used. As described supra, a chimenc antibody is a molecule in which different portions are COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P64 00 0A 98 c N denved from different ammal species, such as those having a variable region derived C from a munne rnAb and a human rimnunoglobulin constant region, humanized Santibodies.

SAlhernatively techniques described for the production of single chain antibodies s Patent No. 4,946,778; Bird, Science 242:423* 42 (1988); Huston et al.. Proc.

Nal. Acad. Sci. USA 85:5879-5883 (1988); and Ward at al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an anuno 0 acid bndge, resulting in a single chain polypcptide. Techniques for the assembly of 0 0 10 functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- S1041 (1988)).

i..

Methods of Producing Antibodies The antibodies of the mvenion can be produced by any method known in the is art for the synthesis of antibodies, in particular, by chemical synthesis or preferably by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, a heavy or light chain of an antibody of the invention or a single chain antibody of the Invention), reqmres construction of an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for prepanng a protein by expressing a polynucleoutde containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors contanmng antibody coding sequences and appropnate transcnptional and translational control signalt. These methods include, for examnplc. n "i;-ro rccombinant DNrtechniques, synthetic techniques, and in wvo genetic recombinaton. The invention, thus, provides replicable vectors compnsing a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chan varable domain, operably linked to a promoter Such vectors may include the COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 00 0 nucleotde sequence encoding the constant region of the antibody molecule (see, e.g..

SPCT Publication WO 86/05807- PCT Publication WO 89101036; and U.S. Patent No.

5.122,464) and the variable domain of the antibody may be cloned into such a vector Sfor expression of the enure heavy or light chain.

s The expression vector is transferred to a host cell by conventonal techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the mvention. Thus, the invention includes host cells containmng a polynucleoude encoding an antibody of the mvention, or a heavy or light chain thereof, C or a single chain antibody of the invention, operably linked to a heterologous promoter.

0 0 10 In preferred embodinents for the expression of double-chained antibodies, vectors o encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire nnmunoglobulin molecule, as detailed below A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles is by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the inventon m situ. These include but are not limited to microorganisms such as bactena E.

coll, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences: yeast Saccharomyces, Pichta) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors cauliflower 2 mosaic virus, CaMV tobacco mosaic virus, TMV) or transformed with recombmant plasmid expression vectors Ti plasmd) contanng antibody coding sequences; or mammalian cell systems COS, CHO, BHK, 293. 3T3 cells) harbonng recombinant expression constructs containing promoters derived from the genome of mammalian cells (te.g. metallothtrane: promocr) nr from mammalian vifiet cL.gl., the adenovirus late promoter the vaccinia virus 7.5K promoter). Preferably bacterial cells such as Eschenchw coli, and more preferably eukaryotic cells, especially for the expression of whole recombiant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 N0.726 966 00 0 2o 0

L

c hamster ovary cells in conjunction with a vector such as the major intermediate SCearly gene promoter element from human cytomegalovius is an effective expression Ssystem for antibodies (Foeckng et al., Gene 45:101 (1986); Cockett et a., B Biorechnology 8:2 (1990)).

s In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed, For example, when a large quantity of such a proten is to be produced, for the generation of phamaceutical composttons of an antibody molecule, vectors whichcl direct the expression of high levels of fuson protein products that are readily purified o 10o may be desirable. Such vectors include, but ar not limited, to the E. coli expression 0 vector pUR278 (Ruthr et al., EMBO J. 2:1791 (1983)), in which the antibody coding 3 sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; plN vectors (Inouyc Inouye, Nucleic Acids Res. 13.3101 3109 (1985): Van Heeke Schuster. J. Biol, Chem. 24:5503-5509 is (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombm or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety In an insect system, Autographa californca nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoprera frugiperda cells. The antibody coding sequence may be cloned ndividually into nonessential regions (for example the polyhednn gene) of the virus and placed under control of an AcNPV promoter (for example the polyhednn promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adcnovirus is used as an expression vector, the antibody coding sequence of nrest may be ligated to an adenovirns transcnptionaislation control complex, the late promoter and trpartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by m vitro or mn vivo recombination. Insenion in a non- essential region of the viral genome region El or E3) will result in a recombinant virus that is viable and capable of expressing the COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/208 16:12 61 2 92586999 4 062837999 N0.726 [67 00 O0 antibody molecule in infected hosts. see Logan Shenk, Proc. Natl. Acad. Set.

t USA 81.355-359 (1984)). Specific initation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiaton codon and adjacent sequences- Furthermore, the nitiation codon must be mi s phase with the reading frame of the desired coding sequence to ensure translation of the enure insert. These exogenous translational control signals and initiaton codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may o be enhanced by the inclusion of appropriate transcnpton enhancer elements, transcption terminators, etc. (see Batter et al., Methods in Enzymol. 153:51 544 00 o to (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the nserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications glycosylaton) and processing cleavage) of protein products may be important for the funcuon of the protein.

i Different host cells have charactensuc and specific mechanisms for the posttranslational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryoc host cells which possess the cellular machinery for proper processing of the pnmary transcript, glycosylation. and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY BHK, Hela, COS, MDCK. 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T HTB2, BT20 and T47D and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the anibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replicaton, host cells can be transformed with DNA controlled by appropnate exnrestsnn control elements prCfC.e, r enhancer, sequences, transcrputiu terminators, polyadenylaton sites, etc.), and a selectable marker. Following the introduction of the foreign DNA. engineered cells may be allowed to grow for 1 2 days m an enriched media, and then are switched to a selective media. The selectable marker in the recombmant plasmid confers resistance to the selection and allows cells COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 61 2 92586999 4 062937999 NO.726 P68 00 l to stably integrate the plasud into their chromosomes and grow to form foci which in t turn can be cloned and expanded into cell lines. This method may advantageously be Sused to engineer cell lines which express the antibody molecule. Such engineered cell lines may be parcularly useful in screening and evaluation of compounds that interact s directly or indirecty with the antibody molecule.

A number of selecuon systems may be used, including but not limited to the herpes simplex virus thymudine kuiase (Wigler et al., Cell 11.223 (1977)), hypoxanlhne-guanne phosphoribosyltransferase (Szybalska Szybalski. Proc. Natl.

o Acad. Sc. USA 48.202 (1992)), and adene phosphoribosyltransferase (Lowy et al., 00 to Cell 22:817 (1980)) genes can be employed in tk hgprt- or aprt- cells, respectively 0 Also, anumetabolite resistance can be used as the basis of selection for the followinmg C" genes: dhfr, which confers resistance to methotrexate (Wiglcr ct al., Natl. Acad. Sci.

USA 77:357 (1980); O'Hare et Proc. Nail. Acad. Sct. USA 78:1527 (1981)); gpt.

which confers resistance to mycophenolic acid (Mulligan Berg, Proc. Natl. Acad- Sct. USA 78:2072 (1981)): neo, which confers resistance to the ammnoglycoside 0-418 Clinical Pharmacy 12:488-505 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 1993, TIB TECH 11 (5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al- Current Protocols in Molecular Biology John Wiley Sons, NY (1993); Knegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli e al. (eds). Current Protocols in Human Genetics, John Wiley Sons, NY (1994); Colberre-Garapn et al.. J. Mol. Biol. 150:1 (1981). which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amnlifir.irnn (fnr a rview see Bebbingtn ad Hentsch!. 'hae us of v;ec. based on geae amplification for the expression of cloned genes in mammalian cells in DNA cloning. Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiablc, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92596999 4 062837999 N0.726 369 00 N amplified region is associated with the antibody gene. production of the antibody will c also increase (Crouse ct al., Mol. Cell. Biol. 3.257 (1983)).

SThe host cell may be co-transfected with two expression vectors of the minvention, the first vector encoding a heavy chai derived polypeptide and the second vector encoding a light chain dernved polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chai polypepudes. Alternatively a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypepudes. In such situations, the light 0 chain should be placed before the heavy chain to avoid an excess of toxic free heavy 00 to chain (Proudfoot, Nature 322:52 (1986): Kohler. Proc. Nail. Acad. Sci. USA 77:2197 o (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody, molecule of the invention has been produced by an animal.

chemically synthesized, or recombmantly expressed, it may be purified by any method IS known in the at for purification of an immunoglobulin molecule, for example, by chromatography ion exchange, affinity particularly by affinity for the specific anigen after Protein A, and sizing column chromatography), centnfugation, differenial solubility or by any other standard technique for the purnfication of proteins. In addition, the antibodies of the present mvention or fragments thereof can be fused to.

heterologous polypeptide sequences described herein or otherwise known in the art. to facilitate punfication.

The present invention encompasses antibodies recombimantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a polypepude (or portion thereof, preferably at least 10, 20. 30.40, 50, 60. 70. 80 90 or 100 amino acids of the polypepude) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antgens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40. 50. 60, 70, 80, 90 or I00 amino acids of the polypcpudc) of the present ;n.cat:on. "or example, antibodies may be used to target the polypepudes of the present invention to particular cell types, either om vitro or in vvo by fusing or conjugating the polypeptudes of the present invention to antibodies specific for partcular cell surface receptors, Antibodies fused or conjugated to the polypepudes of the present invention may also be used in in vitro immunoassays COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 062837999 N0.726 00

O

C and purification methods using methods known in the art. See Harbor et al., t supra, and PCT publication WO 93/21232; EP 439,095; Naramura e al., Immunol.

SLett. 39:91-99 (1994); U.S. Patent 5,474,981 Gillies et al., PAS 89-1428-1432 (1992); Fell et at., J. Immunol. 146:2446-2452(1991), which are incorporated by s reference m ther enireties.

The present invention further includes compositions comprisng the polypepudes of the present inventon fused or conjugated to antibody domains other than the variable regions. For example, the polypepudes of the present mnvention may Cl be fused or conjugated to an antibody Fe region, or porton thereof. The antibody o° in portion fused to a polypeptlde of the present invenion may comprise the constant Sregion, hinge region, CHI domain. CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypepudes may also be fused or conjugated to the above antibody portions to form multmers. For example, Fc portions fused to the polypepudes of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimernc forms can be made by fusing the polypeptides to pormons of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions arc known m the art. See, U.S. Patent Nos. 5336,603 5,622,929- 5,359,046; 5,349,053, 5,447,851 5,112,946; EP 307 434' EP 367 166; PCT publications WO 96/04388; WO 91/06570; Ashkenaz et al., Proc. Nail. Acad. Scs. 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) (said references incorporated by reference in their enureties).

As discussed, supra, the polypeptades corresponding to a polypeptide, polypeptde fragment, or a variant of SEQ ID NO:2 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypepudes or for use an immunoassays using methods known in the art. Further, the polypepudes corresponding to SEQ ID NO:2 may be fused or conjugated to the above antibody portions to facilitate purification. Also as discussed, supra, the polypeptides corresponding to a polypeplde, polypeptide Iragment, or a variant o SEQ ID 'iO. 19 may be fused or conjugated to the above antibody portions to increase the mu viva half life of the polypepudes or for use in mmunoassays using methods known m the art.

Moreover, the polypeptides corresponding to SEQ ID NO' 19 may be fused or conjugated to the above antibody portions to facilitate punfication. One reported COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 16:12 61 2 92586999 4 062837999 NO.726 P71 O0 00 N example describes chimern proteins consisting of the first two domains of the human c CD4-polypeptde and various domains of the constant regions of the heavy or light Schains of mammalian immunoglobulins. (EP 394.827- Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present nventon fused or conjugated to an antibody having disulfide- linked dinnric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomenc secreted protein or protein fragment alone. (Fountoulakts o al., J Blochem. 270:3958-3964 (1995)). In many cases, the Fc part n a fusion protein is beneficial in therapy and 0 diagnosis, and thus can result m, for example, improved phannacokinete properties.

0 0 to (EP A 232.262). Alternatively deletng the Fc part after the fusion protein has been o expressed, detected, and purified, would be desired. For example, the Fc portion may

C

1 hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery for example, human protens, such as hIL 5, have been fused with Fc portions for the purpose of high-throughput screening assays to is identify antagonists of hlL 5. (See, Bennett et al., J. Molecular Recognition 8:52 58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate punfication. In preferred embodiments, the marker ammo acid sequence is a hexa-hisudine pepude. such as the tag provided in a pQE vector (QIAGEN, Inc.. 9259 Eton Avenue. Chatsworth, CA, 91311), among others. many of which are commercially available. As described m Genii et al. Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-hisudine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to. the "HA tag. which corresponds to an epitope derived from the influenza hemagglutmin protein (Wilson et al.. Cell 37-767 (1984)) and the "flag" tag.

The present invenuon further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used dizgn-stcafly to, for exampls. mn cor the developmen.t Or progresionl of a tumor as pan of a clinical testing procedure to, deterune the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prostheic groups, fluorescent materials, luminescent maienals, biolumnmescent materals, COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 19/032929 16:12 61 2 92596999 262837999m.2 P2 NO.?26 9?2 00 0 Clradioactive materials, positron erruruing metals using various positron emission Ct tomographies, and nonradioact~ive paramagnetic metal ions. The detectable, substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or ON indirectly tough an intrmnediate (such as. for example. a linker known in the an) using techniques known in t art. See, for example. U.S. Patent No. 4,741.90D for metal ions wich can be conjugated to anliibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxia se, alkaline phosphatase. beta-galactosadase, or aeylcholinestrase- examples of suitable o prosthetic group complexes include streptavadinlbotfll and avadiilbaorin; examples of 00 to suitable fluorescent mazenials include umbelliferone, fluorescein, fluorescein O ssorhiocyaniate. rhodaxnmc., 4icl~orotriazmnylamine fluorescein. dansyl chloride or Cl phycocryrhnm an example of a luminescent material includes luminol, examples or biolumiunescent materials include lucaferase, lucifenin, and aequormn; and examples of suitable radioactive macna! include 1251, "'In or 'Tc.

Is Further, an antibody or fragmnent thereof may be conjugated to a therapeutic moiety such as a cytotoxin. a cytostatic or cytocidal agent, a thcrapcutwc agent or a radloaCLIVe mectal ion, alpha-emitters such us. for example, 2 3 Ba. A cytotoxin or cytotoxic agent includes any agent that is detimental to cells. Examples include paclitaxol. cytochalasin B, grarmucidin D etbadium bromide, ernetane. mitomycxn, etoposide, tecaposide. vicnstine, vinbiasune. colchicin, doxorubicin, daunorubicin.

dihydrozy anthfracin dionc, flutoxfntrone, iihramycin. acunornycin D I dehydrotestoucrone, gluecocorticotds, procaline. tetracaine. lidocmne. propranolol, and puromycin and analogs; or hornalogs thereof. Therapemtic agents include, but are not limited to, anninetabolites (etg., methotrexate. 6-mercaptopunne, 6-thioguanine, cytarabine. 5-floorouracil decarbzuie). alkyhating agents% mechlorethamiie, thioepa cblorambucil, mincphalan. cairnustine (RSNU) and Jomrusine (CCNU), cyclotbosphamide. busulfan. dibromomannitol. srreptotocrn. mniromycin C. and cisdieblorodiarnine platinum MII (DDP) cisplatan). anthracyclinest daunorubici (formerly daunomrvctnil and dnxnnthiczn). antibioucs .4dzctrniyctn acunarnycan). bleomycin. mithrarnycin. and anthrainycin (AMC)I. and anni-nutotzc agents vinenstine and vinbiasoine).

The conjugates of the invent ion can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to COMS ID No: ARCS-183614 Received by IP Australia: Time 16:38 Date 2008-03-19 19/03/2008 19/032009 16:31 61 2 92596999 4 062837999 N.4 0 NO. 641 [Pol FAX TRANSMISSION No of pageS (inceluding this sit). 7 Level 36. Grosvenor Place 225 George Street Sydney NSW 2000 Australia Bl"ake Dawson PATENT ATTO RN EYS To The Commissioner of Patents IF' Australia F 02 6283 7999 Human Genome Science., Inc New Australian divisional patent application Title: Neutrokine-aipha and neutrokine-aipha splice variant T 012 925860000 F 612 92585699g DX 855 Sydrley Locked Bag No a Grosvenor Place Sydney NSW 2000 Ausra WwWbakedawoson~comn 19 March 2008 Our referarnce 0130 SJ 02 143D 4452 Partner David Clark T 61 2925839 davidolark fbiakadawsoncom PART 4 OF 6 Please o0060K that you havue received tie document in full. If not. please telephone the tender or Call 6i 2 9258 6000.

Conf identiality This documnent is contidential and may contain legally privileged information. It you are not a named or authxelsed recipint you must not read, copy, dlsIuIbuta or act in reliance on It. )I you halve received thi5 document In error.

please telephone our operator immediately on 61 2 925 c 6000 and feturn the document by mail.

Sydney Melbourne Brisbane Perth Canberra 20CI937378_1 COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:31 61 2 92586999 4 062837999 N0.641 P02 00 -oL o classical chemical therapeutc agens. For example, the drug moiety may be a protein or polypeptde possessng a desired biological activity Such proteins may include, for Sexample, a toxin such as abrn, ncm A, pseudomonas exoroxin, or diphthena toxm: a protem such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth S factor, platelet derived growth factor, tissue plasmmogen activator. an apoptotic agent, TNP-alpha, TNF-beta, AIM I (See, nternational Publication No. WO 97/33899).

AIM II (See. International Publication No. WO 97/34911), Fas Ligand (Takahashi ec al., Im. Immunol., 6:1567-1574 (1994)), VEGI (See, Internatonal Publication No. WO 99/23105), CD40 Llgand, a tbrombotic agent or an anti- anglogcnic agent, e.g., 10 angiostatin or endostatn; or, biological response modifiers such as, for example.

0 Slymphokines, Interleukn- ("IL mterleukin-2 ("IL nterleukin-6 ci granulocyte macrophage colony stimulating factor granulocye colony stimulating factor or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immtmnassays or purification of the target anugen. Such solid supports include, but are not limted to, glass, cellulose, polyacrylamade, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutc moiety to antibodies are well known, sec, Amon et al., "Monoclonal Antibodies For Immunotargeing Of Drugs In Cancer Therapy in Monoclonal Antibodies And Cancer Therapy Reisfeld et al.

pp. 243-56 (Alan R. Lass, Inc. 1985); Hellstrom et al., Antibodies For Drug Delivery" m Controlled Drug Delivery (2nd Robinson et al. 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. pp. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody m Cancer Therapy" in Monoclonal Antibodies For Cancer Detection And Therapy Baldwin et al. pp.

303-16 (Academuc Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Prooemes Of Antihndv-Tnxin Cnnjugates Immninol. Rev 62:1 '9-58 ('982).

Alternatively an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676.980, which is incorporated herein by reference m its entirety, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208e 16:31 61 2 92586999 4 062837999 N0. 641 D03 0 cAn antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytolone(s) can be Sused as a therapeutic.

Immunophenotyping The antibodies of the invention may be utilized for immunophenotyping of cell Slines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular 0 marker that is differentially expressed at various stages of differentiation and/or 00 10 maturation of particular cell types. Monoclonal antibodies directed against a specific Seputope, or combinaton of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning wth antibody attached to is a solid matrix plate), and flow cytometry (See. U.S. Patent 5,985,660; and Morrison et Cell, 96:737-49 (1999)).

These techmnques allow for the screening of particular populations of cells, such as might be found with hematological malignancies minimal residual disease (MRD) in acute leukemic patients) and non-sell" cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively these techniques allow for the screening of hematopotetic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found m human umbilical cord blood.

Assays For Antibody Binding The antibodies of the invention may be assayed for immunospecific binding by any method known in the art The immunoassays which can be used, include but are not limited to, competitive and non-compettive assay systems using techniques such as western blots, radioimmunoassays. ELISA (enzyme linked immunosorbent assay), sand'wich ;rmut-uoassays, ;riIrigoprecipuIatIua atssays, pyccipiiii ireachuns, gt:l diffusion precipimn reactions. Immunodiffusion assays, agglutinaton assays, complement-fixaton assays, tmmunoradiometric assays, fluorescent immunoassays.

protein A immunoassays. to name but a few Such assays are routine and well known n the art (see. Ausubel et al. eds, 1994, Current Protocols in Molecular Biology COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:31 61 2 92586999 4 062837999 NO. 641 P04 i Vol. 1, John Wiley Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not Sintended by way of limtation).

Immunoprecipitaton protocols generally comprise lysing a population of cells Smin a lysis buffer such as RIPA buffer NP.40 or Triton X 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease ihibitors EDTA, PMSF aprotnm. sodium vanadate). adding the antibody of interest to the cell O lysae, incubatng for a period of ume 1-4 hours) at 4'C. adding protein A and/or 00 to protein 0 sepharose beads to the cell lysate, mcubating for about an hour or more at 4° C, washing the beads m lysis buffer and resuspending the beads in SDS/sample buffer.

c The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by western blot analysis. One of skill in the an would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an is antigen and decrease the background pre-clearnng the cell lysate with sepharose beads). For further discussion regarding mnmunoprecipitation protocols see. e.g., Ausubel et al, eds, 1994. Current Protocols in Molecular Biology Vol. 1, John Wiley Sons, Inc., New York at 10.16.1 Western blot analysis generally compnses preparing protein samples, electrophoresis of the protem samples in a polyacrylamide gel 20% SDS- PAGE depending on the molecular weight of the antigen), transferring the protem sample from the polyacrylamide gel to a membrane such as ntrocellulose, PVDF or nylon, blocking the membrane in blocking solution PBS with 3% BSA or non-fat milk), washing the membrane m washing buffer PBS-Tween 20). blocking the membrane with pnmary antibody (the antibody of interest) diluted m blocking buffer, washing the membrane m washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody an anti-human antibody) conjugated to an enzymatic substrate horseradish peroxidase or alkaline phusphaiU=,s ur ,adiutuvc nmutscwue ce.g.. "P or dilutea in Dlocing Buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200 16:31 61 2 92586999 4 062837999 NO. 641 905 00 N regarding western blot protocols see, Ausubel et al, eds, 1994, Current Protocols in t Molecular Biology Vol. 1 John Wiley Sons, Inc., New York at 10.8.1_ SELISAs compnse preparing antigen, coaung the well of a 96 well rmcrotuter 0plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of tine, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of o interest) conjugated to a detectable compound may be added to the well. Further, 00 1o instead of coating the well with the antigen, the antibody may be coated to the well. In Sthis case, a second antibody conjugated to a detectable compound may be added followmg the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variatons of ELISAs known in the ar. For further I1 discussion regarding ELISAs see, Ausubel cl al, eds, 1994. Current Protocols in Molecular Biology Vol. 1, John Wiley Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen 'H or with the antibody of interest m the presence of ncreasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competiton with a second antibody can also be determned using radioimmunoassays. In tlus case. the anngen s incubated with antibody of interest conjugated to a labeled compound 'H or in the presence of increasinmg amounts of an unlabeled second antibody Therapcutic Uses The present invention is funher directed to antibody-based therapies which involve admmnisterng antibodies of the invention to an animal, preferably a mammal.

and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/20013 16:31 61 2 925986999 4 062837999 NO.641 P06 00 0 limited to, antibodies of the invenuon (including fragments, analogs and derivautives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and denvatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, minhibit or S prevent diseases, disorders or conditions associated with aberrant expression and/or actvty of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein autoimmune diseases, disorders, or conditions associated with such diseases or disorders, including. but not o lirmted to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, 00 to idiopathic thrombocytopenma purpura. autoimmunocytopenia, hemolyntic anemia, 0 antiphospholipid syndrome, dermatitis, allergic encephalomyelius, myocarditis, c relapsing polychondrnus, rheumautic heart disease, glomeulonephnrus IgA nephropathy), Multiple Sclerosis. Neunus. Uvents Ophthalmia, Polyendocntropathes.

Purpura Henloch-Scoenicn purpura). Reiter's Disease, Stiff-Man Syndrome, is Autonmmune Pulmonary Inflammanon. Guillamin-Barre Syndrome, insulin dependent diabetes mellitis, and autoaimune inflammatory eye, automunmune tbyrolditis, hypothyroidism Hashunoto s thyroditis, systemic lupus erbythematosus, Goodpasture s syndrome, Pemphigus, Recepor autonnimumites such as, for example.

Graves' Disease Myasthenma Gravis, and insulin resistance, autoimmune hemolytic anemra, autoimmune thrombocytopenc purpura rheumatoid arthritis, schlerodermna with anti-collagen antibodies, mixed connective tissue disease, polymyosts/dermaomyoszus, pemnicious anemia, idiopathic Addison's disease, minfertility glomerulonephrms such as prnmary glomerulonephtis and IgA nephropathy bullous pemphigod, Sjogren s syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhoss, other endornne gland failure, vitiligo, vasculits, post-MI, cardiotomy syndrome, urticana, atopic dermaitus, asthma, minflanmmatory myopathies, and other inflammatory granulamatous, degenerative. and atrophsc disorders).

In a specific embodiment, antibodies of the minvenution are be used to treat, inhibit, prognose. diagnose or prevent rheumatoid arnhntis.

In another specific embodiment, antibodies of the invention arc used to treat.

inhibit, prognose, diagnose or prevent systemic lupus eiythcmatosts.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 925eGS99 4 062837999 N0.641 D07 00 0 SThe treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but Sis not limited to. alleviatmg symptoms associated with those diseases, disorders or conditions. The antibodies of the invention may also be used to target and kill cells s expressing Neutrokine-alpha on their surface and/or cells having Neutrokme-alpha bound to their surface. Antibodies of the invention may be provided m pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invemton may be O used therapeutcally includes binding polynucleotides or polypepudes of the present O 0o invention locally or systerically in the body or by direct cytotoxicity of the antibody Sc.g. as mediated by complement (CDC or by effector cells (ADCC). Some of these Sapproaches are described m more detail below Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic. monitonng or therapeutic purposes without undue is expenmenmaion.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoienc growth factors (such as, IL 2, IL 3 and IL for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combinaton with other types of treatments radiation therapy chemotherapy hormonal therapy mmrnunotherapy ant-tumor agents, antibiotics, and immunoglobulin).

Generally administration of products of a species orgmn or species reactivity (in the C case of antibodies) that as the same species as that of the patient is preferred. Thus. m a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypepudes or polynucleotdes of the present invention, fragments or regions thereof, for both :mt.munoassays directed to and therapy of disorders related to polynucleotades or polypepudes, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleoudes or polypepudes of the invention, including fragments thereof. Preferred binding affinites include those with a dissociation constant or Kd COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 DOS 00 0 Sless than 5 X O' M, 10' M,5 X I M, X 10" 7 M 5 X X 10' M. M, 5 X 10 4 M, 10 M, 5 X 10" M, 10 M, 5 X 10 M, 10" M, 5 X I"' 1 M, 10" 2

M,

X 10'L M. 10i M, 5 X 10-" M, 10" 1 4 M, 5 X 10-" M. and o10' M.

Gene Therapy In a specific embodiment, nucleic acids comprisng sequences encoding antibodies or functional derivatives thereof, are administered to treat, hibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy Gene therapy refers to therapy 00 10 performed by the administration to a subject of an expressed or expressible nucleic 0 acid. In this embodiment of the invention, the nucleic acids produce their encoded C protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present nvention, Exemplary methods are described below b For general reviews of the methods of gene therapy see Goldspel e 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 1 1(5);155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described m Ausubel et al. (eds.).

Current Protocols m Molecular Biology John Wiley Sons, NY (1993); and Knegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprses nucleic acid sequences encoding an antibody said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimernc proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutve, and, optionally ussue-specific. In another pantcularembodiment, nucleic acid mole ule arp ued an %whbch the antibody coding sequences arI any orush uesareu sequences are flanked by regions that promote homologous recombinaton at a desired site in the genome, thus providing for mtrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 1903/200e 16:31 GI 2 925BG999 4 OG837999 NO0.641 909 0 0 expressed antibody molecule is a single chain antibody* altematively the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments Sthereof, of the antibody Delivery of the nucleic acids into a patient may be either direct, m which case s the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids nm vrro, then transplanted into the patient. These two approaches are known, respectvely as n vivo or ex vivo gene therapy O In a specific embodiment, the nucleic acid sequences are directly administered 00 10 n m a, where it is expressed to produce the encoded product. Ths can be o accomplished by any of numerous methods known in the art, by constructing them

S

r as par of an appropriate nucleic acid expression vector and administenng it so that they become intracellular, by mfection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286). or by direct injection of naked DNA, or by use of rmcropanrcle bombardment a gene gun; Bolistic. Dupont), or coating with lipids or cell-surface receptors or transfectng agents, encapsulation in liposomes, microparticles, or rmcrocapsules, or by administenng them in linkage to a peptide which is known to enter the nucleus, by administeng it in linkage to a ligand subject to receptor-mediated endocytosis (see, Wu and Wu, J Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acad-ligand complexes can be formed in which the ligand compnses a fusogenmc viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted m vivo for cell specific uptake and expression, by targeting a specific receptor (see, PCT Publications WO 92/06180; WO 92/22635; W092/20316: W093/14188. WO 93/20221). Altermauvely the nucleic acid can be introduced ntraccllularly and incorporated within host cell DNA for expression, by homologous recombmatlon (Koller and Smithies, Proc. Nail. Acad. Sci. USA 86:8932-8935 (1989); Zijlsta e Nature 342:435-438 989),.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an antibody of the invention are used. For example. a retrovral vector can be used (see 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 COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/20028 16:31 61 2 92586999 4 062837999 NO. 641 00 -ZS 0 0 integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used an gene therapy are cloned into one or more vectors, which facilitates Sdelivery of the gene into a patient More detail about retroviral vectors can be found in Boesen et al. Biotherapy 6:291 302 (1994), which describes the use of a retrovial s vector to deliver the mdrl gene to hematopoieuc stem cells in order to make the stem cells more resistant to chemotherapy Other references illustrating the use of retroviral vectors in gene therapy are: Clowes at al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129- S141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3.110-114 C t10 (1993).

00 0 Adenoviruses are other viral vectors that can be used in gene therapy Ci Adenoviruses are especially auractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endorhetial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy Bout et al.. Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovrus vectors to transfer genes to the respiratory epithelia of rhesus monkeys.

Other instances of the use of adenoviruses i gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991): Rosenfeld et al., Cell 68:143- 155 (1992); Mastrangcli et al., J. Clin. Invest 91.225-234 (1993); PCT Publication W094/12649- and Wang. et al.. Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associared virus (AAV) has also been proposed for use n gene therapy (Walsh et al.. Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.

5,436.146).

Another approach to gene therapy involves transferring a gene to cells m tissue culture by such methods s letrnporatorn, lipofectio. caium phosphate rcdiateu transfecton. or viral infecuon. Usually the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062337999 N0. 641 P11 00 0, In this embodiment, the nucleic acid is introduced into a cell pnor to administration in vivo of the resulting recombinant cell. Such introduction can be Scarned out by any method known an the art, including but not limited to transfecuon, electroporatson, mcroinjecuon, infection with a viral or bactenophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, mcrocell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known m the art for the ntroduction of foreign genes into cells (see, Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993): Cohen et al., Meth. Enzymol. 217:618-644 O (1993); Clin., Phanmac. Ther. 29:69-92m (1985) and may be used in accordance with 00 10 the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the C c stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably hertable and expressible by its cell progeny The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells hematopotetic stem or progenitor cells) are preferably administered ntravenously The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled i the art.

Cells ino which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include, but are not limited to, epthelial cells, endothelial cells, keratinocytes, fibroblasts, muscle ceUs, hepatocyces; blood cells such as T lymphocytes, B lymphocytes. monocytes, macrophages, neutrophils, eosmophils. megakaryocytes, granulocytes; various stem or progenitor cells, in partcular hematopoietc stem or progenitor cells, as obtaned from bone marrow umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy nucleic acid sequences encodmg an antibody are mtroduced into tne cells sucn tnal they are expressible by the cells or their progeny and the recombmant cells are then administered m vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintaned in vitro can potentially be used in accordance with this embodiment of the COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 G1 2 925e6999 4 062e37999 NO.641 012 00 0 present inventon (see e.g. PCT Publicaton WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rhemwald, Meth. Cell Bio. 21 A.229 (1980); and Pittelkow and SScott, Mayo Clinic Proc. 61 771 (1986)).

In a specific embodiment, the nuclesc acid to be introduced for purposes of gene s therapy comprises an riducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate induccr of transcrinpion.

O Demonstration of Therapeutic or Prophylactic Activity 0Q 10 The compounds or pharmaceuucal compositions of the mventon are preferably O tested m vuro, and then m vivo for the desired therapeutic or prophylactic activity prior C'q to use mi humans. For example, m vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical compositon include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing 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 invention, n vitro assays which can be used to determine whether administration of a specific compound is indicated, include m viaro cell culture assays a which a patient tssue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample as observed.

Therapeutic and/or Prophylactic Administration and Composition The invennon provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred embodiment, the compound is substantially purfied substantially free from substances that limit its effect or produce undesired side effects). The subject is preseraoy an animai, inluding out nor iimntco to ammals sucn as cows, pigs, inurses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of adminstration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 ND.641 D13 00 '4 additional appropnate formulations and routes of administration can be selected from t |among those described herein below Various delivery systems are known and can be used to administer a compound a> of the invention, encapsulation in liposomes, mtcroparticles, rmcrocapsules, recombinant cells capable of expressmg the compound, receptor-mediated endocytosis K (see, Wu and Wu, J. Biol. Chem. 262:44294432 (1987)), constuction of a Jnucleic acid as part ofa retrovral or other vector, etc. Methods of ntroduction include "t but are not limited to mtradermal, intramuscular, miraperntoneal, mtravenous, O subcutaneous, itranasal, epidural, and oral routes. The compounds or compositions o ]o may be administered by any convenient route, for example by infusion or bolus SIinjection, by absorption through epathelial or mucocutaneous linings oral mucosa, c rect al and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systermc or local. In addition, it may be destrable to introduce the pharmaceutcal compounds or compositions of the invention into the central nervous system by any suitable route, including ntravenincular and intrathecal injction; intraventncular injection may be facilitated by an mtraventcular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary admmnistraton can also be employed, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to adnrumster the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by for example, and not by way of limitation, local infusion dunng surgery topical applicaton, in conjunction with a wound dressing after surgery by injection, by means of a catheter, by means of a suppository or by means of an implant, said implant being of a porous, non-porous, or gelatinous material.

including membranes, such as sialastic membranes, or fibers. Preferably when administering a protein, including an antibody of the nvention, care must be taken lo use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249-1527-1533 (1990); Treat ct at., in Lposonmes i the Therapy of Infectious Disease and Cancer, Lopez-Berestem and Fidlcr Lass. New York. pp. 353- 365 (1989); Lopez-Berestem, ibid., pp.

317-327- see generally ibid.) COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 N0.641 914 00 245 00 In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, Ssupra; Sefton. CRC Cn. Ref. Biomed. Eng. 14.201 (1987); Buchwald et al., Surgery S588:507 (1980): Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another s embodiment, polymeric matenals can be used (see Medical Applications of Controlled Release, Langer and Wise CRC Press. Boca Raton, Flonda (1974); Controlled Drug Boaivailability Drug Product Design and Performance, Smolen and Ball (eds.), Wiley New York (1984); Ranger and Peppas, J, Macromol. Sc. Rev Macromol.

O Chem. 23:61 (1983); see also Levy et al.. Science 228:190 (1985); Dunng et al., Ann.

Sto Neurol. 25:351 (1989); Howard et al.. J.Neurosurg. 71.105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic CN target, the brain, thus requiring only a fraction of the systemic dose (see, e.g..

Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

IS Other controlled release systems are discussed in the review by Langer (Science 249 1527-1533 (1990)), In a specific embodiment where the compound of the invention is a nucleic acid encoding a proteinm, the nucleic acid can be administered n vwvo to promote expression of Ius encoded protein, by constructng it as part of an appropriate nucleic acid expression vector and admnstenng it so that it becomes nmtracellular, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of micropartcle bombardment a gene gun; Biolisuc. Dupont), or coating with lipids or cell-surface receptors or transfectng agents, or by administering it t linkage to a homeobox- like peptide which is known to enter the nucleus (see Joliot el al.. Proc. Natl. Acad. Sc. USA 88:1864-1868 (1991)), etc. Alternatively a nucleic acid can be introduced mtracellularly and incorporated within host cell DNA for expression, by homologous recombmation.

The present invention also provides pharmaceutical compositions. Such compnsit;ons comprse a therapeutically cffecive a iuum ui a compouna, ano a pharmaceutically acceptable carrer. In a specific embodiment, the term pharmaccutcally acceptable means approved by a regulatory agency of the Federal or a state government or listed m the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly mn humans. The term carrier COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 062837999 N0.641 00 0 refers to a diluent, adjuvant, exclpcnt. or vehicle with which the therapeutic is admmistered. Such pharmaceutical earners can be sterile liquids, such as water and C oils, including those of petroleum, animal, vegetable or synthetic orgm, such as peanut oil. soybean oil. mineral oil, sesame oil and the like. Water is a preferred carner when s the pharmaceutical composion is admistered intravenously Saline solutons and aqueous dextrose and glycerol solutions can also be employed as liquid carners, particularly for injectable solutions. Suitable pharmaceutical excipiens include starch, glucose, lactose, sucrose, gelatin, malt, nee, flour, chalk, silica gel, sodium stearate, O glycerol monostearate, talc, sodium chloride, dned skim milk, glycerol, propylene, 00 10 glycol, water, ethanol and the like. The composition, if desired, can also contain minor o amounts of wetting or emulsifying agents, or pH buffering agents. These compositions N _can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository with traditional binders and carners such as tnglycendes. Oral is formulation can include standard carriers such as pharmaceutical grades of manntol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical camers are described m "Remington s Pharmaceuucal Sciences" by E.W Marnt. Such compositions will contain a therapcutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of adinrustration.

In a preferrd embodiment, the composition s formulated tn accordance with routine procedures as a pharmaceutical composition adapted for intravenous adnuistration to human beings. Typically compositions for intravenous admminsuation are solutions in sterile isotonc aqueous buffer. Where necessary the composition may also include a solubilizng agent and a local anesthetic such as lignocame to ease pain at the site of the injection. Generally the ingredients are supplied either separately or mixed together in unti dosage form. for example, as a dry iyophilizcu puwucr or water ree concentrate in a nermetically sealed container sucn as an ampoule or sachette indicating the quantity of active agent. Where the composition as to be administered by infusion. it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200 IG:31 GI 2 925eG999 4 0G2e3?999 N0.641 16 00 0 injection, an ampoule of sterile water for mjecuon or saline can be provided so that the ingrediets may be mixed prior to admmistrauon.

SThe compounds of the invention can be formulated as neutral or salt forms.

SPharmaceutically acceptable salts nclude those formed with anons such as those derived from hydrochloric, phosphorm, acetic, oxalic, tartaric acids, etc., and those formed with catons such as those derived from sodium, potassium, ammonium, Scalcium, femc hydroxides. Isapropylarmne, tnethylamme. 2-ethylammo ethanol, histidinc procaine, etc.

o The amount of the compound of the invention which will be effective in the cO to treatment, inhibiion and prevention of a disease or disorder associated with aberrant 0 expression and/or activity of a polypepude of the invention can be determined by CN standard clinical techniques. in addition, vuro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of adrmnistration, and the seriousness of the is disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose response curves denved from in vitro or animal model test systems.

For antibodies, the dosage adnmnistered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally human antibodies have a longer half-life withm the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration into the brain) of the antibodies by modifications such as, for example, lipidation.

The ivention also provides a pharmaceutical pack or kit comprising one or mliulr cuoliuIcrs fiiiea with one or more or the ingredients or me pnarmaceutical compositions of the invention. Optionally associated with such conainer(s) can be a notice in the form prescribed by a governmental agencv regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human adrinrstration.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 GI 2 925eG999 4 OG2e37999 N0.641 P17 00 0 c Diagnosis and Imaging SLabeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypepuide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or acivity of a polypepude of the inventon. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more O antibodies specific to the polypepude interest and comparing the level of gene 00 10 expression with a standard gene expression level, whereby an increase or decrease in the assayed potypeptide gene expression level compared to the standard expression i level is indicative of aberrant expression, The mvention provides a diagnostic assay for diagnosing a disorder, compnsinmg assaying the expression of the polypoptde of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level ofgene expression with a standard gene expression level, whereby an increase or decrease i the assayed polypepude gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the mvention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill m the art see Jalkanen, et al.. J. Cell. Blol. 101:976-985 (1985); Jalkanen, et J. Cell.

Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, sucn as tme enzyme iinxcu immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the an and include enzvme labels, such as. glucose oxidase; radioistopes, such as iodine 'I25, carbon sulfur triuum indium 1 ln. and technetium 9"Tc), thallium 2 MTi). gallium COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 925eG999 4 06283?999 N0. 641 P18 0 O palladium m pd), molybdenum xenon (IMXe), fluonne 15 "Sm, "Yb, 'UHo, "Y 4 Sc, 'Pr, 0 Rh. "Ru: t lumunescent labels, such as lumrnnol; and fluorescent labels, such as fluoresccm and rhodanune, and bioun, s Techniques known m the art may be applied to label antibodies of the invention.

Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see eg., U.S. Patent Nos. 5,756,065; 5,714,631 5,696,239- 5,652361 5,505,931 5.489 425; 5,435.990; 5.428,139- 5,342,604; 5.274,119- 4.994.560; and 5,808.003, the contents of each of which are hereby incorporated by reference m its to entirety).

00 0 One embodiment of the invention is the detection and diagnosis of a disease or 0 disorder associated with aberrant expression ofa polypeptide of interest in an animal.

preferably a mammal and most preferably a human. Inone embodiment, diagnosis comprises: administering (for example, parenterally subeutaneously or is intrapentoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptde of interest; waiting for a time interval following the administering for permittng the labeled molecule to preferenually concentrate at sites an the subject where the polypeptide as expressed (and for unbound labeled molecule to be cleared to background level); determining background level; and detectig the labeled molecule n the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.

Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determned for a particular system. As described herein, specific embodiments of the invention are directed to the use of the antibodies of the invention to quantlate or qualitate concentrations of cells of B cell lineage or cells of monocytce lineage.

Also as described herein, antibodies of the invention may be used to treat, diagnose, or prognose an ndivioual having an immunoneficiency in a specfic 3o embodiment, antibodies of the invention are used to treat, diagnose, and/or prognose an individual having common vanable immunodeficiency disease (CVID) or a subset of this disease. In another embodiment, antibodies of the invention are used to diagnose, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200 16:31 61 2 92586999 4 062837999 NO.641 319 prognose, treat or prevent a disorder characterized by deficient senum immunoglobulin production, recurrent infections, and/or immune system dysfunction.

SAlso as described herein, antibodies of the invention may be used to treat, diagnose, or prognose an individual having an autoimmune disease or disorder. In a specific embodiment, antibodies of the invention are used to treat, diagnose, and/or prognose an individual having systemic lupus crythematosus, or a subset of the disease.

SIn another specific embodiment, antibodies of the invention are used to treat, diagnose and/or prognose an individual having rheumatoid arthnus, or a subset of tis disease_ o It will be understood m the art that the size of the subject and the imaging 00 O0 system used will determine the quantity of imaging moiety needed to produce Sdiagnostic images. In the case of a radioisotope moiety for a human subject, the c

N

I quantity of radioactivity injected will normally range from about 5 to 20 millicunes of "mTc. The labeled antibody or.antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vtvo tumor imaging is s1 described in S,W Burchiel et al., "Immunopharmacoknmelcs of Radiolabeled Antibodies and Their Fragments. (Chapter 13 m Tumor Imaging: The Radiochemacal Detecuon of Cancer, S.W Burchiel and B. A. Rhodes, eds.. Masson Publishing Inc.

(1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is S to days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after inital diagnosis, six months after mnttal diagnosis, one year after initial diagnosis, etc.

.senc o.f the labeled nolecule can be uc:twtu it ut: pauen using mcihoos known in the an for m vro scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not linuted to, computed tomography whole body COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 N0.641 scan such as position emisslon tomography (PET), magnetic resonance imaging (MRI), and sonography SIn a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical mstrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive Sscanning Instunmen. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography In yet 0 another embodiment, the molecule is labeled with a paramagnetic label and is detected Sto min a patient using magnetic resonance imaging (MRI).

0 cN Kits

I

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprses an antibody of the invention. preferably a purified antibody in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably the kits of the present invention further comprise a control antibody which does not react wrth the polypeptde of interest. In another specific embodiment, the kits of the present invention compnse two or more antibodies (monoclonal and/or polyclonal) that recognize the same and/or different sequences or regions of the polypeptide of the nvention. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymauc substrate, a radioactive compound or a lurmnescent compound, or a second antibody which recogmzes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit as a diagnostic kic for use in screening serum Wontamiiag aiibudies specti i agam lst proulntralve audur cancerous polynucleoudes and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of mnterest. Such a kit may include a substantally isolated polypepude antigen comprising an epitope which is specifically immunoreactive with al least one ant-polypeptde antigen antibody Further, such a kit COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 GI 2 92596999 4 062937999 N0.641 021 00 0 includes means for detecting the bmding of said antibody to the antigen the antibody may be conjugated to a fluorescent compound such as fluorescem or Srhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombiantly produced or chemically synthesized polypeptde antgen.

The polypepnde antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described klt mcludes a solid support to which said polypeptude antigen is attached. Such a kit may also nclude a non-attached reporter-labeled anti-human antibody In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the 0 10 said reporter-labeled antibody In an additional embodiment, the invention includes a diagnostic kit for use in Sscreening serum containing anugens of the polypepide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynuclcotde antigens, and means for detecting the binding of the polynucleoude or polypeptide antigen to the antibody In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody The detecting means of the kit may include a second, labeled monoclonal antibody Alternatively or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.

After binding wth specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bid reporter to the reagent in proportion to the amount of bound antantigen amibody on the solid support. The reagent is again washed to remove unbound labeled antibody and the amount of reporter associated with the reagent is deterrmned.

Typically the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometne, luminescent or colonmetnc substrate (Sigma.

St Louis, MO).

The solid surface reagent in the above assay is prepared by known techniques for ataching protein material to solid support matenral such as polymeric beads. din sticks. 9 6-well plate or filter materal These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 IG:31 61 2 925eG999 4 062e3999 N0.641 922 0 0 0 protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl. hydroxyl. or aldehyde group.

SAlternatively streptavidin coated plates can be used in conjunction with biotnylated antigen(s).

s Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antgen antibody o The invention further relates to antibodies which act as agonists or antagonsts S0to of the polypeptides of the present nvenzon, For example, the present invention o includes antibodies which disrupt the receptor/igand intcractions with the polypepndes Cl of the invention either partially or fully Included are both receptor-specific antibodies and ligand-specfic antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activauon. Receptor activation is signaling) may be determined by technrques described herein or otherwise known m the an. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, included are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand. thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included are antibodies which activate the receptor. These antibodies may act as agonsts for cither all or less rban all of the biological activities affected by ligand-mediated receptor acuvaion. The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herem. Further included are antibodies that bind to Neutrokine alpha and/or Neutrokme-alphaSV irrespective of whether Neutrokme-alpha or Ncutrokmnc-alphaSV is bound to a Neutrokine-alpha Receptor These antibodies act as Neutrokine-alpha and/or Neutrokine-alphaSV agonists as reflected in an increase in cellular proliferation in response to binding of Neutrokne-alpha and/or Neutrokinea.p..ha to a 'Ne.utroki-alpha rcceptor in the presenuc of tist atiiibudi'e. The auovc antibody agonists can be made using methods known in the ar. See WO 96/40281 US Patent 5,811.097' Deng, B et al., Blood 92(6):1981 1988 (1998); Chen, 7. ct al., Cancer Res. 58(16):3668-3678 (1998): Harrop, J.A. et al., J. Immunol.

161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58(15):3209-3214 (1998): Yoon, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2o08 16:31 61 2 92596999 4 062837999 NO.641 023 D Y ect, J. Immunoj. 160(7):3j70-3179 (1998); Pros. M. et al.. j. Cell. Set.

IlII(Pt2-);237-247 (1998). Piclard, V et al., j. lmxunol. Methods 205(2):177-190 (1997); Lautard, J. et 41,, Cytokmnde 9(4):233-241 (1997); Carison, N.G. et at.. J. Esol.

Chem, 272(17):.11295-11301 (1997); Taryman, R.E. et at., Neuron 14(4):755-762 s (1995): Muller, Y.A. et al., Structure 6(9):!1153-1167 (1998); Bartunek. P et at., Cytokine 14-20 (1996) (sid references incorporated by reference in their entireties).

At least fourteen monoclonal antibodies have been generated against Neutrokine-alpha. These monoclonal antibodies arm designated: 12D6, 2FE5. 9B6, IDA, i o 5F4,9A5, 10012, 11032, 16D4,3D4, 16C9 13D)5, 15CID, and 1205. Preliminary analysis of these antibodies indicates that each bind's Neurolne-alpha protein in a Western blot analysis and when Neurrokine-aipha protein is bound to an ELISA plate.

However, further analysis of antibodies 12136, 2E5, 9H6, 1iB8. 5J'4, 9A5, 100 12, 110 12, and 1 6B4 indicates chat only the antibodies designated 12D)6. 9B6. t, 100G12. 9A5, and I I3G12 bind a memibrane-bound forma of Neurrokmne-alpha. Thus, a subset of the monoclonal antibodies generated against Neucrokine-aipha have been detrmined to bind only the memnbrane-bound formn of Neutrokmec-alpha this subset does not bind the soluble form of Neutrolune-alpha corresponding to amino acids 134 to 285 of SEQ ID NO:2). which as discussed hcrein, is primarily limited to 2o expression on manocytes and dendnczic cells.

Antibody 9B6 has been found to bind specifically to the membrane bound form of Neutrokine-alpha, but not to rhe soluble fonn of Neurrokine-aipha.

Eparope mapping of antibody 9B6 has mndkcated that this antibody binds specifically to an ammno acid sequence contained in anuno acid residues from about Scr- 171 to about Ph(;-194 of SEQ ID) NO:2. More particularly epntope mapping has indicated that antibody 9B36 binds specifically to a peptide comrising amino acid residues Lys-173 to Lys-ISS of SEQ ID NO:2.

In contrast, antibodies 16C9 and 15CIO have been found to hind the soluble brm OfNc.~..u. tL (amznso acids- 134 ow 285 of SEQ IDJ NO.2) anu tu iilbi 3a Neutrokcine-alplia-mediated proliferation of B cells. See for example, Example 10. The ISCIO antibody has also been found to inhibit binding of Neuctrokine-aipha to its receptor. Epntope mapping of antibody 15CCW has indicated that this antibody bands specifically to an amino acid sequence contained in amino acid residues fraom about COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/20088 16:31 61 2 92586999 4 062837999 NO. 641 U24 SGlu-223 to about Tyr-246 of SEQ ID NO:2. More paricularly epitope mapping has indicated that antibody 15C10 binds specifically to a pepude comprising ammo acid t residues Val-227 to Asn-242 of SEQ ID NO:2. Antibody 15C10 also binds specifically to a peptide compsing amino acid residues Phe-230 to Cys-245 of SEQ ID NO:2.

As described above, anti-Neutrokine-alpha monoclonal antibodies have been prepared. Hybrtdomas producing the antibodies referred to as 9B6 and 15CI0 have been deposited with the ATCC and have been assigned deposit accession numbers SPTA-1158 and PTA-1159 respectively In one embodiment, the antibodies of the Snvention have one or more of the same biological characteristics as one or more of the t antibodies secreted by the hybndoma cell lines deposited under accession numbers SPTAI-158 or PTA-159 By "biological chracterstics" is meant, the mn vtro or in 0, vivo actuvttes or properties of the antibodies, such as, for example, the ability to bind to Neutrokinc-alpha the polypeptide of SEQ ID NO:2, the mature form of Neutrokine*alpha, the membrane-bound form of Neutrokine-alpha, hbc soluble form of 1i Neutrolkne-alpha (amino acids 134 to 285 of SEQ ID NO:2). and an antigenic and/or epitope region of Neutrokine-alpha), the ability to substantially block Neurrokinealpha/Neutrokine-alpha receptor binding, or the ability to block Neutrokne-alpha mediated biological activity stimulation of B cell proliferation and immunoglobulin production). Optionally the antibodies of the invention will bind to the same epitope as at least one of the antibodies specifically referred to herein. Such epitope binding can be routinely determined using assays known in the art.

Thus, in one embodiment, the invention provides antibodies that specifically bind the membrane-bound form of Neutrokne-alpha and do not bind the soluble form of Neutrokne-alpha. These antibodies have uses which include, but are not limited to, as diagnostic probes for identifymg and/or isolating monocyte lineages expressing the membrane bound form of Neutrokine-alpha. For example, the expression of the membrane bound form of Neutrokme-alpha is elevated on activated monocytes. and accordingly antibodies encompassed by the invention may be used to detect and/or iiuaLIUC• m Ivezs ui acuivmeu iut.ucy.. udiuunl uiy utibudi that uiy U11" membrane bound form of Neutrokne-alpha may be used to target toxins to neoplastic, prencoplastic. and/or other cells that express the membrane bound form of Neutrolnealpha monocytes and denditic cells).

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200 16:31 61 2 92586999 062837999 NO.641 00 C0 In another embodiment, antibodies of the invention specifically bind only the soluble form of Neutroknae-alpha (armno acids 134 to 285 of SEQ ID NO:2). These Santibodies have uses which include, but are not lirmted to, uses such as diagnostic Sprobes for assaying soluble Neutrokne-alpba m biological samples, and as therapeutic agents that target toxins to cells expressing Neutrokne-alpha receptors B cells).

and/or to reduce or block in vitro or in vivo Neutrokine-alpha mediated biological Sacitivty stimulation of B cell proliferation and/or immunoglobulin production).

The invention also provides for antibodies that specifically bind both the membran-bound and soluble form of Neutrokme.-alpha.

000 o As described above, the invention encompasses antibodies that inhibit or reduce Sthe ability of Neutrokme-alpha andlor Neutrokne-alphaSV to bnd Neutrokine-alpha c receptor and/or Neurokme-alphaSV receptor in vitro and/or in vivo. In a specific embodiment, antibodies of the invention inhibit or reduce the ability of Neutrokme alpha and/or Neutrokne-alphaSV to bind Neutrokne-alpha receptor and/or Neutrokne-alphaSV receptor m vitro. In another nonexclusive specfic embodiment, antibodies of the invention inhibit or reduce the ability of Neutrokne-alpha and/or Neutrokine-alphaSV to bind bind Neutrokine-alpha receptor and/or NeutroknealphaSV receptor in vivo. Such inhibition can be assayed using techniques described herein or otherwise known in the art.

The invention also encompasses, antibodies that bind specifically to Neutrokne-alpha and/or Neutrokne-alphaSV but do not inhibit the ability of Neutrokme-alpha and/or Neutroklne-alphaSV to bind Nentrokine-alpha receptor and/or Neutrokine-alphaSV receptor in vitro and/or in vivo. In a specific embodiment, antibodies of the invention do not inhibit or reduce the ability of Neutrokine-alpha and/or NeutrokmnealphaSV to bind Ncutrokme-alpha receptor and/or NeutroklnealphaSV receptor in vitro. In another nonexclusive specific embodiment, antibodies of the invention do not inhibit or reduce the ability of Neutrokme-alpha and/or Neutrohlne-alphaSV to bind Neutrokiun-alpha receptor and/or Neutrokme-alphaSV As described above, the invention encompasses antibodies that inhibit or reduce a Neutrokne-alpha and/or Neutrokie-alphaSV-mediatcd biological activity m vitro and/or in vvo. In a specific embodiment, antibodies of the invention inhibit or reduce Neurokmne-alpha- and/or Neutrokine-alphaSV.mediated B cell proliferation in vitro.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 925eG999 4 062e3?999 N0.641 D26 00 O Such inhibition can be assayed by routnely modifying B cell proliferation assays SC described herein or otherwise known m the an. In another nonexclusave specific embodiment, antibodies of the invention inhibit or reduce Neutrokme-alpha- and/or Neutrokne-alphaSV-mediated B cell proliferation m vivo. In a specific embodiment, the antibody of the Invention is 15C 10, or a humanized form thereof. In another preferred spectfic embodiment, the antibody is 16C9 ora humanized form thereof.

Thus, m specific embodiments of the invention, a 16C9 and/or 15C10 antibody or humanized forms thereof, are used to bind soluble Neutrokme-alpha and/or Ncutrokne- O alphaSV and/or agonsts and/or antagonists thereof and thereby inhibit (either pamally 0 10 or completely) B cell proliferation.

O Alternatively the invention also encompasses, antibodies that bind specifically t o a Neutrokme-alpha and/or Neutrokme-alphaSV but do not inhibit or reduce a Neutrokme-alpha and/or Neurokinc-alphaSV-mediated biological activity in vtro and/or m vivo sumulaion of B cell proliferation). In a specific embodiment, antibodies of the nvention do not inhibit or reduce a Neutroklne-alpha and/or Neutrokne-alphaSV-mediated biological activity in vitro. In another nonexclusive embodiment, antibodies of the invention do not mhibit or reduce a Neutrokme-alpha and/or Neutrokme-alphaSV mediated biological activity n vivo. In a specific embodiment, the antibody of the invention is 9B6, or a humanized form thereof.

As described above, the invention encompasses antibodies that specifically bind to the same epitope as at least one of the antibodies specifically referred to herein, in viro and/or in vivo.

In a specific embodiment, the antibodies of the invention specifically bind to an ammo acid sequence contaned in amino acid residues from about Ser-171 to about Phe-194 of SEQ ID NO:2, in vitro. In another specific, non-exclusive embodiment, the antibodies of the invention specifically bind to an ammo acid sequence contamed in ammo acid residues from about Ser-171 to about Phe 194 of SEQ ID NO:2, n vivo. In another specific, non-exclusive embodiment, the antibodies of the ivention specifically omo to an armno acio sequence contaIncu in aninu aucau rU jdUc from i .ys- 173 to Lys188 of SEQ ID NO:2, in vitro. In another specific, non-exclusive embodiment, the antibodies of the mvention specifically bind to an amino acid sequence contained in amino acid residues from Lys-173 to Lys-188 of SEQ ID NO:2.

in vivo.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 iq/w/2ooe8 16:31 61 2 92586999 4 062837999 N0.641 927 00 In an additional specific embodiment, the antibodies of the invention specifically bind to an amino acid sequence contained in ammno acid residues from Sabout Glu-223 to about Tyr-246 ofSEQ ID NO:2, in vitro. In another specific, nonexclusive embodiment, the antibodies of the invention specifically bind to an amino acid sequence contained in anmno acid residues from about Glu-223 to about Tyr-246 of SEQ ID NO:2 m vivo. In another specific, non-exclusive embodiment, the antibodies of the nvention specifically bind to an amino acid sequence contained m amino acid residues from Val-227 to Asn-242 of SEQ ID NO:2. In vitro. In another O specific, non-exclusive embodiment, the antibodies of the invention specifically bind to 00 10 an ammino acid sequence contained in amino acad residues from Val-227 to Asn-242 of SSEQ ID NO:2, m vivo. In another specific, non-exclusive embodiment, the antibodies Sof the inventon specifically bind to an amino acd sequence contained in amino acid residues from Phe 230 to Cys-245 of SEQ ID NO:2, in vitro. In another specific, nonexclusive embodiment, the antibodies of the invention specifically bind to an amino lb acid sequence contained in amino acid residues from Phe 230 to Cys-245 of SEQ ID NO:2, in vivo.

The invention also provides antibodies that competitively inhibit the binding of the 9B6 monoclonal antibody produced by the hybndoma deposited as PTA- 1159 to a polypepude of the invention, preferably the polypepude of SEQ ID NO:2, more preferable to a polypcpide having the amno acid sequence of residues Ser 171 to Phe- 194 of SEQ ID NO:2. Competirve inhibition can be determined by any method known n the art, for example, using the competitive binding assays described herein. In preferred embodiments, the antibody compenavely inhibits the binding of 9B6 monoclonal antibody by at least 95%, at least 90%, at least 85%. at least 80%. at least 75%, at least 70%, at least 60%, at least 50%, to the polypeptide of SEQ ID NO:2, or more preferable w a polypeptide having the amino acid sequence of residues Ser 171 to Phe-194 of SEQ ID NO:2.

The invention also provides antibodies that competitively inhibit the binding of the 15CI monoclonal antibody proaucca by in nyonoonia olpospu u a.r FPT-. .5 to a polypeptide of the invention, preferably the polypeptide of SEQ ID NO:2, more preferable to a polypeptide having the amino acid sceuence of residues Glu-223 to Tyr- 246 of SEQ ID NO:2. In preferred embodiments, the antibody compeitively inhibits the binding of b1CIO monoclonal antibody by at least 95%. at least 90%, at least COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 928 00 233 0 at least 80%, at least 75%, at least 70%, at least 60%. at least 50%, to the polypeptude of SEQ ID NO:2, or more preferable to a polypeptide having the amino acid sequence of residues Glu-223 to Tyr-246 of SEQ ID NO:2.

Additonal embodiments of the invention are directed to the 9B6 antibody and t o the hybdoma cell line expressing this antibody A hybndoma cell line expressng Antibody 9B6 was deposited with the ATCC on January 7 2000 and has been assigned SATCC Deposit No. PTA-1159 In a preferred embodiment, antibody 9B6 is humanized.

0 Additional embodiments of the nvention are directed to the 15CI0 antibody Oo 10 and to the hybndorna cell line expressing this antibody A hybndoma cell line expressing Antibody 15CIO was deposited with the ATCC on January 7 2000 and has N been assigned ATCC Deposit No. PTA- 1158. In a preferred embodiment, antibody 15C10 is humanized.

In a specific embodiment, the specific antibodies described above are humanized usmg techniques described herein or otherwise known in the art and then used as therapeutics as described heremn.

In another specific embodiment, any of the antibodies listed above are used in a soluble form.

In another specific embodiment, any of the antibodies listed above are conjugated to a toxin or a label (as described infra). Such conjugated antibodies are used to kill a particular population of cells or to quanutate a particular population of cells. In a preferred embodiment, such conjugated antibodies are used to kill B cells expressing Neutrokmealpha receptor on their surface. In another preferred embodiment, such conjugated antibodies are used to quantitate B cells expressing Neutrokine-alpha receptor on their surface.

In another specific embodiment, any of the antibodies listed above are conjugated to a toxin or a label (as described mnfra). Such conjugated antibodies are used to kill a particular population of cells or to quanutate a particular population of Xli. L, u piCfereaTu t'lbudiJim such LV.jugaicu aiodic are usu i kiil no.nocye cells expressing the membrane-bound form of Neutrokine-alpha. In another preferred embodiment, such conjugated antibodies are used to quantisate monocyte cells expressing the membrane-bound form of Neutrokine-alpha.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208 16:31 G1 2 925eG999 4 OG2837999 NO.641 929 00 o The antibodies of the mInvention also have uses as therapeutics and/or prophylactics which include, but are not limnted to. in activating monocyes or blockinmg monocyte activation and/or killing monocyte lineages that express the membrane bound form of Neutrokme-alpha on their cell surfaces to treat, prevent, and/or diagnose myeloid leukemias, monocyte based leukemias and lymphomas, monocytosis, monocytopenia, rheumatoid arthritis, and other diseases or conditions associated with activated monocyes). In a specific embodiment, the antibodies of the invention fix complement In other specific embodiments, as further described herein, the antibodies of the minvention (or fragments thereof) are associated with heterologous polypeptides or o00 nucleic acids toxins, such as, compounds that bind and activate endogenous 0 cytotoxic effecter systems, and radioisotopes; and cytotoxic prodrugs).

C In another embodiment, one or more monoclonal antibodies are produced wherein they recognize or bind Neurokine-alpha and/or a mutein thereof, but do not recognze or bind Neutrokme-alphaV and/or a mutein thereof. In a related Is embodiment, one or more monoctonal antibodies are produced wherein they recognize or bmind Neutrokinc-alphaSV and/or a mutem thereof, but do not recognize or bind Neutrokne-alpha and/or a mutein thereof.

As discussed above, antibodies to the Neutrokine-alpha and/or Neutroklne-alpha SV polypeptides of the invention can, n turn, be utilized to generate anti-adioaype antibodies that mimic the Neutrokane-alpha, using techniques well known to those skilled in the art. (See, Greenspan Bona, FASEB J 7(5):437-444 (1989), and Nissinoff, J Inmmunot 147(8):2429-.2438 (1991)). For example, antibodies which bind to Neutrokne-alpha and/or Netrokine-alpha SV and competitively minhibit the Neutrokne-apha and/or Neutrokmne-alpha SV mulimenzaon and/or binding to ligand can be used to generate anti-idiotypes that rmimic the Neutrokine-alpha

TNF

munmenzanon and/or binding domain and, as a consequence, bind to and neutralize Neutrokine-alpha or Neutrokline-alpha SV and/or us ligand. Such neutralizing antiidiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic repmens to acurralize Neutrokinc-alpha liganu. F exanpae, sucn ann-idiotypic antibodies can be used to bind Neutrokne-alpha and/or Neutrokme-alpha SV or to bmd Neutrokne-alpha and/or Neutrokine-alpha SV receptors on the surface of cells of B cell lineage, and thereby block Neutrokinc-alpha and/or Neutrokine-alpha SV mediated B cell activation, proliferation, and/or differcntanon COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 925e6999 4 062e3?999 NO. 641 00 0 3 SImmune System-Related Disorder Diagnosis C Neutrokmne-alpha is expressed in kidney lung, peripheral leukocyte, bone marrow T cell lymphoma, B cell lymphoma, activated T cells, stomach cancer, smooth muscle, macrophages, and cord blood tissue, and particularly cells of monocytic lineage. Moreover, Neutrokme-alphaSV as expressed in pnmary dendrnic cells.

Additionally Neutrokine-alpha is expressed on the cell surface of the following nonhematopoietic tumor cell lines. Colon carcinomas HCT 116 (ATCC Accession No. 0 CCL 247) and HT 29 (ATCC Accession No. HTB-38); Colon adenocarcnomas Caco- 00 10 2 (ATCC Accession No. HTB-37), COLO 201 (ATCC Accession No. CCL 224), and o WiDr (ATCC Accession No. CCL 218); Breast adenocarcinoma MDA-MB-23 I (ATCC Accession No. HTB-26); Bladder squamous carcinoma SCaBER (ATCC Accession No. HTB-3); Bladder carcinoma HT 1197 (ATCC Accession No. CRL 1473); Kidney carcinomas A-498 (ATCC Accession No. HTB-44), Caki-1 (ATCC s1 Accession No. HTB-46), and Caki-2 (ATCC Accession No. HTG-47); Kidney Wilms tumor SK-NEP I (ATCC Accession No. HTB-48); and Pancreas carcinomas 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 a number of immune system-related disorders, substantially altered (increased or decreased) levels of Neutrokine-alpha and/or Neutrokne-alphaSV gene expression can be detected in immune system tissue or other cells or bodily fluids sera, plasma, urine, synoval fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" Ncutrokme-alpha and/or Neutrokme-alphaSV gene expression level, that is.

the Neutrokne-alpha and/or Neutiokme-alphaSV expression level in immune system tissues or bodily fluids from an individual not having the immune system disorder.

Thus, the ivention provides a diagnostic method useful dunng diagnosis of an immune system disorder, which involves measuring the expression level of the gene encoding the Neutrokine-alpha and/or Neutroinke-alphaSV polypeptide in immune system tissue or other cells or body fluid from an individual and companng the measured gene expression level with a standard Neutrokine-alpha and/or Neutrokine-alphaSV gene expression level, whereby an incrase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder or normal activation, proliferation, differentjauon, and/or death.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 D31 o 00 Cl In particular, a is believed tha certain tissues in mammals with cancer of cells ct or tissue of the immune system express sigmficantly enhanced or reduced levels of the Neutrokmne-alpha and/or Neutrokine-alphaSV polypcpude and mRNA encoding the Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide when compared to a s corresponding "standard" level. Further. it is believed that enhanced or depressed levels of the Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide can be detected in certan body fluids sera, plasma, unne, and spinal fluid) or cells or tissue from mammals with such a cancer when compared to sera from mammals of the same O species not having the cancer.

00 10 For example, as disclosed herein, Neutrokine-alpha is highly expressed in cells Sof monocytc lineage. Accordingly polynucleotldes of the invention C polynucleoude sequences complementary to all or a portion of Neutrokne-alpha mRNA and/or Neutrokme-alphaSV mRNA) and antibodies (and antibody fragments) directed against the polypeptidcs of the invention may be used to quantiate or qualitate concentrations of cells of monocytc lineage monocyuc leukemia cells) expressing Neutrokine-alpha on their cell surfaces. These antibodies additionally have diagnosuc applications m detecting abnonnalities m the level of Neutrokine-alpha gene expression, or abnormalities m the structure and/or temporal, tissue, cellular, or subcellular location of Neutrokine-alpha and/or Neutrokine-alphaSV These diagnostic assays may be performed in vivo or m vitro, such as. for example, on blood samples.

biopsy tissue or autopsy tissue.

Additionally as disclosed herein, Neutrokine-alpha receptor is expressed primarily on cells of B cell lineage. Accordingly Neutrokme-alpha polypepudes of the Sinvention (including labeled Neutrokne.alpha polypeptdes and Neutrokine-alpha fusion proteins), and anti-Neutrolkne-alpha antibodies (including anu-Neutrokme-alpha antibody fragments) against the polypeptldes of the invention may be used to quanttate or qualitate concentrations of cells of B cell lineage B cell related leukemias or lymphomas) expressing Neutrokine-alpha receptor on their cell surfaces. These Neutrokine-aipna polypeptiaes ana antibodies audinonally nave diagnosaiu appli;caion in detecting abnormalities n the level of Neutrokme-alpha receptor gene expression. or abnormaliles in the structure and/or temporal. tissue, cellular, or subcellular location of Neutrokine-alpha receptor and/or diagnosing activity/defects in signalling pathways associated with Neutrokine-alpha. These diagnostic assays may be performed in vivo COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/20oe 19/03/008 :31 G1 2 92586999 4 06237?999h.61 2 NO. 641 P32 0 or in vitro, such as, for example, on blood samples or biopsy tissue using techniques descibed herein or otherwise known in the art.

In one embodiment, Neutrolcane-alpha and/or Neutrolcme-alphaSV polynucleotides or polypeptides or Neutrokine-aipha and/or Ncuzrokznc-alphaSV s agomists, or antagonists anu-Neurrokine-alpha and/or anui-Neucrokine-aIphaV antibodies) of the invention ame used to treat, prevent, diagnose, or progpose an individual having an immunodeficiency luuinunodaficiencws, that may he treated, prevented, diagnosed, and/or o prognosed with the Neutrokane-aipha and/or Neurrokane-alphaSV polynuicleotades or 00 10 poiypeptides or Neuirokmne-alpba and/or Neurroline-alpbaSV agomsrs or antagonists o (eWg., anti-Neuirokine-alpha and/or anzi-Neuirokzne-alphaSV antibodies) of the Cl invention, include, but are not limited to one or more immunodeficienctes selected from: severe combined immunodeficiency (SCID)-X linked, SCID-auosomal, adeosine deamwuasc deficiency (ADA deficiency). X-Uinked agazumaglobulinemia i s (XLA), Bruton s disease. congenital agammaglobulinerrua, X-linked infantile agamxawglobinenrua, acquired agarniaglobuliosmnia. adult onset agaxnmaglobulinrnma. late-onset Againmaglobulinemnia. dysgamraglobulincxrua.

hypogammaglobul inernua, tr-ansient hypogammaglobulinenrua of infancy unspecified hypogarnmaglobulineia, agammaglobniinemia, common variable inmmunodeficiency (CVII)) (acquired), Wiskott-Aldnich Syndrome (WAS), X-linkcd inununodeficiency with hyper 1g*M, non X-Iinked inmnunodeficiency with hyper 1gM, selective IgA deficiency IgG subclass deficiency (with or without IgA deficiency). antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency 8 coil lyrnphoproliferauve disorder (ELPO).

selective IgM inmmunodeficiency recessive aganunaglobulinenna (Swiss type), reticular dysgenesis. neonatal neutropenia, severe congenital leukopenial, thymnic alymphoplasma-aplauia or dysplasia with imunodeficiency atax a-telaingicctasia short limbed dwarfism, X-linked lymphoproliferauive syndrome Nezejof syndromernr--bn-d i un. nnOd e fic; en 1 w i rh I gs. punn ae raucicusidc Phtnujbpuziust1a UC: i vi cflLy (PNP), MHC Class 11 deficiency (Bare Lymphocyte Syndrome) and severe cominied immnunodeficiency According to this embodiment, an individual having an immunodeficiency expresses aberrantly low levels of Neutrokine-alphA and/or Nenirokine-aipha SV when COMS ID No: ARCS-i 83623 Received by IP Australia: Time (1-tm) 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 D33 00 zsj 0 0 compared to an individual not having an immunodeficiency Any means described heremn or otherwise known in the art may be applied to detect Neutrokine-alpha and/or C Neutrokine-alphaSV polynucleotides or polypepudes of the minvention FACS analysts or ELISA detection of Neutrokinealpha and/or Neutrokine-alpha3V polypepudes of the minvention and hybridizaton or PCR dtecuon of Neutrokme-alpha and/or Neutrokine-alphaSV polynucleotzdes of the anventwon) and to determie the expression profile ofNeutrokinme-alpha and/or Neurokne-alphaSV polynucleobdes and/or polypeptides of the Invention in a Iolgical sample.

A biological sample of a person afflicted with an immunodeficiency is to characteried by low levels of expression of Neutrokinealpha and/or Neutrokzne- 0 alphaSV when compared to that observed in individuals not havming an immunodeficency Thus, Neutrobkne-alpha and/or Neutrokmne-alphaSV polynucleoudes and/or polypepndes of the. invention, and/or agonists or antagonists thereof. may be used according to the methods of the wvenuon in the diagnosis and/or is prognosis of an inmmunodeficency For example, a biological sample obtained from a person suspected of being afflicted with an immunodefictency ("the sublect") may be analyzed for the relative expression level(s) of Neutrokine-alpha, andlor Neutrokine alphaSV polynucleotides and/or polypeptides of the invention. The expression level(s) of one or more of these molecules of the invention as (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with an immunodeficiency A significant difference in expression level(s) of Neutrokine-alpha, andlor Neutrokine-alphaSV polynucleotides and/or polypepudes of the invenon, and/or agomusts and/or antagonists thereof, between samples obtained from the subject and the control suggests that the subject is afflicted with an immunodeficieney In another embodiment, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleoides or polypeptides or Neutrokine-alpha and/or Neutroktne-alphaSV agonsts or antagonists anti-Neutrokme-alpha and/or anti-Neutrokne-alphaSV antibodics, of the imvention are usru tu tirea, diagnoswor ponose an mdivinal having common variable immunodeficiency disease (CVID' also known as acquired agammaglobulinemia and acquired hypoganmmnaglobulinemia" or a subset of this.

disease. According to this embodiment, an individual having CVID or a subset of mindividuals having CVID expresses aberrant levels of Neucrokine-alpha and/or COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92596999 4 062837999 NO. 641 U34 00 0 0 Neutrokine-alpha Receptor on ther B cells and/or monocytes, when compared to mdividuals not having CVID. Any means described herein or otherwise known in the art may be applied to detect Neutokmune-alpha polynucleondes or polypeptides of the invenhlon and/or Neutrolun-alpha Receptor polypeptides FACS analysts or s ELISA detection of Neutrokme-lpha andlor Neutrokne-alphaSV polypeptides of the mvenion and hybndization or PCR detecton of Neutrolue-alpha and/or NeutrokinealphaSV polynucleotdes of the mnvenuon) and to determine differentially the expression profile of Neurokune-alpha and/or Neutrokne-alphaSV polynucleotides or o polypeptides of the invention and/or Neutrobne-alpha receptor polypeptides in a 00 to sample containiong at least monocyte cells or some component thereof RNA) as 0 compared to a sample containing at least B cells or a component thereof RNA).

Cl In the instance where a sample containing at least monocyte cells or some component thereof(e.g., RNA) as determined to reflect Neutrokne-alpha andlar NeutrokinealphaSV polynuclotade or polypeptade expression and a sample containing at least B is cells or a component thereof(e.g., RNA) is determined to reflect less than normal levels of Neutrokmine-alpha receptor polynucleotzde or polypeptde expression, the samples may be correlated with the occurrence of CVID acquired agammaglobulinea or "acquired hypogammaglobulinemia").

A subset of persons afflicted with CVID are characterntzed by high levels of expression of both Neutr6lane-alpha and the Neutrokne-alpha receptor min peripheral or circulatinmg B cells when compared to that observed in individuals not having CVID In contrast, persons who are not afflicted with CVID are typically charactzed by low levels of Neutrokme-alpha expresston and high levels of NAR expression n peripheral or circulating B cells. Thus. Neutrokmne-alpha, Neurokute- alphaSV polypepudes, and/or NAR polypepudes, polyntiucleotides and/or polypepudes of the minvention, andlor agonirsts or antagonists thereof, may be used according to the methods of the mnvenaton an the differential diagnosis of this subset of CVID For example, a sample of penriphenal B cells obtained from a person suspected of being afrfiied with CVID ("ute subjecf) may be anaiyzec ror tne relative expression aeveqs) of Neutirokmne-alpha, Neutrokmne-alphaSV and/or NAR polynucleotides and/or polvpeptides of the invention.. The expression levells) of one or more of these molecules of the minvention is (are) then compared to the expression level(s) of the same molecules of the iavenuon as expressed an a person known not to be afflicted with COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200B 19/032008 16:31 61 2 92586999 4 062837999m.4 Q3 NO. 641 00 240 0 CVII) ("the controJ*). A significant difference in expression level(s) of Neutrokinealpha and/or Neutrione-alphasv polynuicleoudecs or pelypeptides Of the invention, and/or NAR polypeptides. and/or agoniss and/or antagomsrs thereof, between samnples obt:z:e from the subject and the conrol suggests that the subject is afflicted with tis Neutrokute-alpha, and/or ntI-Neutrobin-aphaSV,antij)de3s) are used to diagnose o pregnose, tret or prevent a disorder characterized by deficient serum aminnoglobulin 00 o production, recurrent infections, and/or immune system dysfunction. Moreover.

0 Neutrbkne-alpta. an/o Ncutmbne-alpbasv polyniz-leoids or polypeptie, or agornsts or antagonists thereof anti-Neuarokine-alpha and/or anti-Neutrokine alphaSV antibodies) may be used to diagnose, prognose, treat, or prevent infections of the joints, bones, skin, andl/or parozid glands, blood-borne infections sepsls, meninitis, septic arthrius, and/or osteomyclitis), autozmnmune diseases those disclosed herein), iniflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but nor limiuted to. CVID other pnimary immune deficiencies.

HIV disease, CLL, rectuent bronchitas, sinusitis, otitis mnedia 1 conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster severe herpes tester), and/or pneumnocystis camnii.

In Mnother embodiment, Neutrokmne-alpha and/or Netroktne-alpbaSV polynucleotidcs or polypepuides or Neutrokine-aipha and/or Neutrokine-aiphaSV agonists or antagonists anti-Neucrokne-aspha and/ar anzi-Neurroine-alphasV antibodies) of the invention are used to treat, diagnose, or prognose an individual having an auroinunune disease or disorder Autoirnmune diseases or disorders that may be treated, diagnosed, or propnosed using Neutrokine-aipha and/or Neulrokzne-ralphaSV polynucicoudes or polypeptides; or NeuLmoknne lpha tnd/or tgUE11SUr t aigonisis te-g., anui- Neurrolone-alpba and/or antz-Neutrokine-alphasv antibodies) of the invention include, but are not limited to, one or more of the following; aruoinunune hemoivujc anemia.

autoimmune neonatal throinbocytopenia. idiopathic ilhrombocytopenia purpura, autoimmunocytopernia, hemolytic anemia. antiphospholipid syndrome, dermatitis, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 19/032029 16:31 61 2 925136999 4 062937999 h.4 3 NO. 641 P36 0 0 allergic encephalomyelibs, myocarditas, rela1png pelychondritis. rheumatic beant disease, glomerulonephrzuis JgA nephropaihy), Multiple Sclerosis, Neuritts, Uveits Ophthalia, Polyenidocninopalhnes, Purpura Henloch'Scoenlein purpura), thyroiditis, systemic lupus erhythematosus, Goodpasrure s syndrome, Pemphagus, ri- Reiceptor auronmmunitzes such as, for example, Graves' Disease Myasthensa o Gravis, and insulin resistance, autoummune hemolytic anemita, autoiwaimune 00t 10 thomhocytopenlc purpina rheumatoid arthritis, schleroderma with aim-collagen 0 antibodies, mixed connective tissue disease, polymyosilisidermalomnyositis. perniciobus ci anemia, idiopathic Addison's disease, infertility glomeruloncphritis such as primary glomerulonephnztis and %gA nephrapathy bullous pemrphigoid. Sjogren's syndrome, diabetes niilitus. and adrenergic drug- resistance (including adrenerglc drug resistance is with asthma or cystic fibrosis), chronic active hepatitis, prmary biliary cirrhosis, other endocrine gland falure. vatiligo. vasculiuis. post-MI, cardioromy syndrome, urticaria, acopic dermatitis, asthma, inflarmatory myopathies. and ocher intlamrnaoiy granulamazous, degenerative, and atrophic disorders.

According to this embodiment, an individual having an autoimunie disease or 2o disorder expresses aberrantly high levels of Neuzroiune-alpiha, Neutrokine-alpha

SY

and/or MAR when compared to an individual not having an aulotaunune disease or disorder Any means described herein or otherwise known in the art may be applied to detect Nemtrokmne-alpha, and/or Neuxrokine-alpbaSV polynuclcoudes or polypeptides of the invention and/or NAP. polypep~des FACS analysis or EUSA detection of 2s Neutrobne-alpha andWar Neuirakmne-alphaSV polypeptides of the invention and hybridization or PCR detection of Neurrokine-alpha and/or Netrokznc-alphasV polynucleoudes of the invention) and to determine the ezpression profile of Neutrolrme-alpha and/or Neiitrokmne-alpbaSV polynucleotides and/or pulypepoides of the inwetibon avid'or MAR pelyp=eptides~ lia biol1ogican tupie.

A biological sample of persons afflicied with an autolmmune disease or disorder is characterized by high levels of expression or Neutrokmne-alpha. NeutrokinealphaSY and/or ['AR when compared to chal observed in individuals not hiving an autoimmune disease or disorder. Thus. Neutrokine-aipha and/or Neurrokine-.alphaSV COMS ID No: ARCS-183623 Received by IP Australia: Time (F-Pm) 16:56 Date 2008-03-19 19/03/2009 16:31 61 2 92586999 4 062837999 NO.641 P37 00 polynucleotudes and/or polypepudes of the nvention, and/or agosts or antagonists thereof, may be used according to the methods of the invention in the diagnosis and/or prognosis of an auotmmune disease or disorder. For example, a biological sample obtamned from a person suspected of being afflicted with an autoimmune disease or s disorder ("the subject") may be analyzed for the relative expression level(s) of Neutrokne-alpha and/or Neutrokine-alphaSV polynucleotides and/or polypepudes of the invention and/or NAR polypepudes. The expression level(s) of one or more of these molecules of the mvention is (are) then compared to the expression level(s) of-the same molecules of the mivention as expressed in a person known not to be afflicted to with an autoimmune disease or disorder. A significant difference in expresison level(s) 00 o of Neutrokine-alpha, andlor Neutrokmine-alphaSV polynucleontdes and/or polypeptdes of the invention, and/or agonists and/or antagomnists thereof, and/or NAR polypeptdes between samples obtained from the subject and the control suggests that the sutbject is afflicted with an autommnmune disease or disorder in another embodiment. Neutrokine-alpha and/or Neutrokne-alphaSV polynucleotades or polypeptides or Neutrokzne-alpha and/or Neurrokmine-alphaSV agonists or antagonists ant-Neutrokme-alpha and/or anu-Neutrokme-alphaSV antibodies) of the invention are used to treat, diagnose, or prognose an individual having systemic lupus erythematosus or a subset of this disease. According to this embodiment, an individual havming systemic lupus erythematosus or a subset of individuals having systemic lupus erythematosus expresses aberrantly high levels of Neutrolunre-alpha andlor Neutrokine-alpha SV when compared to an mindividual not havming systemic lupus erythemaosus or this subset of systemic lupus erythernatosus.

Any means described herein or otherwise known in the art may be applied to detect Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides or polypeptides of the minvenution FACS analysts or EUSA detection of Neutrokne-alpha andlor Neutrokine-alphaSV polypeptides of the invention and hybridizaton or PCR detection of Neutrokine-alpha and/or Neutrokme-alpiaSV polynucleotides of the invention) and to determmne the exoression nronllA of Neutrok!ne-a-!ph and/ct Neutroksn-alphaS" polynucotides and/or polypeptides of the invention in a biological sample A biological sample of persons afflicted with systemic lupus erythematosus is characanrzed by high levels of expression of Neutrokine-alpha and/or NeutrokinealphaSV when compared to that observed in individuals not having systemic lupus COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 338 00 Serythematosus. Thus, Neutrokme-alpha and/or Neutrokine-alphaSV polynucleoudes and/or polypeputdes of the invention, and/or agorusts or antagonists thereof, may be Sused according to the methods of the nvention in the diagnosis and/or prognosis of systeinc lupus erythematosus or a subset of systemic lupus erythematosus. For example, a biological.sample obtaned from a person suspected of being afflicted with systemic lupus erytheamatosus ("the subjeci') may be analyzed for the relative Sexpression level(s) of Neutrokme-alpha, and/or Neutrokine-alphaSV polynucleotides and/or polypeptides of the invention. The expression level(s) of one or more of these o molecules of the invention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with o systemic lupus erythematosus. A significant difference in expression level(s) of ci Neutrokine-alpha, and/or Neutrokine.alphaSV polynucleoudes and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, between samples obtained from the subject and the control suggests that the subject is afflicted with systemic lupus erythematosus or a subset thereof.

Furthermore, there is a direct correlation between the seventy of systemic lupus erythematosus. or a subset of this disease, and the concentranon of Neutrokine.alpha and/or Neutrokine-alphaSV polynucleotides (RNA) and/or polypeptides of the invention. Thus, Neutrokme-alpha and/or Neutroktne-alphaSV polynucleoudes, (RNA), polypeptdes and/or agonists or antagonists of the invention. may be used according to the methods of the invention m prognosis of the seventy of systemic lupus erythematosus or a subset of systemic lupus erythematosus. For example, a biological sample obtained from a person suspected of being afflicted with systemic lupus erythematosus ("the subject") may be analyzed for the relative expression level(s) of Neutrokine-alpha, and/or Ncutrokne-alphaSV polynucleoudes and/or polypeptides of the mvention. The expression level(s) of one or more of these molecules of the mvention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a panel of persons known to represent a range in seventies of this disease. According to this method, the match of expression level iwht a characterized member of the panel indicates the seventy of the disease.

In another embodiment, Neutrokme-alpha and/or Neutrokane-alphaSV polynucleoides or polypeptides or Neutrokme-alpha and/or Neutrokme-alphaSV agomsts or antagonists anu-Neutrokine-alpha and/or ant-Neutrokine-alphaSV COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 D39 00 0 0 antibodies) of the invention are used to treat, diagnose, or prognose an individual having rheumatoid arthrits or a subset of this disease. According to this embodiment, San individual having rheumatoid arthritis or a subset of individuals having rheumatoid arthritis expresses aberrantly high levels of Neutrokine-alpha and/or Neutrolune-alpha s SV when compared to an individual not having rheumatoid arthntis or this subset of rheumatoid arthntis. Any means described herein or otherwise known in the ar may be applied to detect Neurokme-alpha and/or Neutrokme-alphaSV polynucleotdes or polypeptides of the invention FACS analysis or ELISA detection of Neutrokne- O alpha and/or Neutrokme-alphaSV polypeptades of the invention and hybndizauon or 00 10 PCR detection of Neutrokin-alpha and/or Neutrokine-alphaSV polynucleoudes of the Sinvention) and to determine the expression profile of Neutrokane-alpha and/or C -Neutrokme-alphaSV polynucleotides and/or polypeptldes of the nvention m a biological sample A biological sample of persons afflicted with rheumatoid arthnus is characterzed by high levels of expression of Neutrokine-alpha and/or NeutroktnealphaSV when compared to that observed in individuals not having rheumatoid arthrnts. Thus, Neutrokme-alpha and/or Neutrokin-alphaSV polynucleoudes and/or polypeptdes of the invention, and/or agonists or antagonists thereof, may be used according to the methods of the invention in the diagnosis and/or prognosis of rheumatoid arthrnts or a subset of rheumatoid anhrts. For example, a biological sample obtained from a person suspected of being afflicted wih rheumatoid arhnus ("the subject") may be analyzed for the relative expression level(s) of Neutrokmealpha, and/or Neutrolkne-alphaSV polynucleotides and/or polypepudes of the invention. The expression level(s) of one or more of these molecules of the inventon is (are) then compared to the expression level(s) of the same molecules of the inventon as expressed in a person known not to be afflicted with rheumatoid arthnus. A signficant difference i expression level(s) of Neutrokme-alpha, and/or NeutrokanealphaSV polynucleoutdes and/or polypeptides of the invention, and/or agonists and/or aniagonmss thereor, Deiween samples oolaineo irom the suoject anu die contro suggests that the subject is afflicted with rheumatoid anhntis or a subset thereof.

Thus, the invention rovides a diagnostic method useful during diagnosis of a immune system disorder, including cancers of this system, and immunodeficiencies andlor autonmmune diseases which involves measuring the expression level of the gene COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200 16:31 61 2 92586999 4 062837999 NO.641 040 0 0 0 encoding the Neurokine-alpha andlor Neutrokne-alphaSV polypeptide im mmune k system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard Neutrookne-alpha and/or Neutokine-alphaSV gene expression level, whereby an increase or decrease i the gene expression level compared to the standard is indicative of an immune system disorder.

4 Where a diagnosis of a disorder in the immune system, including, but not Slimited to, diagnosis of a tumor, diagnosis of an immunodeficiency and/or diagnosis of o an autommune disease, has already been made according to conventional methods, the C to present invention is useful as a prognostic indicator whereby patients exhibiting o enhanced or depressed Neutrokine-alpha and/or Neutrokme-alphaSV gene expression :will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

By analyzing or determining the expression level of the gene encoding the is Neutrokme-alpha and/or Neutrokme-alphaSV polypeptide is intended qualitatively or quantitatively measuring or estmating the level of the Neurokme-alpha and/or Neutrokme-alphaSV polypeptide or the level of the mRNA encoding the Neutrokine-alpha and/or Neutrokmne-alphaSV polypeptde in a first biological sample either directly by determining or estmating absolute protein level or mRNA level) or relatively by comparing to the Neutrokme-alpha and/or Neutrokine-alphaSV polypeptide level or mRNA level in a second biological sample).

Preferably the Neutokine-alpha and/or Neutrolne-alphaSV polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard Neurrokmne-alpha and/or Neutrokine-alphaSV polypeptde level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determned by averaging levels from a population of individuals not having a disorder of the immune system. As will be appreciated in the art, once a standard Neutrokine-alpha and/or Neutrokne-alphaSV polvpeptude or mlRNe level is known, it can be useu repicazcuy An a Sniuairu tor comparison.

By "biological sample is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains Neutrokme-alpha and/or Neutrokin-alphaSV polypepude or mRNA. As indicated, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 941 00 2 0 o biological samples include body fluids (such as sera, plasma, urane, synovial fluid and spinal fluid) which contain free extracellular domains of the Neurrokine-alpha and/or SNeutrokine-alphaSV polypepude, immune system tissue, and other tissue sources found to express complete or free extracellular domain of the Neutrokine-alpha and/or Neutrolune-alpaSV or a Neutrolkine-alpha and/or Neutrokmne-alphaSV receptor.

Methods for obtaining tissue biopsies and body fluids from mammals are well known m the ar. Where the biological sample is to intclude mRNA, a tissue biopsy is the preferred source.

The compounds of the present invention are useful for diagnosis, prognosis, or treatment of varous immune system-related disorders in mammals. preferably humans.

o Such disorders inmelude, but are not limited to tumors B cell and monocytic cell 0 -C leukermas and lymphomas) and tumor mestasis, infections by bacteria. viruses and other parasites, immunodeficencics. inflammatory diseases, lymphadenopathy autoimmune diseases rheumatoid arhtris, systemic lupus erythamatosus. Sjogren syndrome, mixed connective tissue disease, and inflammatory myopathlies). and graft venrsus host disease.

Total cellular RNA can be isolated from a biological sample usming any suitable technique such as the smingle-step guamdinium-th ocyanate-phenol-chloroform method described in Chomezynskl and Saeuhi. AnaL Biochem. 162.156-159 (1987). Levels of mRNA encoding the Neutrokne-alpha and/or Ncurokine-alphasV polypepude are then assayed using any appropriate method. These include Northern blot analysts, SI nuclease mapping, the polymerase chain reaction (PCR), reverse transcription min combination with the polymerase chain reaction (RT-PCR). and reverse transcnption in combmination with the ligase chamin reaction (RT-LCR).

Assayming Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide levels in a biological sample can occur using antibody-based techniques. For example, Neutrokine-alpha and/or Neutrokine-alphaSV polypeptide expression in tissues can be studied with classical immunohistological methods (Yalkanen, et at, J. Cel. Biol.

101-96-985 Jalkanen, e J. Cell BiL 105:30187-3 096 95). 0Jr anlibody-based methods usethl for detecting Neutrokmine-alpha and/or Neurokne-alphaSV polypeptide gene expression include irmmunoassays. such as the enzyme linked rnmunosorbent assay (ELISA) and the radiotmmunoassay

(RIA).

Suitable antibody assay labels are known in the art and include cnzymine labels, such as.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 042 00 O glucose oxidase, and radioisotopes, such as iodine T1, 2 l, carbon 4

C),

sulfur tnunm indium "In, "'In and techneuum ITce), t thallium gallium palladium molybdenum xenon 3 sXe), fluonne '0Gd, 'Pm, 'La, "Y "Sc. "'Re, 'Pr, "Ru; luminescent labels, such as lumnol; and fluorescent labels, such as fluorescein and rhodarmne, and biout.

Techniques known m the art may be applied to label antibodies of the invention.

Such techniques include, but are not lirrted to, the use of bifuncuonal conjugating Sagents (see U.S. Patent Nos, 5.756,065; 5,714,631. 5.696,239- 5,652,361 SoI 5,505,931 5,489 425- 5,435.990; 5,428,139- 5,342,604; 5274,119- 4.994,560; and 0 5,808,003, the contents of each of which are hereby incorporated by reference in its entirety).

The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the NeutrokAne-alpha gene (such as. for example, cells IS of monocytic lineage) or cells or tssue which are known, or suspected, to express the Neutrokme-alpha receptor gene (such as, for example, cells of B cell lineage and the spleen). The protein isolation methods employed herein may for example, be such as those described in Harlow and Lane (Harlow E. and Lane, 1988, Antibodies:

A

Laboratory Manual" Cold Sprng Harbor Laboratory Press, Cold Spring Harbor, New York), which s incorporated herein by reference in is enurety The isolated cells can be denved from cell culture or from a patient. The analysis.of cells taken from culture may be a necessary step in the assessment of cells that could be used as pan of a cellbased gene therapy technique or, altemanavely to test the effect of compounds on the expression of the Netrokine-alpha gene or Neutrokne-alpha receptor gene. For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quanttatuvely or qualitatively detect the presence of Neutrokmealpha gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescendy labeled antibody couped with light microscopic, flow cyto.Minr u fluonrmetnc detection.

The antibodies (or fragments thereof) or Neutrokine-alpha polypepudes or polypepudes of the present invention may additionally be employed histologically as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 N0.641 P43 00 2Aif 0 Sin situ detection of Neutrokne-alpha gene products or conserved variants or peptide Sfragments thereof, or for Neutrokine-alpha binding to Neutrokme-alpha receptor. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or Neutrokine-alpha polypeptde of the s present invention. The antibody (or fragment) or Neutrokine-alpha polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the Neutrokine-alpha gene product, or conserved variants or peptide fragments, or Neutrolkm-alpha polypeptide binding, but also its distribution m the C 10 examined tissue. Using the present mvention, those of ordinary skill will readily Sperceive that any of a wide variety of histological methods (such as staining r procedures) can be modified in order to achieve such in situ detection.

Immunoassays and non-nmmunoassays for Neutrokine-alpha gene products or conserved variants or peptide fragments thereof will typically comprse ncubanng a is sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated m cell culture, in the presence of a detectably labeled antibody capable of identifyng Neurokime-alpha gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.

Immunoassays and non-immunoassays for Neutrokine-alpha receptor gene products or conserved variants or peptide fragments thereof will typically comprse incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells.

or lysates of cells which have been incubated in cell culture, in the presence of a detectable or labeled Neutrokmne-alpha polypeptide capable of identfying Neutronkealpha receptor gene products or conserved variants or peptide fragments thereof, and detectng the bound Neuttrokne-alpha polypeptde by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilized onto a solid uhase sunnort or eaner such as mtrocellulose, or othr solid suppor which ;s capable of immobilizing cells, cell paricles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled anti- Neutrokine-alpha antibody or detectable Neutrokme-alpha polypeptide. The solid phase support may then be washed with the buffer a second time to remove unbound COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208 16:31 61 2 92586999 4 062837999 NO. 641 44 00 0 0 antibody or polypeptde. Optionally the antibody is subsequently labeled- The amount of bound label on solid support may then be detected by conventional means.

C By "solid phase support or carrier" is intended any support capable of binding an antgen or an antibody Well-known supports or carrers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyaerylarmdes, gabbrs, and magnette. The nature of the carrer can be either soluble to some extent or insoluble for the purposes of the present invention. The support matenal may have vrtually any possible structural configuraton so long as the Scoupled molecule is capable of binding to an antigen or antibody Thus, the support 0 1to configuraton may be spherical, as n a bead, or cylindrical, as in the inside surface of a Stest tube, or the external surface of a rod. Alternatively the surface may be flat such as C. a sheet, test stnp, etc. Preferred supports include polystyrene beads. Those skilled n m the art will know many other suitable earners for binding antibody or antigen, or will be able to ascertain the same by use of routine expenmenation.

The binding activity of a given lot of anti-Neutrokine-alpha antibody or Neutrokme-alpha polypepude may be determined according to well-known methods.

Those skilled in the art will be able to determine operative and optimal assay conditions for each determnnmaon by employing routine expenmentation In additon to assaying Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, Neutrokine-alpha and/or Neuzrokine-alphaSV polypeptides or polynucleotides can also be detected in vivo by tmaging. For example, in one embodiment of the invention, Neutrokine-alpha and/or Neutrokmne-alphaSV polypeptide and/or anu-Neutrokim-alpha antibody is used to image B cell lymphomas.

In another embodiment, Neutrokme-alpha and/or Neutroknme-alphaSV polypeptdes and/or antt-Neulrokine-alpha antibodies and/or Neutrokne-alpha polynucleoudes of the invennon polynucleotides complementary to all or a portion of Neutrokme-alpha and/or Neutrokme.alphaSV mRNA) is used to image lymphomas monocyte and B cell lymphomasl Antibody labels or markers for m vvo imagmg of Neutrokne-alpha and/or Neutrokine-alphaSV polypeptide include those detectable by X-radiography NMR.

MR. CAT-scans or ESR. For X-radiography suitable labels include radioisotopes such as barium or cesium, which errt detectable radiation but are not overtly harmful COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:31 61 2 92586999 4 062837999 N0D.641 00 o to the subject. Suitable markers for NMR and ESR include those with a detectable charactensuc spin, such as deutenum. which may be incorporated into the antibody by t labeling of nurients for the relevant hybndoma. Where m wvo imaging is used to detect enhanced levels of Neutrokme-alpha andlor Neutrokmne-alphaSV polypeptide for diagnosis in humans, it may be preferable to use human antibodies or -humanized" chimcnc monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the an. For example methods for producing chimenc antibodies are known in the art. See, for review Morrson, Science 229-1202 o (1985); Ot et al., BioTechmques 4:214 (1986); Cabilly et al., U.S. Patent No.

4.816,567- Tamguchi et al., EP 171496; Morson et al., EP 173494; Neuberger ct al., 0 WO 8601533; Robinson et al., WO 8702671 Bouliann et aL. Nature312:643 (1984): C Neuberger el aL, Nature 314:268 (1985).

Additonally any Ncutrokine-alpha polypepude whose presence can be detected, can be administered. For example, Neutroune-alpha polypeptides labeled is with a radio-opaque or other appropnate compound can be administered and visualized m vivo, as discussed, above for labeled antibodies. Funher such Neutrokine-alpha polypeptides can be utilized for in vtro diagnostic procedures.

A Neutrokine-alpha and/or Neuroknme-alphaSV polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 2 carbon sulfur tntum indium In, and technetum ("Tc.

thallium gallium "6Ga), palladium molybdenum xenon fluonne 'Sm, "Lu, IWGd, "4La, 'Yb, "Y 4 7Sc.

2 Re, 'Pr, 'Rh, a radio-opaque substance, or a maenrial detectable by nuclear magnetic resonance, is introduced (for example, parenterally subcutaneously or intrapentoneally) mio the mammal to be examined for immune system disorder It will be understood m the ar that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radio=solope rc:crt for a human subject, te quamuily u radiuacLu vty injected will normally range from about 5 to 20 millicunes of The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain Neutrokiuc-alpha protein. hn viv tumor imaging is described in S.W Burchiel et al., "Immunopharmacokmetics of Radiolabeled Antibodies and Their COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/3/200e 16:31 61 2 92586999 4 062837999 NO.641 P46 0251 Fragmentso (Chapter 13 in Tumor Imagmng: The Radiochemacal Detection of Canmcer S.W Burchiel and B. A. Rhodes, eds., Masson Publishinmg Inc. (1982)).

t With rspect to antibodies, one of the ways in which the anti-Neutroklne-alpha antibody can be detectably labeled is by linking the same to an enzyme and using the linked product min an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)" 1978, Diagnostic Horizons 2:1 7 Microbiological Associates Quarterly Publication. Walkersville, MD); Voller et al., J. Clin. Pathaol.

31:507-520 (1978): Butler. Mn/h. Enymol. 73:482 523 (1981); Maggio, E. 1980, Enzyme Immunoassay CRC Press, Boca Rarton, FL,, Ishikawa, E.et al., (eds.), 1981. Enzyme immunoassay Kgaku Shorn, Tokyo). The enzyme which is bound to 00 o the antibody will react with an appropriate substrate, preferably a chromogenice 0 substrate, in such a manner as to produce a chemncal moiely which can be detected, for example, by spectrophotomemc, fluonmertic or by visual means. Enzymes which can be used'to detectably label the antibody include, but are not imited to. malate is dehydrogenase, staphylococcal nuclease, dela-5-sterod Isomerase. yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase. tnose phosphate Isomerase, horseradish peroxidase, alkaline phosphazase, asparaginase, glucose oxadase, betagalactosudas. ribonuclease. urease, catalase, glucose-6-phsphate dehydrogenase, glucoamylase and acetylcholincstcrase. Additionally the detection can be accomplished by colonmetric methods which employ a chromogenae substrate for the enzyme. Detection may also be accomplished by visual comparson of the extent of enzymatic reaction of a substrate m comparison with similarly prepared standards.

Detection may also be accomplished using any of a vanety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, It is possible to detect Neurrokme-alpha through the use of a radionnmunoassay (RIA) (see, for example. Wemtraub, Princiaples of Radionmmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocnrine Society March, 1986, which is incorporated by reference herein). The radioactive Isotope can be detected by means mincluding, but nut iiniLui a gamman counter, a scintillation counter. or autoradiography It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave-length. its presence can then be detected due to fluorescence. Among the most commonly used COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e8 16:31 61 2 92586999 4 062937999 NO.641 947 00 fluorescent labeling compounds are fluorescemn isothiocyanate, rhodamine.

phycoerythnn. phycocyanrn, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence cemamg metals such as or others of the lanthanzde series. These metals can be attached to the S antibody using such metal chelatung groups as diethylenetnrammepentacetjc acid (DTPA) or ethylcnediammtetraaeuc acid (EDTA).

The antibody also can be detectably labeled by coupling It to a chemiluminescent compound, The presence of the chemilummiaenscgrt-tagged antibody as then determined by detecting the presence of luminescence that arises during the to course ofa chenmical reaction. Examples of particularly useful cemiluminescent 0 labeling compounds are lumnol, isolummol, theromatrc, acndinium ester, imadazole, S2acrndinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemilurnnescence found in IS biological systems inm, which a catalytic protein increases the efficiency of the chemilnunescent reaction. The presence of a biolumnescent protein is deterruned by detecting the presence of lumminescence. Important bioluminescent compounds for purposes of labeling include, but are not limited to, lucifenn, lucferase and aequorm.

Treameat of Immune System-Related Disorders As noted above. Neutrokne-alpha and/or Neutrokne-alphaSV polynucleoudes and polypeputdes, and anti-Neutrokine-alpha antibodies, are useful for diagnosis of conditions involving abnormally high or low expression of Neutrokine-alpha and/or Neutrokne-alphaSV activities. Given the cells and tissues where Neutrokme-alpha and/or Neutrokine-alphasV is expressed as well as the activities modulated by Neutrokine-alpha and/or Neutrokine-alphaSV t is readily apparent that a substantially altered (increased or decreased) level of expression of Neutrokine-alpha and/or Ncutrokane-alphV min an individual compared to the standard or normal" level produces r"h.gicaul condii"os related to the budily sysummusu in wnaen Neuurokane-alpha and/or Neutroktne-alphaSV is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since the Neutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the invention are members of the TNF family the extracellular domains of the respective proteins may COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208 16:31 61 2 92586999 4 062837999 NO.641 P48 00 obbe released In soluble form from the cells which express Neutrokme-alpha and/or Neuroknc-alpbaSV by proteolytic cleavage and therefore, when Neutrokne-alpha and/or Neutrokme-alphaSV polypeputide (particularly a soluble form of the respective extracellular domains) is added from an exogenous source to cells, tissues Or the body Sof an mindividual, the polypeptide will exert its modulatinmg activiies on any of its target cells of that Individual. Also, cells expressing this type II transmembrane protein may be added to cells, tissues or the body of an mindividual whereby the added cells will binmd to cells expressang receptor for Neutrokine-alpha andlor Newrokne-alphaSV whereby the cells expressmg Neutrokine-alpha and/or Neutrokne-alphaSV can cause actions proliferation or cytotoxicity) on the recepror-bearng target cells.

00 In one embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention compositions containing Neurtmkne alpha and/or NeurokmealphaSV polypeptdes or ann-Neutrokine-alpha and/or anti- Neutrokine-alphaSV antibodies associated with hererologous polypepudes.

iD heterologous aucleic acids, toxins, or prodrugs) to targeted cells, such as, for example, B cells expressing Neutroknme-alpha and/or Neutrokine-alphaSV receptor, or monocytes expressing the cell surface bound form of Neutrokine-alpha and/or Neutrokine-alphaSV Neutrokine-alpha and/or Neutrokne-alphaSV polypeptides or anri-Neutrokne-alpha and/or anu-Neutroklne-alphaSV antibodies of the invention may be associated with heterologous polypepudes, heterologous nucleic acids, toxins, or produgs via hydrophobic, bydrophilic, ioneic and/or covalent interactios.

In one embodiment, the invention provides a method for the specific delivery of compositions of the Invention to cells by administering polypeptides of the invenion Neutrokmne-alpha and/or Neutrokine-alphaSV polypeptides or ani-Neutrokmes alpha and/or anti-Neutrolne-alphaSV antibodies) that are asociated with heterologous polypepudes or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the minvention provides a method for delivenring a single stranded nuclic acid anusense or ribozymes) or double stranded nucllc acid DNn that caln integrate into ic VCcll's genome or replicate epasomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells the destruction of tumor cells) by administering polypepudes of the Invention Neutrokine-alpha and/or Neutrokme-alphnSV polypepudes or COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/032008 16:31 61 2 92586999 4 062837999 NO.641 949 00 anu-Nctroknc-alpha and/or anti-Neutrokne-alphaV antibodies) in association with toxins or cytotoxic prodrugs.

In a specific embodiment the invention provides a method for the specific destruction of cells of B cell lineage B cell related feukermas or lymphomas) by admmastermng Neutrokrne-alpha and/or Neutrokne-alphaSV polypeptdes min association with toxins or cytoloxic prodrugs.

In another specific embodiment, the invenuon provides a method for the specific destructon of cells of monocyteic lineage monocytc letukeminas or lymphomas) by adminwstering ant-Neutrokne-alpha and/or anti-Neutrokine-alphaSV io antibodies m association with toxms or eytotoxic prodrugs.

o By "toxan is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxmins, catalytic subunits of toxins, cytotoxms (cyotoxic agents), or any molecules or enzymes not normally present In or on the surface of a cell that under defined conditions cause the cell's death.

1i Toxins that may be used according to the methods of the invention include, but are not liutred to, radioisotopes known in the art, compounds such as. for example, antibodies (or complement fixming containing porons thereof) that bind an inherent or induced endogenous cytotoxuc effector system. thymidine kmnase, endonuclease, RNAse, alpha toxin, neun. abnn, Pseudomonas exotoxin A, diphtheria toxin, saporin. momordin, gelonin, pokeweed antiviral protem alpha-sarcin and cholera toxin. "Toxin also includes a cytostatic or cytoidal agent, a therapeutic agent or a radioactive metal son, alpha-emitters such as, for example, or other radioisotopes such as, for example, 'Pd, Xe. MGe, Co "Zn, "Sr. "P 9 Y 1 "Sm, Gd, 'Yb. "Cr, K'Mn, "Se, Wttnum, "'Tin, t Rhenium, '"Holmum, and "'Rhenum: luminescent labels, such as lumunol; and fluorescent labels, such as fluorescemn and rhodamne, and bouin.

Techniques known an the art may be applied to label antibodies of the bnvenuon.

Such techsniques include, but are not limited to, the use ofl bfunctional conjugaung agents U.S. ritent Nos. 5256,065- 5,714.631 5,696,239- j,65236A 5,505,931 5,489 425; 5.435,990; 5,428,139- 5,342,604; 5,274.119- 4,994,560: and 5.808,003 the contents of each of which are hereby incorporated by reference in its entirety). A cytooxn or cytotoxic agent includes any agent that is detrimental to cells.

Examples include pacliwaol, cytochalmin B, grarrucidin D chidium bromidc, emetine, COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 19/032008 16:31 61 2 92596999 4 062937999 I.4 NO. 641 P50 0 o mitomycin. eloposide, tenoposide. vuncnisu noe vinblastane, colebiern, doxorubacin, C daunorubicin, dibydroxy azubracin dione, nuzoxantrone, nuibrmina aeninorayem D ct 1~~~~~-debydrozestosrerone. glucocortcogas, procaine. ttaae ioanpornll n puromycin and analogs or bornologs thereof. Thenpeuuic agents include, but are not s limited to, antimerabolites methcnrexnc, 6-mnercaptopunne, 6-duoguanine, cynarabmne, 5-fluorouracil decarbazmne), aikylating agents rnecblorediamne, 4 tioepa chiorambucil, meiphalan. cannusame (BSNU) and lomustne (CCNU), cyclodiosphamide. busulfan, dibromomannatol, streptozoocan, rmtomycin C, and clsdichlorodiarnu platinum (11) (flop) clisplatin), anthracyclines; daunontbicin io (formerlIy daunomycmn) and doxorubuicin). antibiotics dactanotnycin (fbrnuarly 0o actmnomycin). bleomycin. rnthramycin, and arnbramnycmn and anui-nuwutc 0pi vinenistne and vrnblastuie). By cytoboxic prodrug is rmant a non-toxic compound that is convened by an enzyme, normally present in t cell, into a cyroroxic compound. Cytozoxac prodrugs is that may be used according to the methods of the invention include, but are not limited to. glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate: denivatives of etoposade or mitomycin C, cytosinc arabiroside, daunorubisun, and phecnoxyacetarrude derivatives of doxorubican.

It will be appreciated that conditions caused by a decase in the standard or normal level of Neuurokzne-alpba and/or Ncuurokine-alphaSV activity in an individual, particularly disorders of the inmnune system, can be (meated by administration of Neutrokcine-alpna andfor Neutrokine-alpimsv polypeptide (in the form of soluble entracellular domain or cells expressing the complete protein) or agonist. Thuts. the invention also provides a method of treatment of an individual in need of an increased level of Neurrokine-alpha and/or Neutrokine-alphaSV activity comprising admirustcnng to such an individual a pharmaceutical composition comprising an amount of an isolated Neurrokmine-alpha and/or Neuilrokine-alphaSV polypeptide of the invention, or agOnist thereof, effective to increase the Neutiokine-alphai and/or Meutrokine-alphasv acti-iay level ifn such an indi--idual.

It will also be appreciated that conditions caused by a increase in the standard or normal level of Neutrobcne-alpha and/or Ncutrokine-alpbaSV activity in an individual.

particularly disorders of the immune system, can be treated by administration of Neutrokine-alpha andtor Neuhrokine-alphaSV polypeptides (in the form of soluble COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/20ee 19/032009 1:31 61 2 92598999 4 082937999 .81 NO. 641 951 00 O extracdllular domain or cells expressing the complete protein) or antagonist an anui-Neurrolune-alpha antibody). Thus, the invention also provides a method of trcatment of an individual in need of an deminsed level of Neurrokumc-alpha. and/or Neutrokine-alphaWv activity compnisig administering to such an individual a s pharmaceutical composition comprising an amiount of an isolated Neutrokine-alpha and/or Neuzraksne-alphaSV polypeptide of the invention, or antagonist thereof, effective, to decrease the Neulrokune-alpha and/or Neutrokie-alphaSV activity level in such an individual.

In of Neutrokine-alpba and/or Neuirokinc-alphaSV polynucleotides or polypeptides 00 o the invention, or agonists of Neuirokine-alpha and/or Neutrokine-alhaSV can be O used in the treatment of infectious agents. For example, by increasing the immnune response, particularly increasing the proliferation and differentiation of B cells.

infectious diseases may be treated- The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.

is Alternatively Neutrokine-alpha and/or Neutrokinc-alphaSV polynucleolides or polypeptides, or agontists of Neutrokine-alpta and/or Neutrokine-alphaSV may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptomns that can be treated by Neutrolcine-alpha and/or Neuzrokine-alphaSV 2o polynucleocades or polypeprides, or agonuis of Ncurrokine-alpha and/or NeutrokinealphaSV Examples of viruses, include, but are not limited to the following DNA and RNA viruses and vial) families: Arbovrs, Adenovindac, Arenavindac, Artenvurus, Bimaviridie. Buiiyavandae, Calicividae, Circovindae, Coronavinidae. Dengue, EBV HIV Flavavindae. Hepadnavindie (Hepatitis), Herpesvandae (such as, 2s Cytoinegalovwrus, Herpes Simplex. Herpes Zoster), Mononegavirus Paramyxoviridac. Morbiliyirus, Rhabdoviridae), Orlhcnnyzovindae Influenza A, Influenza B, and prainfluenza), Papiloma virus, Papovaviridac. Purvovyindae.

Picornavindae, Poxvaridae (such as Smallpox or Vaccinia), Reovirrdae RuiAVAst4,ij RcuvcinA-uiau C(ftL Y-A. "ItL.Y-1i, Lcuuivarus;, idata TIUsavillsc 3o Rubivwus). Viruss falling within these families can cause a variety of diseases or symnptoms, including, but not limited to: arthritis, bronchiollitts. respiratory svncvuial virus, encephalitis, eye inkcuions conjunctaviiis, k'eratmts). chronic fatigue syndrome, hepatitis B. C, F_ Chronic Active, Delta), Japanese B encephalitis. .Junin.

COMS ID No: ARCS-183623 Received by IP Australia: Time (Him) 16:56 Date 2008-03-19 19/03/200e 19/03~08 1:31 61 2 925136999 4 062837999hC41 NO. 641 IP52 00 oChikungunya, Rift Valley fever, yellow fever, memungmts, oppontunistic infections AIDS). pneumnonia, Burkin's Lymphorna, chickenpox, hemorrhagic fever. Measles, Mumps, Parazntluenz, Rabics, the common cold, Polio, leukcnrua, Rubella, sexually trasnuucd diseases, skin diseases Kaposi's, warts), and viremit. Neuvrokrnealpha ad/or Neuzrokmne-alphaSV polynuicleouides or polypepuides. or agomisrs or antagonists of Neutrokine-alpha and/or Ncuzrokine-aiphaSV can be used to treat, prevent, diagnoearid/or detect any of these symptoms or diseases. In specific embodiments, Neutrokiuc alpha polynuiclcooides, polypeptzdcs, or agonists ame used to o treat, prevent, and/or diagnose: meningitis, Dengue, EBV and/or hepatitis Cl 10 hepaitis In an additional specific embodiment Neurrokine alpha polynuclezcscs.s 00 o polypepudes, or agonasts are used to treat patiernts nlonresponsive to one or more other Cl commercially available hepatitis vaccines. In.a further specific embodiment, Neutrokmne alpha polynucleouides, polypeptides. or agonisw are used to treat, prevent, anid/or diagnose AIDS. In an additional specific embodiment Neinrokxnc-alpaa and/or is Neutrokine-alphaSV and/or Neurrokine-aipha Receptor polynucleondes, polypeptides, agornsts, and/or antagonists are used to treat, prevent. and/or diagnose patients wit cryptosporidiosis.

Similarly bacterial or fungal agents that can cause disease or symptoms and that can be treated by Neutrokine-alpha and/or Neuvrokine-alphaSV polynucleotides or polypeptides. or agomisgs or antagonists of Neutrokane-alpha and/or NeutrokmnealphaSv include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacteria) families and fungi: Actinomycetales Corymebactcnium, Mycobactrnum, Norcardia), Cryptococcus neoformaus, Aspergillosis, Racillacese Anthrax, Closticliurn), Bacteroidacese, Blastomycosis, flordewella, Borrelia Borrelia burgdorfen, Brucellosis, Candidasms, Campylobacter, Coccsdioldomycozs.

Cryptococcosas, Dermatacycoses, E. coli Enterotoxigemuc E. ccli and Enternhemonthagic E. cobi), Ernerobacteraaceae (Klebsiella, Salmonella (e.g- Salmonella typhr, and Salmonella paratyphi), Serrauia, Yersinia), Erysipelothnix.

Helicobacter, Leelonel losis. I .eninsprois, Lester:: (e.g.-4m L&ttcn noncyj-ogc~esP.

Mycoplasmiawles, Mycobactrinum leprae, Vibno cholerac, Neissenaceae Aemnezobacter, Gonorrhea, Menigococcal), Meissenia memingmtdis, Pasieurellacen Infections Aciunobacillus, Heamophilus Heamoptilus influenza type B3), Pasteurella), Pseudoinonas, Rwckettsiaceae, Chiamydiwace, Syphilis, Shigella spp., COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92596999 4 062837999 NO. 641 P53 00 Staphylococcal, Memngiococcal, Pneumococcal and Streptococcal Strepococcus pneumoniac and Group B Streptococcus). These bactenal or fungal families can cause the following diseases or symptoms, including, but not limited to: baeterema, endocardius, eye infections (conjunctivits; tuberculosis, uvems). ginglvltis, S opportumstc minfections AIDS related infections), paronychia, prosthesis-related znfections, Renter's Disease, respiratory tract infections, such as Whooping Cough or Empyema. sepsis, Lyme Disease, Cat-Scratch Disease. Dysentery Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis mengins types A and Chlamydia, Syphilis, Diphthenria, Leprosy Paratuberculosis, Tuberculosis, 0o Lupus, Botulism, gangrene, tetanus, impetigo, Rheumateic Fever, Scarlet Fever, 0 sexually transmitted diseases, skin diseases cellulitis, dermatocycoses) toxemia urinary tract inmfecuons, wound infections. Neutroke-alpha and/or NeutrokinealphaSV polynucleoudes or polypeptides, or agonists or antagomists of Neutrokine alpha and/or Neutrokne-alphaSV can be used to ncat, prevent. diagnose. and/or detect is any of these symptomns or diseases. In specific embodiments. Neutrokino alpha polynucleondes, polypeputides, or agomsts thereof are used to treat, prevent, and/or diagnose: tetanus, Dipthbena, botulism, and/or mcningitis type B.

Moreover, parasitic agents causinmg disease or symptoms that can be treated by Neutrokinc-alpha and/or Neutrokine-alphaSV polynucicotides or polypeptides, or agonsts of Neutrokine-alpha and/or Neutrokine-alphaSV include, but not limited to, the following families or class: Amebtasts. Babesiosis, Coccidiosis, Cryptospondiosts, Dientamoebrasts, Dournne, Ectoparasiuc, Giardiasis, Helrunthasis Leishmaniasis, Theilenasis, Toxoplasmosis. Trypanosomlasis, and Tnrichomonas and Sporozoans Plasmodium virax, Plasmodium falcapanum, Plasmodium malanac and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms. including, but not limuted to: Scabics, Trombiculiasjs, eye minfections, Intestinal disease dysentery glardissis), liver disease, lung disease, opportursutic minfections AIDS related), malaria, pregnancy complications, and toxoplasmosis. Neutrokine-alpha and/or Neutrokine-alohaV nnlmnucetUdes or polypeptdcs, or agornsts or untaguisLs or Neutrokne-apha and/or Neutrokine-alphaSV can be used to rreat, prevent, diagnose, and/or detect any of these symptoms or diseases. In specific embodiments Neutrokine alpha polynucleoudes, polypeptudes, or agonims thereof are used to treat, prevent.

and/or diagnose malaria.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.G41 P54 00 o In another embodiment, Neutrolone-alpha and/or Neutrokine-alphaSV polynucleotudes or polypeptedes of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose inner ear minfection (such as, for example, otius media), as well as other minfections characterized by infection with Streprococcus pnemwonaac and other pathogenice organisms.

In a specific embodiment, Neutrolkne-alpa and/or Neutroklne-alphaSV polynucleoudes or polypeptides, or agonists or antagolss thereof anti- Neutrokane-alpha. and/or anu-Neutrokzne-alphaSV antibodies) are used to treat or prevent a disorder charactrized by deficenr serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, Neutrokine-alpba.

Sand/or Neutrobne-alphaSVpolynucleotides or polypeptdes, or agonists or antagonsts Sthereof anti-Neutrolon-alpha, and/or ant-Neutrokine-alphaSV antibodies) may be used to treat or prevent mfecnons of the joints, bones, skin, and/or parotid glands.

blood-bomrne infections sepsis, meningitis, septic arthrais, and/or osteomyelitis), is autommuwne diseases those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) mcluding, but not lirmted to, CVID, other pnrimary imunmune deficiencies, HIV disease, CLL, recurrent bronchitis.

sinusis, otitis media, conjunctivutis, pneumonia, hepatitis, meningitis, herpes zoster severe herpes zoster), and/or pheumocystis carnii.

Neutrokine-alpha and/or Neutrokme-alphaSV polynucleotides or polypeptides of the invenuon, or agonists or antagonists thereof, may be used to diagnose. prognose.

treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: pnrimary Immuodeficienctes, immune-mcdiated thrombocytopenma, Kawasaki syndrome, bone marrow transplant recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection adult or pediatric HIV minfection), chronic inflammatory demyelinaung polyneuropathy and post-transfusion purua.

.ddiunaluy Ncutrokine-alpha andior Ntuiroki e-mpmS'v puiynueieuues or 3o polypeptides of the invention, or agonists or antagonists thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders.

or conditions associated therewith. CGuillain-Barre syndrome, anemia anemnia associated with parvovirus B 19 patients with stable mutliple myeloma who are at high COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 00 oC risk for minfection recurrent Infection), autoimmune hemolytic anema warm-type autoammune hemnolytic anemia). thrombocytopenia neonatal thrombocytopenisa). and immune-mediaed neutropenza), transplantation (e.g, cytamegalovirus (CMV)-negative recipients of CMV-positive organs).

hypoganunagobalinmna hypoammaglobulinemc neonates with nsk factor for infection or morbidity), epilepsy intractable epilepsy), systemic vasculitiuc syndromes, myasthemua gravs decompensaion in myasthenta gravis), dermatomyositss, and polymyosits.

Additional preferred embodiments of the invention include, but are not limited in to, the use of Neutrokinc-alpha and/or Neutrokme-alpha SV polypptldes, Neutrokrne 0 alpha and/or Neutroksne-alpha SV polynucicotides, and functional agonists thereof. in the following applications; Adminstration to an animal mouse, rat, rabbit, hamster, guinea pig. pigs, micro-pig. clucken, camel, goat, horse, cow sheep, dog, cat, non-human primate, and I> human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies IgG, IgA, IgM, and IgE), to. induce higher affinitly antibody production IgG, IgA, IgM, and IgE), and/or to increase an immune response. In aspecific nonexclusive embodiment, Neutrokme-alpha polypeptides of the minvention, and/or agonists thereof, are administered to boost the immune system to produce increased quantities of IgG. In another specific nonexclusiva embodiment. Neutrokine-alpha polypepudes of the invention and/or agomsts thereof, are administered to boost the immune system to produce increased quantines of IgA. In another specific nonexclusive embodiment, Neutrokzne-alpha Q polypeptides of the invention and/or agonists thereof. are administered to boost the immune system to produce increased quantities of IgM.

Adminmistration to an imal (including, but not limited to, those listed above, and also including transgenzc anmunals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capabi of producing humnt.. un aurnuutuuin uiuimnulcs uy nmes bo al reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893. W1O/9634096. WO/9633735 and W019110741).

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/o3/2ooe IG:31 61 2 92586999 4 062837999 NO. 641 956 00 o A vaccine adjuvant that enhances unmune responsiveness to specific antigen.

In a specific embodiment, the vaccine adjuvant s a Neutrolune-alpha and/or t Neutrokine-alphaSV polypeptide described herein. In another specific embodiment, the vaccine adjuvant is a Neutrokme-alpha and/or Neutrolkn-alphaSV polynucleotide SS described heren the Neutrolkne-alpha and/or Neutrokme-alphaSV polynucleotde is a genetic vaccine adjuvant). As discussed hereim, Neutrokine-alpha and/or Neutrokine-alphaSV polynucleotides may be administered using techniques known in the art, ncluding but not limited to, liposomal delivery recombinant vector delivery injection of naked DNA, and gene gun delivery An adjuvant to enhance tumor-specific immune responses.

o An adjuvant to enhance anti-viral immune responses. Ann-viral immune 0 responses that may be enhanced using the composiions of the invention as an adjuvant, include, but ait not limited to. virus and vius associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, menmngs,. Dengue, EBV and hepatiis hepatitis In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a vrus, disease, or symptom selected from the group consisting of: HV/AIDS, Respiratory syncytal virus, Dengue, Rotavirus. Japanese B encephalitis, Influenza A and B, Paramfluonza. Measles, Cytomegalovirus, Rabies, Juntn, Chikungunya. Rift Valley fever. Herpes simplex, and yellow fever In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bactenal or anti-fungal immune responses. Antbacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the an.

In sntcfic embodients, the compos:tons of the invenion aie ucu as a adjuvant to enhance an immune response to a bactena or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningtis type B. In another specific embodiment, the compositions of the invention ar used as an adjuvant to enhance an Immune response to a bacteria or fungus, disease, or symptom selected COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 062837999 NO. 641 P57 00 o from the group consisting of: Vibno cholere, Mycobacienum leprae, Salmonella typh, Salmonella pararyphi, Messenra menmgnmdis, Streptococcus pncumonmae, Group B streptococcus, Shigella spp., Enteroroxigeic Echenchita coli. Enterohemorrhagic

E.

coi, Borrelia burgdorfer, and Plasmodium (malana).

An adjuvant to enhance anti-parasitinc Immune responses. Anu-parasltc immune responses that may be enhanced using the compositions of the mnventon as an adjuvant, mnclude parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the ilventtion are used as an adjuvant to enhance an ummune response to a parasite. In another N 10 specific embodiment, the compositions ofthe minvention are used as an adjuvant to 00 o enhance an immune response to Plasmodium (malana).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual pnor to their receipt of Immunosuppressive therapies.

is As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrautons.

As an agent to accelerate recovery of immiunocompromised individuals.

As an agent to boost nmmunoresponsiveness among aged populations.

As an unmune system enhancer pnor to, during, or aftrr bone marrow transplant and/or other transplants allogeneic or xenogeneic organ transplantation). With respect to transplantation. compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are adnunistered after transplantation, prior c. to the bginning of recovery of T-cell populaions. In another specific embodiment, compositions of the invention are first admunstered after transplantaton after the beginning of recovery of T cell populatons, but pnor to full recovery of B cell populations.

As an agent to boost zmmunoresponsiveness among B cell immunodeficment individuals, such as. for examle. an individnal who has undergone Parnal or.

complete splenectomy B cell immuriodeficicncies that may be ameliorated or treated by administenring the Neurrokmne-alpha and/or Neutrokinc-alphaSV polypeptides or polynucleotides of the invention, or agonists thereof, minclude, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal. adenosine COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 19/032008 16:31 61 2 92596999 4 062837999 N.4 NO. 641 950 00 Odeammuase deficiency (ADA deficiency), K-linked agammtraglobulinemnia

(LA),

Bruton s disease, congenital Aganmaglobulinewna, K-linked infantile agamimglobulinejpja, acqwired agammaglobulinemia, adult onset aganunaglobulincnualate-onset agammgiobulinemia, dysgammaglobulineia, s hypogamnlaglobulincuuia, transient hypogammaglobulngmga of infancy unspecified hypogarmmagibunma aga ninaglobulinema,, common variable imnmunodeficiency (CVII)) (acquired), Wiskont-Alinch Syndrome (WAS), K-linked immunodeficiency with hyper ISM, non X-llnked immnunodeficiency with hyper 1gM, selective JgA o deficiency igO subclass deficiency (with or without IgA deficiency), antibody Cl 1 deficiency with normal or elevated Igs, immunodeficiency with thymoma, IS heavy O0 chain delezions, kappa chain deficiency B cell lymphoproliferative disorder

(BLPD).

0 selective I gM immurnodeficiency recessive agamrnagiobulinemita (Swiss typie).

Cl-reticular dysgenlesis, neonatal neutropenia, severe congenital leukopenia, thyic alymphoplasia-aplasia or dysplasia with immuinodeficiency ataxia-celangaectasia, short is limbed dwarfism, X-linked Iymphoproiiterauve syndrome (XLP), Nezelof syndrome combined immunodeficiency with Igs, panne nucleoside phosphorylase deficiency (PNP). MI-C Class U deficiency (Rare Lymphocyte Syndrome) and severe combined immnunodeficiency As an agent to boost immunorcsponsivcness among individuals having an acquired loss of 13 cell function. Conditions resulting in an acquired loss of B cell function chat may be ameliorated or created by adnuristrnng the Weutrolcjne-alpha and/or Neutrokine-alph4Sv polypeptides or polynucleotides of the invenon, or gofliSs thereat, include, but are not limited to, HI'. Infection, AIDS, bone marrow trarisplant, and B cell chronic lymphocytec leukema (CII). (1 As an agent to boost immunorcsposmens among individuals having a temporary immune deficiency Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by adnunistenog the Neutrokine-alpha and/or Neutrokine-alphasv polypeptides or polynucleotjdes of the mnvention, or agonists thereoft include, but are not lizmtea to. recovery irom varnaa jiaceljons IFg.-iflucaza).

conditions associated with malnutriton, recovery from infectious miononucleosis, or conditions associated with stress, reccwcrv from measles, recovery from blood transfusion, recovery frm surgery.

COMS ID No: ARCS-i 83623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92596999 4 062B3?999 N0.641 D59 00 oD 2f SAs a regulator of antigen presentauon by monocytes, dendnce cells, and/or B-cells. In one embodiment, Neutrokme-alpha and/or Neutrokne-alphaSV C polypeptides (in soluble, membrane-bound or transmembrane forms) or polynucleotides enhance antigen presentation or antagonize antigen presentation an s vtro or in vivo. Moreover, in related embodiments, this enhancement or antagonizaton of antigen presentation may be useful an ant-tumor treatment or to Smodulate the immune system.

As a mediator of mucosal Immune responses. The expression of Neutrokmealpha by monocytes and the responsiveness of B cells to this factor suggests that it may 00 to be involved in exchange of signals between B cells and monocytes or their differentiated progeny This activity is in many ways analogous to the CD40-CD154 N signaling between B cells and T cells. Neutrokne-alpha may therefore be an important regulator of T cell independent immune responses to environmental pathogens. In particular, the unconventional B cell populations (CD5+) that are associated with mucosal sites and responsible for much of the innate immunity i humans may respond to Neutrokine-alpha thereby enhancing an individual's protective immune status.

As an agent to direct an individual's immune system towards development of a humoral response (ie. TH2) as opposed to a THI cellular response.

As a means to induce tumor proliferation and thus make it more susceptible to auu-neoplasuc agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all ant-neoplastc regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.

As a B cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplasuc B cell populations.

As a means of detecting B-lineage cells by virtue of its specificity This application may require labeling the protein with biotin or other agents as described herein) to afford a means of detection.

A a umulator of B cell producr, n patholgies such as %iDS, chrorme lymphocyte disorder and/or Common Variable Immunodificiency As part of a B ccll selection device the function of which is to isolate B cells from a heterogenous mixture of cell types. Neutrokine-alpha could be coupled to a solid support to which B cells would then specifically bind. Unbound cells would be COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208 16:31 61 2 92596999 4 062837999 NO. 641 960 00 0 washed out and the bound cells subsequently elured. A nonlimuting use of this selection would be to allow purgmg of tumor cells from, for example, bone marrow or penrpheral blood prior to transplant.

As a therapy for generation and/or regeneration of lymphod tissues following surgery trauma or gentic defect.

As a gene-based therapy for genetically inherited disorders resulting in Immuno-nmcompetence such as observed among SCID patients.

As an antigen for the generation of antibodices to inhibit or enhance Neutroknealpha mediated responses.

00 10 As a means of activating monocytes/macrophages to defend against parasitic 0 diseases that effect monocytes such as Leshmama.

Ci As pretreatment of bone marrow samples pror to transplant. Such treatment would increase B cell representation and thus accelerate recover As a means of regularng secreted cytoknes that are elicited by Neurrolkecalpha.

Neutrolkne-alpha or Neutroukme-alphaSV polypeptides or polynucleondes of the invention, or agomists may be used to modulate IgE concentrations in vitro or min VIvo.

Additionally Neutroluneo-alpha and/or Neutroknc-alphaSV polypeptides or polynucleotides of the Invention, or agomsts thereof, may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhmtnrs, and eczema.

In a specific embodiment, Neutrokine-alpha and/or Ncutrolkine-alphaSV polypeptides or polynuleotides of the invention, or agonusts thereof, is admministered to treat, prevent, diagnose, and/or ameliorate selective IgA deficiency In another specific embodiment, Neutrokine-alpha andlor Neutrokmne-alphaSV polypepides or polynucleondes of the invention, or agonirsts thereof, as administered to treat, prevent, diagnose, andlor ameliorate ataxa-telangiectasaa.

In Anther specific cmbodimzen, Neutmre!-i-phna tor rtrk&cin.- phas' polypeptides or polynucleotrdes of the invention, or agonsts thereof, Is administered to treat, prevent, diagnose, and/or ameliorate common variable Immunodeficiency COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 925936999 4 062937999 NO.6CA1 P61 0 polypepnie rlnceond eofteinvention, or agonwsts thereof, is adirusrered to Ct ftet, prevent, diagnose. and/ar ameliorate X-linked agamrnaglobulinemi& In another specific embodime~nt, Neutrokrne-alpha and/or Neutroiune-aipiusv s polypepudes or polynucleotirjes of the in vention. or agorists thereof, is administered to treat, prevent, diagnose, and/or ameliorahe severe combined immunodeficiency

(SCID).

In another specific embodiment. Neutrokmne-aipha and/or Neuhrokrne-alphasv polpepide orpolynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate Wiskout-Aldnch syndrome.

In another specific emobodinment. Newmrkane-alpbia and/or Neutrokmne-alphaSV 00 ~polypeptides or Polynucieotides of the invention, or agonists thereof, is administered to O treat, prevent. diagnose, and/oir ameliomcce X-linked Ig deficiency with hyper 1gM.

In anothier specific embodimnt. Neutrokane-alpha and/or Neuirokxne-alphasv polypepoides or polynuicleotades of the invention, or agoiszs or antagonists antiis Netrokinc-alpha antibodies) thereof, is administered to treat, prevent, and/or diagnose chronic myclogenous leukemia, acute myclogenous leukemia, leukemia. bysniocyuic leukemia, momocytne leukemia acute monocyuic leukemia), leukemnic reuiculosas, Shilling Type monocytac leukemia, and/or other leukerma2s denived from mnonocytes and/or monocyhic cells and/or tissues.

In another specific embodiment. Neurrokmce-alpha and/or Neutrokmne-alphasv polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate monocyuic leukemnoid reaction. as seen, for examnple, with tuberculosis.

In another specific embodiment, Neinrokrne-alplia and/or Neutrokine-alphasv polypepdes or poiynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate monocyzic leukocytosis, monocyho leukopenia, monocytopenia, and/or monocyiosis.

in a specific ernbodiment. Neutroakune-aapha, and Neuzrokine-aWphaSV polynucleouides or nolynenuilpsr, nf the invention, and/or anu-4eutrobine"alpha antibodies and/or agorusts or antagonists thereof, are used to treat, prevent, detect.

and/or diagnose primary B lymphocyte disorders and/or diseases, and/or conditions associated therewith. In one embodiment, such primary B lyniphocyie disorders, diseases, and/or conditions are charactenzedi by 4 complete or partial loss- of humoral COMS ID No: ARCS-183623 Received by IP Australia: Time (I-Em) 16:56 Date 2008-03-19 19/03/2009 19/032008 16:31 61 2 92586999 4 062837999 N.4 6 NO. 641 [PG2 00 o -i I 0 imamunity Primary B lymphocyte disorders, diseases, and/or conditions associated therewith that are characterized by a complete or partial loss of bumoraliimmuiy and that may be pre vented, treated, detected and/or diagnosed with compositions of the invenltion include, but are noe limited to, X-Lxnlced Agarmaglobuinernia

(XLA).

S severe combinted immunodeficiency disease (SCID), and selective IgA deficiency In a preferred embodiment, Neuzrokzne-alpha and Neuurokine-alpliasv __polynudeoudods, polypepaides, and/or agomasts and/or anlagoinszs thereof are used to treat, prevent, and/or diagnose diseases or disordeirs affecting or conditions associated o with any one or more of the vanous mucous. membranes of the body Such diseases or Cl o disorders include, but are not limited to. for example, mucosins, mulcoclasis, 00 inucocolitis, mucocumaneous leisbmanaasis (such as. for example. American Cl leashmaniasis, Icishmaniasis amtricana, nasopharyngeal leashmaniasis, and New World leislunaniasis), mucocutanicous lymph node syndrome (for example. Kawasaki disease), mucoenteritis, mucoepidermoid cacinomra, mucoepidennoid tumor, is mucoepithelial dysplasia, mucoid adenacarcanoma, mucoid degeneration, mnyxoad degeneration; myxomaxous degeneration; mnyromaxosas, mucoad medial. degeneration (for example, cystic medial necrosis). mucolipidosis (including, for example, mucolipidosis 1, mucolipidosis U. inucolipidosis 11I, and mucolipidosas TV), mucolysms disorders, mucomenbranous enenuis, mucoentenuis, mucopolysacchazdosis (such as, 2o1 for example, type I mucopolysacchandosis Hurlers syndrome), type IS mucopolysaccndosus Scheic's syndrome or type V mucopol ysaccharidosi s), type 11 mueopolysacchandosas Hunters syndrome), type III mucopolysacchnridosis Sanfilippo's syndrome), type IV mucopolysacchndws (iLe., Morqwro's syndrome), type Vi mucopoiysacchanidoszs Maroieaux-Laxny syndrome), type VII mucopolysaccharadosis mucopoiysaccharndosws due to betaglucuronid=s def icienicy), and mucosulamdosws), mucopolysaceharaduna, inucopumulent cofljunctivlus maucopus, miucormycosis zygomycosis), mucosal disease bovine virus diarrhea), mucous colitis (such as, for example, mucocolitis In yx d nn ic koz suC' as, fOr eXrApile, cysti fibrosis, cystic fibrosis of the pancreas, Clarke.Hadfield syndrome, fibrocystic disease of the pancreaS, mucoviscidosis, and viscidosis). In at highly preferred embodiment, Neutrokie-.lpha, and/or Neutrokine-alphaSV polynuicleotides, polypeptades, and/or COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 U63 00 co agonsts and/or antagonists thereof are used to treat, prevent, and/or diagnose mucositis especi ally as associated with chemotherapy In a preferred embodiment, Neutrokine-alpha, and/or Neutrokine-alphaSV polynucleioudes, polypeptides, and/or agoatusts and/or antagomsts thereof are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with sinusitis.

An additional condition, disease or symptom that can be treated, prevented.

and/or diagnosed by Neutrolune-alpha and/or Neutlrokine-alphaSV polynucleotides or polypeptdes or agomists of Neutrokne-alpha and/or Neutrolune-alphaSV is 00 Ia osteomyelius.

An additional condition, disease or syrptom that can be treated, prevented, and/or diagnosed by Neutrolune-alpha andfor Neutrokne-alphaSV polynuccotdes or polypepudes, or agonists of Neutrokine-alpha and/or Neutrokine-alphaSV is endocarditis.

All of the above described applications as they may apply to vetennary medicmine.

Antagomsts of Neutrokine-alpha include binding and/or inhibitory antibodies, anutsense nucleeic acids, ribozymes, and Neutrokine-alpha polypepudes of the invention.

These would be expected to reverse many of the acavitiecs of the ligand described above as well as find clinical or practical application as: A means of blocking vanous aspects of immune responses to foreign agents or self. Examples include autoimmune disorders such as lupus, and arthritis, as well as Immunoresponsiveness to skin allergies, inflanmation, bowel disease, injury and C pathogens. Although our current data speaks directly to the potential role of Neutrokine-alpha in B cell and monocyte related pathologies, it remains possible that other cell types may gain expression or responsveness to Neutrolmne-alpha. Thus, Neutrokine-alpha may like CD40 and its ligand, be regulated by the starus of the immune system and the rmroenvironment in which the cell is located.

A rherapy for prevenung the B cell proliferatoc and g seCretiou neousb ateu with autoimmune diseases such as Idiopathic thrombocytopequ purpura. systemic lupus erythematosus and MS.

An minhibitor of graft versus host disease or transplant rejection.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 IN.641 64 00 o A therapy for B cell malignancies such as ALL. Hodgkins disease, non.

Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple c myeloma, Burkit's lymphoma, and EBV-transformed diseases.

A therapy for chronc hypcrgammaglobulinemela evident m such diseases as Is monoclona l gammopathy of undetermined sgnificance (MGUS), Waldenstrom s disease, related idiopathic monoclonalgammopathies, and plasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cell LympoDmas.

A means of decreasing the mvolvement of B cells and Ig associated with o Chronic Myelogenous Leukenua.

An Immunosuppressive agent(s).

Neutrokie-alpha or Neutrokine-alphaSV polypeptdes or polynucleoides of M |the invention, or antagonists may be used to modulate IgE concentrations in vitro or m vivo.

In another embodiment, administraton of Neutrokine-alpha or Neutrokne alphaSV polypeptides or polynucleoudes of the invention, or antagonists thereof, may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions including, but hot limited to, asthma. rhiitis, and eczema.

An inhibitor of signaling pathways involving ERKI, COX2 and Cyclin D2 which have been associated with Neutrokine-alpha induced B cell activation.

The above-rccited applications have uses in a wide variety of hosts. Such hosts include, but are not lirmted to, human, murne, rabbit, goat, guinea pig. camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow sheep, dog, cat, non-human Sprimate. and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig. sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with a pharmaceutically acceptable carer, as described herein.

The antagonists may be employed for instance to inhibit Neutrokine-alphaincdiated and/or Newioknc-alphaS -mcdiaicu lelnuituaxs ai autvatuln uj macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, activated and CD8 cvtooxe T cells and natural killer cells, in certain auto-immune and chronic nflammatory and infective diseases. Examples of auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes. The COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO.641 00 o0 2? 0 antagonists may also be employed to treat, prevent, and/or diagnose infectious diseases including silicosis, sarcodosIs, idiopathic pulmonary fibrosis by preventing the Srecruitment and activation of mononuclear phagocytes. They may also be employed to treat, prevent, and/or diagnose idiopathic hyper-osinophilic syndrome by preventing coasmophil production and migration. Endotoxic shock may also be treated by the antagonists by preventing the migrauon of macrophages and their production of the SNeutrokme-alpha and/or Neutrokme-alphaSV polypeptdes of the present inventon.

The antagonists may also be employed for treating atherosclerosis, by preventing Smonocyte infiltralon n the artery wall. The antagonists may also be employed to treat.

00 10 prevent, and/or diagnose hlstammne-mediated allergic reactions and Immunological 0 disorders including late phase allergic reactions, chronime urticarna, and atopic dermatitis (1 by inhibiting chemokne-induced mast cell and basophil degranulation and release of histamine. IgE-mediated allergic reactions such as allergic asthma, rhmnits, and eczema may also be treated. The antagonists may also be employed to treat, prevent, and/or diagnose chronic and acute inflammation by preventing the attracton of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung. Antagonists may also be employed to treat, prevent and/or diagnose rheumatoid atnhnts by preventmg the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role m the pathogenesis of both degenerative and nflammatory arthropathzes. The antagonists may be employed to interfere with the deleterous cascades attributed primarily to IL 1 and TNF which prevents the biosynthesis of other inflammatory cytokmes. In this way the antagonists may be employed to prevent inflammation. The antagonists may also be employed to inhibit prostaglandin-mdependent fever induced by Neutrokine-alpha and/or Neutrolune-alphaSV The antagonists may also be employed to treat, prevent, and/or diagnose cases of bone marrow failure, for example, aplasuc anemia and myeloayspasuic synorome. The antagusts may au be ciuspoyeu tu treat, prevent, and/or diagnose asthma and allergy by preventing cosinophil accumulation in the lung.

The antagonists may also be employed to treat. prevent, and/or diagnose subepithelial basement membrane fibrosis which is a prominent feature of the asthmauc lung. The COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/208 16:31 61 2 925986999 4 062837999 NO.641 I66 00 antagonists may also be employed to treat, prevent, andlor diagnose lymphomas one or more of the extensive, but not limiting, list of lymphomas provided herein).

SAll of the above described applications as they may apply to veternary medicine. Moreover, all applications described herem may also apply to veennary S medicine.

Neutrokmne-alpba and/or Neutrokme-alphaSV polynucleotides or polypepudes of the invention and/or agorusts and/or antagomsts thereof, mnay be used to treat, prvent, and/or diagnose various Immune system-related disorders and/or conditions associated wath these disorders, in mammals, preferably humans. Many autoirmune ci 10 disorders result from inappropnrate recognition of self as foreign maternal by immune cells. This inapproprate recognition results in an immune response leading to the destruction of the host tssue. Therefore, the admnistration of Neutroktne-alpha and/or Neutrokine-alphaSV polynucleondes or polypeptides of the invention andor agonists and/or antagonists thereof that can inhibit an immune response, particularly the i proliferation of B cells and/or the production of umunoglobulins, may be an effective therapy in treatming and/or preventing auteummune disorders. Thus. in preferred embodiments, Neutroklune-alpha and/or NeutrokanealphaSV antagonists of the minvention polypeptde fragments of Neutrokine-alpha and/or Ncutroklnc-alphaSV and ant-Neutrokmine-alpha antibodies) are used to treat, prevent, and/or diagnose an autoammune disorder.

Autoimmune disorders and conditions associated with these disorders chat may be treated, prevented, and/or diagnosed with the Neutrokane-alpha polynucleoudes, polypeptides, and/or antagonimst of the invention anti-Neutrokine-alpha antibodies), include, but are not limited to, autoimmune hemolytic anemia, autoinmmune 2s neonatal thrombocytopenica, idiopathic thrombocytopenia purpura, autoanunmmunocytopenia, hemolytic anemia, anltiphospholipid syndrome, dermatits, allergic encephalomyelits, myocardius. relapsing polychondritis, rheumatic heart discase, glomerulonephntas IgA nephropathy), Multiple Scleross, Ncuntais, Uvens. Onhthalmia, PolvQtidocnnopathr=,. Durpua Henloch-Scocnei purpura;, Reter's Disease, Stiff-Man Syndrome, Auroimmune Pulmonary Inflammanon, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:31 61 2 92586999 062837999 NO. 641 D67 00 Additional autoimmune disorders (that are highly probable) that may be treated, prevented, and/or diagnosed with the compositins of the ivention include, but are not Slimnted to, amuounmune thyroiditis, hypothyroadism Hashimoto's thyroiditis) (often charactenrzed, by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterzed, by circulating and locally generated immune complexes), Goodpassure's syndrome (often characterized, by antibasement membrane antibodies), Pemphigus (often characterized, by epidermal acantholytic antibodies), Receptor autoimmunmues such as, for example, Graves' Disease (often characterized by TSH receptor antibodies), Myasthenia Gravis o0 (often characterzed, by acetylcholine receptor antibodies), and insulin O resistance (often characterized, by insulin receptor antibodies), autoimmune SC hemolyuc anema (often characterized, by phagocytosis of antibody-sensitized RBCs). autoimmune thrombocytopenic purpura (often characterized, by phagocytosis of antibody-sensutized platelets.

Is Additional autounmune disorders (that are probable) that may be treated, prevented, and/or diagnosed wah the compositons of the invention include, but are not limited to, rheumatoid arthnns (often characterzed, by immune complexes in joints), schleroderma with anti-collagen antibodies (often characterized, by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, by antibodies to extractable nuclear anugens ribonucleoprotein)), polymyosits/dermatomyosts (often characterized, by nonhistone ANA), pernicious anemia (often characterized. by antpaneal cell, mucrosomes, and ntrinsic factor antibodies), idiopathic Addison's disease (often r characterized, by humoral and cell-mediated adrenal cytotoxicny infertility (often characterzed, by anttspermatozoal antibodies), glomerulonephnis (often characterized, by glomerular basement membrane antibodies or immune conmplxes) such as primary glomerulonephntis and IgA nephropathy bullous pemphigold (often characterized, by IgG and complement in basement memhrane. Singren vndrnme inften characterized. by mul.ripl ts.ue antibodies, and/or a specific nonhistone ANA diabetes millitus (often characterized. by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cysuc fibrosis) (ofien charamenzed, by bela-adrcnergic receptor antibodies).

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2988 i i i?

I

r it; i' i> i- 16:31 61 2 92586999 4 062837999 NO. 641 D68 Additional autormmnune disorders (that are possible) that may be trated, prevented, and/or diagnosed with the compostuons of the ivention include, but are not limuted to, chronic active hepatitis (often characterized, by smooth muscle antibodies), primary biliary irrhosis (often charactezed, by mitchondnal s antibodies), other endocrine gland failure (often characterized, by specific tissue antibodies in some cases), vitiligo (often charactrnzed, by melanocyte antibodies), vasculits (often charactenzed, by Ig and complement in vessel walls and/or low scrum complement), pos-MI (often characterized, by myocardial antibodies), cardioomy syndrome (often characterized, e.g. by myocardial antibodies), urticana (often characterized, by IgG and IgM antibodies to IgE). atopic dermattis (often characterzed, by IgG and IgM antibodies to IgE), asthma (often characenzed, by IgG and IgM antibodies to IgE), inflammatory myopathles, and many other inflammatory granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autmmmune diseases and disorders and/or 5i conditions associated with the diseases and disorders recited above are treated, prevented, and/or diagnosed using anti-Neutrokme-alpha antibodies and/or anti- Neutrokne-alphaSV In a specific preferred embodiment, rheumatoid arthrns is treated, prevented, and/or diagnosed using anu-Neutrokme-alpha antibodies and/or anti-Neutroklne alphaSV antibodies and/or other antagonist of the invenuon.

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

In a specific preferred embodiment. nephritis associated with lupus is treated, prevented, and/or diagnosed using anu-Neutrome-alpha antibodies and/or anti- Neutrokne-alphaSV antibodies and/or other antagonist of the invention.

In a specific embodiment, Neutrolune-alpha and/or Neutrokme-alphaSV polynuclcotides or polypeptides. or antagonist thereof anti-Neutrokme-alpha and/or anti-Neutrokne-alphaSV antibodies) are ni.lr tno nt or ~preent ~S--,Cupus erythematosus and/or diseases, disorders or conditions associated therewith. Lupus.

associated diseases, disorders, or conditions that may be treated or prevented with Neutrokine-alpha and/or Neutrokne-alphaSV polynucleotdes or polypeptdes, or antagonists of the mvention, include, but are not limited to. hematologic disorders COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:31 61 2 92586999 4 062837999 NO. 641 969 00 0 hemolyte anerma, Iukopenia, lymphopena, and thrombacytopenia). Immunologic disorders anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity oral ulcers, arthnus, fever, fatigue, weight loss, serosnis pleuntus (pleuncy)), renal disorders nepbntis). neurological disorders seizures, penpheral s neuropathy CNS related disorders), gastronsstsinal disorders, Raynaud phenomenon, and pencarditis. In a preferred embodiment, the Neutmkine-alpha and/or NeutrokinealphaSV polynucleotides or polypepudes, or antagonists thereof anti-Neutrokine alpha and/or anu-Neutrokne-alphaSV antibodies) are used to treat or prevent renal disorders associated with systemc lupus erythematosus In a most preferred 00 Io embodiment, Neurroktne-alpha and/or Neutrolune-alphaSV polynucleotides or 0 polypepudes, or antagonists thereof ann-Neutrokine-alpha and/or anti- I Neutrokine-alphaSV antibodies) are used to treat or prevent nephnius associated with systemic lupus erythemarosus..

Similarly allergic reactions and conditions, such as asthma (particularly allergic 11 asthma) or other respratory problems, may also be treated by NeutrokmLne-alpha and/or Neutrnmune-alphaSV polynucleoudes or polypeptdes of the invention and/or agonists and/or antagonists thereof. Moreover, these molecules can be used to treat, prevent, and/or diagnose anaphylaxis, hypersensiuvity to an anugenic molecule, or blood group incompatibility Neutrokaine-alpha and/or Neutrokine-alphaSV polynuelcoudes or polypepuides of the invention and/or agoniasts and/or antagonists thereof, may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly an immune response as also minvolved min GVHD but, in this case, the foreign transplanted immune cells destroy the host tissues. The adminstration of Neutroknc-alpha and/or Neutrokne-alphaSV polynucleades or polypeptides of the invention and/or agomsts and/or antagonists thereof, that inhibits an immune response, particularly the proliferaton, diffmrenuagn, or chemotaxas of "-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly Neutrokine-alpha and/or Neutroklne-alphaSV polynucleotides orat polypepudes of the invention and/or agonists and/or antagonists thereof, may also be used to modulate inflammation. For example, Neutrokine-alpha and/or Neutrokine- COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:31 61 2 92586999 4 062837999 N0.641 070 00 o 2% c alphaSV polynucleoudes or polypeptdes of the nvention and/or agonists and/or antagonists thereof. may ihibit the proliferation and differentuaon of cells involved I C an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory condions, both chronic and acute conditons, mcluding chronic prostatils. granulomatous prostatus and malacoplakia, inflammation associated with infecton sepuc shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischenua-reperfusion injury endotoxm lethality arthritis, complement-mediated hyperacute rejecuon. nephrtis, cytokme or chemokine induced O lung injury mflammatory bowel disease. Crohn's disease, or resulting from over 00 to production of cytokines TNF or IL 1.) In a specific embodiment, anti-Neutroktne-alpha antibodies and/or antcN Neutrokme-alphaSV antibodies of the invention are used to treat, prevent, modulate, detect, and/or diagnose inflammaton.

In a specific embodiment, anti-Neutrokme-alpha antibodies and/or anti- Ncutrokine-alphaSV antibodies of the invention are used to treat, prevent, modulate, detect, and/or diagnose mflamatory disorders.

In another specific embodiment, anti-Ncutrokme-alpha antibodies and/or anti- Neutrokine-alphaSV antibodies of the invention are used to treat, prevent, modulate.

detect, and/or diagnose allergy and/or hypersensitivty Antibodies against Neutrokine-alpha and/or Neutrokine-alphaSV may be employed to bind to and inhibit Neutrokine-alpha and/or Neutrokine-alphaSV activity to treat, prevent, and/or diagnose ARDS, by preventing infiltration of neutrophils inio the lung after injury The agonists and antagonists of the instant may be employed in a composition with a pharmaceutcally acceptable carrier. as described hereinafter. Neutrolkne-alpha and/or Neurokme-alphaSV and/or Neutrokine-alpha receptor polynucleotpdes or polypepttdes of the nvenuon and/or agomsts and/or antagonists thereof. are used to treat, prevent, and/or diagnose diseases and disorders of the pulmonary system bronch such as. for example, smopulmonary and bronchial infecuuus anu coudiulL. assucia&tu with such discac.s auu di.srders anu other respiratory diseases and disorders. In specific embodiments, such diseases and disorders include, but are not limited to. bronchial adenoma, bronchial asthma.

pneumonia (such as, bronchial pneumonia, bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps.

COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2009 19/3/209 16:31 G1 2 92596999 4 062937999 NO. 641 9?1 0 0 bronchiecasma (such as 1 bronchaectasia sicca, cylindrical bronchiciasis. and saccular bronchzeciaszs), bronchioar adonocarcinomna, bronchiolar carcinoma, bronchioliuis (such as, exudative broncluoliuts. bronchiolitis fibrosa obliterans, and proliferative bronchioliuis), bronchiole-alveolar carcinoma. bronchitic astluna, s bronchitis (such as, asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, croupous bronchitis, fibnnous bronchitis, hemnorrhagic bronchitis, infectious avian bronchitis, obliterative bronchitis, plastic bronchitis, pseudlomembranois bronchitis. putid bronchitis, and verrmous bronchitis), bronchoceninc ogrdnulomnatosas. bronchoedema, bronchoesophageal fistula, bronchagemecacnoa 00io bronchogenic cyst, bronchol~libiaszs, bronchomalacma, bronchomycosis (such as. e.g., 0 obmonchopuilmonary asporgillosis), broncliopilmay wprohetosus, hemorrhagic C] brnchits. broncbcrrhea. bronchospasm, bronchostaxis, bronchoscenosas. Biafs respiration, bronchial respiration, Kussmnaul respiration, Kussmaul-Kien respiration.

respiratory acidosis, respiratory alkalosis, respiratory distress syndrome or the ts newborn, respiratory insufficiency respiratory sclerotna, respiratory syncytial virus, and the like.

lIn a specific embodiment, Ncuwrobne-alpha. and/or Neuirokine-alphaSV polynucleouides or polypeptides of the invention and/or agonists and/or antagonists thereof, are =sd to treat, prevent, anid/or diagnose chronic obstructve pulmonary disease (COPD).

In another embodiment, Neutrokine-aipha and/or Ncutroktne-alpbaSV polynnicleocides or polypeptides of the invention and/or agonists and/or antagonists thereof. are used to treat, prevent, and/or diagnose fibroses and conditions associated with fibroses, such as, for example, but not limited to. cystic fibrosis (including such fibroses as cystic fibrosis of the pancreas, Clarke-Midfield syndrome. fibrocystic disease of the pancreas, mucoviscidos is, and vicidosas), endcomyocrd ia! fibrosis.

idiopathic retroperironeal fibrosis, lepomeningeal fibrosis, mediastinal fibrosis, nodular subepiderrnal fibrosis, pencentral fibrosis, peinmuscular fibrosis, pipestern fiumr,'pnauxinii (ibrUSIS, SUOHICa11Uvenu ribruszs, wiu Syinsu1cab ay PpcStCm fibrosis.

The TNP family ligands are known to be among the most pleiotropic cyzokines, inducing a large number of cel lular responses, including cytoroxicaty anti-viral acUtity anwZnur~lIOry ACtLivticS, and te rranscnpuionai regulation of several COMS ID No: ARCS-183623 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 19/3/208 16:39 BLAKE DAWSON WALDRON LAWJYERS 4 0629D37999 NO.iso rDoi Level 38, Grosvenor Place 225 George Street Sydney NSW 2000 Australia FAX TRANSMISSION No of Pages including this sheet). 1 Blake Dawson PATENT ATTORNEYS

TO

The Commissioner of Patents IP Australia F 02 6283 7999 Human Genome Sciences, Inc New Australian divisional patent application Title: Neutroklne-alpha and neutrokine-aiphs splice variant PART 5 OF 6 T 612 925886000 F 61 292588999 OK 255 Sydney Locked sag NO 6 Grosvenor Place Sydney NSWV 2000 Australia WWW.012kkilawson.com 19 March 2D308 Our ref aenasc 060 8d 02 1430 4482 Penner David Clark T 61 2*0298 8829 dlavictark O~btskedawson.oom Please check that you have received this document in full. If niot. please telephone t Senaeror call 61 2 2258 6000.

Coefldentlallty Thie document is confidential and may contain legafly privileged information. If you ore not a namred or aulthorised recipient you rmut not read, copy, dis~tribute or act in reliance on It. It you have received this document in error, Please telephone our operator immed~iely on rpi 2 9258 6000 and return the document by mail.

Mebourne Brigbane Perth Canberra 2030S73711 COMS ID No: ARCS-i 83628 Received by IP Australia: Time (1-Pm) 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 0G6237999 N. 198 P02 00 c genes (D V Goeddel re al, "Tumor Necrosis Factors: Gene Strucure and Biological ^C Actvities, Symp. Quant. BioL 51:597- 609 (1986). Cold Spnng Harbor. B. Beutler and G A. Ceramt, Annu. Rev. Biochem. 57:505-518 (1988); LJ. Old, Sea. Am. 258:59-75 S(1988); W Fiers, FEBS Lett. 285-199-224 (1991)). The TNF-family ligands. including Neutrokine-alpha and/or Neutrokine-alphaSV of the present invention, induce such various cellular responses by binding to TNF-family receptors. Neutroklnc-alpha and/or Neutrokane-alphaSV polypeptdes are believed to elicit a potent cellular response including any genotypic, phenotypic, and/or morphologic change to the cell, o cell line, tissue, tissue culture or patient As indicated, such cellular responses include 00 Io not only normal physiological responses to TNF-family ligands, but also diseases o associated with increased apoptosis or the inhibition of apoptosis. Apoptosisprogrammed cell death-is a physiological mechanism involved in the deletion of penpheral B and/or T lymphocytes of the immune system, and its disregulation can lead to a number of different pathogenic processes Armesen, AIDS 8:1197 1213 (1994); P.H. Krammer ec Curr Open. Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition of apoptosis that may be diagnosed, treated, or prevented with the Neutrokme-alpha and/or Neutrokine-alphaSV polynucleoudes or polypepudes of the invention, and agomsts and antagonists thereof, include cancers (such as follicular lymphomas, carcinomas with p 5 3 mutations, and hormone-dependent tumors, including, but not limited to. colon cancer, cardiac tumors, pancreatic cancer, melanoma, retnoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastora, myxoma, myoma. lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chandrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus crythemalosus and immune-related glomeulonephntis rheumatold arthntUs); viral infections (such as herpes viruses, pox vruses and adenoviruses); mflammation; graft vs. host disease; acute graft rejection and chronic graft rejection. Thus, in preferred emooiiments iveurorinc-aipna anmor rieutroKine-aipnaSv poiynuc uu or polypeptides of the invention abd/or agonists or antagonists thereof, are used to treat, prevent, and/or diagnose autoimmune diseases and/or inhibit the growth, progression.

and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukermas (including, for example, MLL and COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2009 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO. 198 P03 00 0231 c chronic lymphocync lcukerma (CLL)) and follicular lymphomas. In another emnibodiment Netrokne-alpha and/or Neutroke-alphaSV polynucleoides or polypepudes of the invention are used to acivac, differentiate or proliferate cancerous cells or tissue B cell lineage related cancers CLL and MLL), lymphocyuc leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy chemotherapy or radiation therapy).

Moreover, in other embodiments, Netrokne-alpha and/or Neutrokne-alphaSV polynuclcoudes or polypeptides of the invention or agonists or antagonsts thereof, are Sused to inhibit the growth, progression. and/or metastases of malignancies and related 00 10 disorders such as leukemia (including acute leukenias acute lymphocytac O Icukema, acute myelocyne leukemia (including myeloblasuc, promyelocytic.

myelomonocyuc, monocytic, and erythroleukomia)) and chronic leukemias chronic myelocytc (granulocytic) leukemia and chronic lymphocync leukemia)), polycythemia vera, lymphomas Hodgkin's disease and non-Hodgkn's diseasc).

is muluple rityeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, Jiposarcoma, chondrosarcomna. osteogenic sarcoma, chordoma, angosarcorna, endotheliosarcoma, lymphangiosarcoma, lymphangioendtheliosarcoma, synovioma, mesothelioma Ewming's tumor, eIiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovaran cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcmioma, papillary carcinoma, papillary adenocarcminomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma. hpazoma, bile duct carcinoma, chonocarcinoma, semmoma. embryonal carcinoma, Wilm's tumor, cervical cancer, testcular tumor, lung carcinoma, small cell lung carcznoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytorna. inedulloblastoma. cramopharyngioma, ependymoma, pinealoma, hemangoblastoia, acoustic neuroma. oligodendroglioma, .eicnangioaua, melanoma. reaurobiasrumn, afnt reunobinsaumu.

Diseases associated with increased apoposis apoptosis that may be diagnosed, treated, or prevented with the Neutrokine-alpha and/or Neutrokirne-alphaSV polynucleotides or polypeputdes of the minvention., and agonists and antagonists thereoflnclude AIDS, neurodegeneranve disorders (such as Alzhelmer's disease.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 004 00 8 2%? cl Parkinson s disease, Amyotrophic lateral scleross, Retmnits pigmentosa, Cerebellar t degeneration); myelodysplastic syndromes (such as aplasue anenna). ischeme m njury (such as that caused by myocardial infarction, stroke and reperfusion njry), toxin- Ominduced liver disease (such as that caused by alcohol), septic shock, cachexia and s anorexia. Thus, In preferred embodiments Neutrokne-alpha and/or Neutrokine-alphaSV polynucleotides or polypepudes of the invention and/or agonmsts or antagomsts thereof, are used to treat, prevent, and/or diagnose the diseases and disorders listed above.

o In preferred embodiments, Neutrokine-alpha and/or Neutrokne-alphaSV 00 to polypeptides of the inrvention and/or agonasts or antagonists thereof antio Neatrokine-alpha antibodies) inhibit the growth of human histiocytic lymphoma U-937 cells in a dose-dependent manner. In additional preferred embodiments.

Neutrokmne-alpha and/or Neutrokine-alphaSV polypeptides of the invenution and/or agonrsts or antagomsts thereof ani-Neutrokine-alpha antibodies) inhibit the growth of PC 3 cells, HT 29 cells. HeLa cells, MCF-7 cells, and A293 cells. In highly preferred embodiments, Neutrokme-alpha and/or Neutrokine-alphaSV polynucicoudes or polypeputdes of the invention and/or agonasts or antagonists thereof ann- Neutrokine-alpha antibodies) are used to inhibit growth, progression, and/or metastasis of prostate cancer, colon cancer, cervical carcinoma, and breast carcinoma.

Thus, in additional preferred embodiments, the present invenution is directed to a method for enhancing apoptosts induced by a TNF-family ligand. which involves admnzstenng to a cell which expresses a Neutroiine-alpha and/or Neutrolkine-alphaSV receptor an effective amount of Neutrokine-alpha and/or Neutrokne-alphaSV or an agomst or antagonist thereof, capable of increasing or decreasing Neutrokine-alpha and/or Neutrokne-alphaSV mediated signaling. Preferably Neutrokine-alpha andlor Neutrokine-alphaSV mediated signaling is increased or decreased to treat, prevent, and/or diagnose a disease wherein decreased apoptosis or decreased cytokine and adhesion molecule expression as exhibited. An agonist or antagonist can include sotutne rorms or veutrogLne-aipna anioor Neutrorane-alpnaSv ancia monocional 3o antibodies directed against the Neurokme-alpha and/or Neutrokine-alphaSV polvpeptide.

In a further aspect, the present invention is directed to a method for inhibiting apoptosis induced by a TNP-family ligand, which minvolves administering to a cell COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 00 C' which expresses the Neutrokmne-alpha and/or Neutrokne-alphaSV receptor an effective c amount of an agomst or antagonist capable of increasing or decreasing SNeutroknc-alpha and/or Neutrokkne-alphaSV mediated signaling. Preferably SNcutrokinc-alpha and/or Neutroktc-alphaSV mediated signaling is increased or s decreased to treat, prevent, and/or diagnose a disease wherein increased apoptosis or NF-kappaB expression is exhibited. An agonist or antagonist can include soluble forms of Neutrokine-alpha and/or Neutrokne-alphaSV and monoclonal antibodies directed against the Neutrokine-alpha and/or Neutrokane-alphaSV polypeptude.

C- Because Neurokme-alpha and/or Neutrokmc-alphaSV belong to the TNF 0 0 i1 superfamily. the polypeptides should also modulate angiogenesis. In addition, since Neutrokine-alpha and/or Neutrokie-alphaSV ihibit immune cell functions, the Q polypeptides will have a wide range of anu-inflammatory activities. Neutrokne-alpha and/or Neutrokine-alphaSV may be employed as an ant-neovasculanzing agent to treat, prevent, and/or diagnose solid tumors by stimulating the invasion and activation ts of host defense cells, cytotoxic T cells and macrophages and by inhibitmg the angiogenesis of tumors. Those of skill in the art will recognize other non-cancer indications where blood vessel proliferation is not wanted. They may also be employed to enhance host defenses against resistant chronic and acute infections, for example, myobactenal infections via the auracton and activanon of rmcrobicidal leukocytes.

Neutrolne-alpha and/or Neutrokne-alphaSV may also be employed to inhibit T-cell proliferation by the mhibition of IL 2 biosynthesis for the treatment of T-cell mediated auto-immune diseases and lymphocytic leukemias (including, for example, chronic lymphocyic leukemia Neutrokne-alpha and/or Neutrokine-alphaSV may also Q be employed to stimulate wound healing, both via the recruitment of debris clearing and connectve tissue promoting inflammatory cells. In this same manner, Neutrokne-alpha and/or Neutrokne-alphaSV may also be employed to treat, prevent, and/or diagnose other fibrouic disorders, including liver cirrhosis, osteoarthnus and pulmonary fibrosms. Neutrokine-alpha and/or Neurokmne-alphaSV also increases the presecS"e of eass.apils that have the disAinc::v fcuor of killing the rarvae o parasites that invade tissues, as in schistosomiasis, tnchinoss and ascanasis. It may also be employed to regulate hematopoxesis, by regulatng the activation and differentiation of vanous hematopoietic progenitor cells, for example, to release mature leukocyies from the bone marrow following chemotherapy ie., in stem cell COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 Q06 00 C mobilization. Neutrolune-alpha and/or NeutroknmealphaSV may also be employed to C treat, prevent, and/or diagnose sepsis.

Polynucleotides and/or polypeptides of the invention and/or agomsts and/or antagonists thereof are useful m the diagnosis and treatment or prevenuon of a wide s range of diseases and/or conditions. Such diseases and conditons include, but are not limited to, cancer immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorecial cancer, 0C non-small cell carcinoma of the lung. small cell carcinoma of the lung, stomach cancer, 0 0 1o etc.). lymphoproliferative disorders lymphadenopathy). nmcrobial viral.

O bacterial, etc.) infection HIV I infection, HIV 2 infection, herpesvirus ifection (including, but not limited to. HSV I HSV 2, CMV VZV HHV-6. HHV 7 EBV), adenovrus infection, poxvirus infection, human papilloma virus infection, hepatitis infection HAV HBV HCV etc.), Helicobacter pylon infection, invasive is Staphylococcia, etc.), parasitic infection, nephrtis, bone disease osteoporosis), atherosclerosis, pamn, cardiovascular disorders neovascularzation, hypovasculanzatton or reduced Circulation ischemic disease myocardial nfarction, stroke, AIDS, allergy inflammation, neurodegenerative disease Alzhcnmer s disease, Parkinson's disease, amyotrophic lateral scleross, pigmentary retinitis, cerebellar degeneration, etc.), graft rejection (acute and chronic), graft vs. host disease, diseases due to osteomyelodysplasia aplasuc anemia, etc.), joint tissue destruction in rheumatism, liver disease acute and chronic hepatits, liver injury and cirrhosis), autoimmune disease multiple sclerosis, rheumatoid arthnts.

systemic lupus erythematosus, immune complex glomerlonephnus, autoimmune diabetes, autoimmune thrombocytopenme purpura. Grave s disease, Hashimoto s thyrolditis, etc.), cardiomyopathy dilated cardiomyopathy), diabetes, diabetic complications diabetic nephropathy diabetic neuropathy diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephnrs. septic shock, and ulcerative colitis.

Pnlvnnem.lnldes andler polypeptides of the minWtia andoon ador agriiis aiu u.

3o antagonists thereof are useful in promoting angtogenesis. wound healing wounds, bums, and bone fractures). Polynucleotides and/or polypeptdes of the invention and/or agonists and/or antagonsts thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen, anti-viral immune responses.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2098 16:39 BLAKE DAWSON WALDRON LAWYERS 4 0652837999 NO. 198 P07 00 More generally polynucleotdes and/or polypepuades of the minvention and/or agonists and/or antagonists thereof are useful in regulating elevating or reducing) immune response. For example, polynuclectides and/or polypeptides of the invention may be useful min preparation or recovery from surgery trauma, radiation therapy chemotherapy and transplantation, or may be used to boost immune response andlor recovery in the elderly and smmunocompronused individuals. Alternatively polynucleoutdes and/or polypeptides of the mvenuon and/or agomsts and/or antagonists thereof are useful as immunosuppressive agents, for example in the treantment or C prevention of autoimmune disorders. in specific embodiments, polynucleotides and/or 00 o ito polypepudes of the invention are used to treat or prevent chronic inflammatory allergic 0 or autoimmune conditions, such as those described herein or are otherwise known in C the a.

Preferably treatment using Neutrokmne-alpha and/or NeutrokmealphaSV polynucleaotides or polypeptides. and/or agonists or antagonists of Neutroklne-alpha is and/or Neutrokne-alphaSV anut-Neutrokine-alpha antibody). could either be by administering an effective amount of Neutrokmc-alpha andlor Neutrokne-alphnSV polypeptide of the invention ,or agonmst or antagonist thereof, to the pautient, or by removing cells from the patient, supplyming the cells with Neutrokmine-alpha and/or Neutrokme-alphaSV polynucleotide,. and returng the engineered cells to the patient (ex vivo therapy). Moreover, as further discussed herein, the Neurrokine-alpha and/or Neutrokine-alphaSV polypeptide or polynucleotde can be used as an adjuvant in a vaccine to raise an immune response agamnst infectious disease.

Formulations andAdmunutration The Neutrokinme-alpha and/or Neutrokne-alphaSV polypepude composition (preferably containing a polypeptide which'is a soluble form of the Neutrorine-alpha and/or Neutrokine-alphaSV extracellular domamns) will be formulated and dosed in a fashion consistent with good medical practice, takming into account the clinical condition of the individual patient t especally the stde effect of ucancrA wi-th Neutrokii-ipaz and/or Netutrokinme-alphaSV polypeptide alone), the site of delivery of the Neutrokine-alpha andlor Neutrokl ne-alphaSV polypeptide composition, the method of administraton, the scheduling of adnunistranon, and other factors known to COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/208 16:39 BLAKE DAWSON WAILDRON LAWYERS 4 062837999 No. 198 Do 00 0 o 293 SS3 practitioners. The effective amount" of Neutrokme-alpha and/or Neutrokmne-alphaSV polypeptide for purposes heren as thus deteruned by such considerations.

As a general proposton, the total phamaneutically effective amount of Neutrokine-alpha andlor Neutrokne-alphaSV polypepude admministered parenterally s per dose will be w the range of about I mrcrogramn/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion.

More preferably this dose is at least 0.01 mg/kg/day and most preferably for humans between about 0.01 and 1 mg/kg/day Ci In another embodiment, the Neutrokine-alpha and/or Neutrokrne-alphaSV 00 to polypepude of the invention is administered to a human at a dose betweeen 0.0001 and o 0.045 mg/kg/day preferably at a dose between 0.0045 and CLD45 mg/kg/day and more preferably at a dose of about 45 microgram/kg/day in humans; and at a dose of about 3 mg/kg/day in ruce.

If given continuously the Neutrokine-alpha and/or Neutrokme-alphaSV is polypeptide is typically adminimstered at a dose rate of about I rmerogran/kg/bour to about 50 micrograms/kghour. either by 1-4 mjecuous per day or by continuous subcutaneous minfusions, for example. using a muni-pump. An intravenous bag solution may also be employed.

The length of treatment needed to observe changes and the interval followmg treatment for responses to occur appears to vary depending on the desired effect.

In a specific embodiment, the total pharmacutcally effective amount of Neutrokmne-alpha and/or Neutrokinme-alphaSV polypepude administered parenterally per dose will be in the range of about 0.1 microgramlkgday to 45 inerograms/kg/day of patient body weight, although. as noted above, thi will be sublect to therapeutic discretion. More preferably this dose is at least 0.1 microgram/kg/day and most preferably for humans between about 0.01 and 50 micrograms/kg/day for the protein.

Neutrolune-alpha and/or Neurrokne-alphaSV may be administered as a continuous ifusion, multiple dicreet injections per day three or more times daily or twice aiiy). sinagle injectuion per cay or as discreet ijCcuuus ivwn iAtfiuiClmidly icg., twice daily once daily every other day twce weekly weekly biweekly monthly bimonthly and quartcrvl). If given continuously the Neutrokmne-alpha and/or Netroline-alphaSV polypeptide is typically admnistered at a dose rate of about 0.001 COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 N0.198 D09 00 C to 10 mncrogram/kg/ hour to about 50 micrograms/kg/hour. either by 1-4 injections per c day or by continuous subcutaneous infusions, for example, using a mnum-pump.

Effective dosages of the compositions of the present invention o be Sadministered may be determined through procedures well known to those m the art which address such parameters as biological half-life, bioavailability and toxicity Such determination is well within the capability of those skilled m the art, especially in light of the detailed disclosure provided herein.

Bioexposure of an organism to Neutrokme-alpha and/or Neutrolne-alphaSV 0 polypeptide during therapy may also play an mnportant role in determining a 00 to therapeutically and/or pharmacologically effective dosing regime. Vanatons of dosing O such as repeated adminstrations of a relatively low dose of Neutrokme-alpha and/or Neutrokm -alphaSV polypepude' for a relatively long period of tame may have an effect which is therapeutically and/or pharmacologically distmgushable from that achieved with repeated adrmnistrations of a relatively high dose of Neutrokme-alpha and/or Neutrokne-alphaSV for a relatvely short period of time. See, for instance, the serum immunoglobulin level experiments presented in Example 6.

Using the equivalent surface area dosage conversion factors supplied by Freireich, E. et al. (Cancer Chemotherapy Reports 50(4):219-44 (1966)), one of ordinary skill in the art is able to conveniently convert data obxained from the use of Neutrokme-alpha and/or Neutrokne-alphaSV in a given expenmental system into an accurate estimation of a pharmaceutically effective amount of Neutrokine-alpha and/or Neutrokne-alphaSV polypeptide to be administered per dose n another experimental system. Expenmental data obtained through the administration of Nemrokine-alpha n mace (see, for instance, Example 6) may converted through the conversion factors 2s supplied by Freireich, et aL, to accurate esumates of pharmaceutically effective doses of Neutrokme-alpha in rat, monkey dog, and human. The following conversion table (Table III) is a summary of the data provided by Freireich, et al. Table III gives approximate factors for convening doses expressed in terms of mg/kg from one species to an eouivalent surface area doprssed a.,s M ingg i another specie taibuiar-u.

Table III. Equivalent Surface Area Dosage Conversion Factors.

To- Mouse Rat Monkey Dog Human COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 16:39 BLAKE DFAWSON WALDRON LAWYERS 4 062937999 NO.198 00 (N -FROM-- (20' (150e f3.5kg(8kS~ (6Oka) t Mouse 1 1/2 1/4 116 1/12 Rat 2 1 1/2 1/4 1/7 SMonkey 4 2 1 3/5 1/3 S Dog 6 4 5/3 1 1/2 Human 12 7 3 2 1 SbThus, for example, using the conversion factors provided in Table III, a dose of mg/kg in the mouse converts to an appropriate dose of 12.5 mg/kg in the monkey 00 a1 because (50 mg/kg) x 12.5 mg/kg. As an additional example, doses of 0.02, 0 S0.08, 2. and 8 mg/kg in the mouse equate to effect doses of 1.667 micrograms/kg, C 6.67 micrograrnskg, 66.7 micrograms/kg. 1667 nucrograms/kg, and 0.667 mg/kg, respectively in the human.

Pharmaceutical compostions containing Neutrokne-alpha and/or is Neutrokne-alphaSV polypepudes of the invention may be admimstered orally rectally parenterally subcutaneously intracistemally intravaginally mntrapentoneally topically (as by powders, ointments, drops or transdermal patch), bucally or as an oral or nasal spray via nhalauon of a vapor or powder). In one embodiment, "pharmaceuncally acceptable carrier" means a non-toxic solid, semisolid or liquid filler, diluent, encapsulating materal or formulaton auxiliary of any type. In a specific embodiment. "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed m the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly humans.

Nonlimiting examples of suitable pharmaceutical carriers according to this embodiment are provided m "Remngton s Pharmaceutical Sciences" by E.W Martn, and include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, nuneral oil, sesame oil and the like.

Water is a preferred carner when the pharmaceuucal composltion is administered inraenously Saline solutions and aqeous dextrose and ;gyccrol solutions can be employed as liqmd carriers, parucularly for injectable solutions. The compositon, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustaned-release formulations and the like.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 ig/o3/2ooe 19/032009 16:39 BLAKE DAWJSON WJALDRON LPAYERS 4 062837999 N.9 1 No. ige Pil 00 Cl The term parentcraV' as used herein refers to modes of administration which ct include intravenous. intrunuscular. intraperitoneal, intrasternial, subcutaneous and inltfraaicuW~ injctionl and infusion.

In a preferred embodiment. Neurrokine-aipha and/or Neuirokut-alphaSV s compositions of the invention (including polypepides. polynucleoudes. and antibodies and agomists and/or antagonists thereof) are adinistered subcutaneously In another preferred embodiment Neuwrokme-alpha and/or Neutrokine-alphaSV compositions of the mvenuion (including polypepuides, polynucleonides, and antijbodies, o and agenitss and/or antagonists thereof) are administered intravenously 00 t0 Ncutrokine-alpba andor Neunrobine-alphaSV comnpositions of the invention are o also suitably adrminstered by susiaamed-release systerms. Suitable examples of Clrsutied-r1nas compositions include suitable polymeric materials (such as, for oxample, semn-pern-eable polymer matrices in the form of shiaped articles, filmis.

or Funrocapsules), suitable hydrophobic materials (for example as an emulsion in an is acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as. for example, a sparingly soluble salt).

Sustained-release mnatrices, include polylacodes Pat. No. 3,773,919 EP 58.481), capolymers of L-glucamic acid and gamma-ethyl-L-glutamnate (Sidman, U et Osopolwners 2:547 556 (1933)), poly (2 hydroxyerhyl methacrylate) (Rt. Langecr etaW.,) L homed Mater Res. 15-167-277 (198 and R. Langer, Chemn- Tech.

12:98-105 (1982)). ethylene vinyl acetate (It. Langer etiat., Id.) or poly-Dfl-3-hydroxybucync acid (EP 133.988).

Sustained-release compositions also include liposonally entrapped compositions of the invention (see generally Langer, Science 249- 1527-1533 (1990); Treat el al., in Japosonues in the Therapy of Infectious Disease and Cancer Lopez Berestein and Filler Liss, New York. pp. 317 327 and 35 3-365 (1989)).

Laposoines; containing Neutrokine-aipha and/or Neuirokine-alphaSV palypcptide my be prepared by methods known per se: DE 3,218,121 Epstein el al., Proc. Nazi. Acad.

So. (USA) a2:3688-3692 (19851: Hwan et 21., -oal.Acad. Sci. (Us^) 77h4030-4034 (1980); EP 52,322; EP 36.676; EP 88,046:, EP 143,949, EP 142,641 Japanese Pat. App!. 83-118009; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102.324. Ordinarily the liposoines; are of the small (about 200-800 Angstroms) unilardlar type in which the lipid content us greater than about 30 ruol. percent COMS ID No: ARCS-i 83628 Received by P1 Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 198 112 00 Scholesterol, the selected proportion being adjusted for the optimal Neutrokine-alpha c and/or Neutrokme-alphaSV polypeptade therapy G In another embodiment systamed release compositions of the inventon include O crystal formulations known m the art.

s In yet an additional embodiment, the compositions of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Cnt. Ref. Biomed. Eng.

14:201 (1987); Buchwald et Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med.

321:574 (1989)).

O Other controlled release systems are discussed m the review by Langer (Science 00 10 249-1527 1533 (1990)).

O For parenteral admmistration, m one embodiment, the Neutrokme-alpha and/or Neutrokne-alphaSV polypeptide is formulated generally by mixing it at the desired degree of purity in a unit dosage injectable form (solution, suspension, or emulsion).

with a pharmaceutically acceptable carner, one that Is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides.

Generally the formulations are prepared by contactng the Neutrokme-alpha and/or Neutrokne-alphaSV polypeptde uniformly and intimately with liquid carriers or finely divided solid camers or both. Then. if necessary the product is shaped into the desired formulation. Preferably the carner is a parenieral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carner vehicles include water, saline. Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herem, as well as liposomes. The carier suitably contains minor amounts of additives such as substances that enhance isotonicty and chemical stability Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate.

citrate, succmate, acetc acid, and other organic acids or their salts: antaoxidants such as ascorbic acid; low molecular weight (less than ahonu ten residues polypeptides, e.g..

polyargnmne or tnpeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids.

such as glycme, glutamic acid, aspartic acid, or arginme: monosacchandes, disacchardes, and other carbohydrates including cellulose or its derivatives, glucose.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/20013 19/032800 16:39 BLAKE DAWJSON WAPLDRON LAWJYERS 4 082=3999mi9 P1 NO. 19e P13 00 oCzg 0 Cl manage, sucrose, or dextrins; chelating agents such as EDTA, sugar alcohols such as Ct marnrol or soritol;- counterions such as sodium; preservatives, such as cresol, phenol, cblorobutanol, benzyl alcohol and parabens, and/or nonionic surf actaucs such as polysorbates, poloxamers, or PEG.

The Neutrokine-alpha and/or Neutrolrjnc-alphaSV polypopuide is typically formulated mn such vehicles at a concentration of about 0.001 mg/rn] to 100 mg/mi. or 0.1 mg/uiilto 100rng/ml, preferably 1 10 mg/mi or 1 10 mg/mk at a pHof about 3to in, or 3to 8. more preferably"5-, most preferably 6-7 It will be understood that thr.

0 use of certain of the foregoing excipicowt. camrers, or stabilizers will result in the 00 l~~o formation of Neurroknealpha andWar Neutrokine-alphaSV polypepude sls Neutokie-aphaand/or Neutrokine-alphaSV polypepude to be used O Cl Ctherapeutic admnistration mast be sterile. Sterility is readily accomplished by filuranlon through sterile filtration membranes 0.2 micron membranes).

Therapeutic Neutrobne-alpha and/or Newtrokine-alphaSV polypeptide compositions is generaly are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Neutrolune-alpba and/or Neutrokine-alphaSV polypeptide ordinarily will be stored in unit or multi-dose conainers. for example, sealed ampoules or vials, as an 2o aqueous solution or as a lyophilized formulation for reconstiution. As an example of a lyaphilized formulation, 10-mi vials are filled with 5 mrl of sterilc-lilcrd 1% (w/v) aqueous Neurrokmie-alpha and/or Neutroline-aiphaSV polypeptide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstiutinmg the lyophilized Newrokine-alpha and/or Neutrokmec-alphaSV polypepude using 2s bactenosrauc Waterfor-lnjecuion.

Alternatively Neutrokine-alpha andlor Neuirokine-alphaSVpolypepnide is stored in single dose containers in lyophilized form. The infusion selection isq reconstituted using a sterile canrer for injection.

The invention aMs rVwdes p b~ncuiapck or kit coimpriingil Lie ui more containers filled with one or more of the ingredients of the pharnaccutical compositions of the invention. Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the rnanufacture. use or sale of pharmaceuticals or biological products, which notice reflects, approval by the COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/20088 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 D14 00 0 agency of manufacture, use or sale for human administration. In addition, the c polypeptides of the present ivention may be employed in conjunction with other Stherapeutic compounds.

M The composiions of the invention may be adminstered alone or m combinaton s with other adjuvants. Adjuvants that may he administered with the composiuons of the invention include, but are not limited to, ahun, alum plus deoxycholate (ImmunoAg), MTP-PE (Bocmne Corp.), QS21 (Genentech. Inc.), BCG, and MPL. In a specific embodiment, compositions of the invention are administered in combination wit alum.

C In another specific embodiment, compositions of the invention are administered m 00 to combination with QS-21. Further adjuvants that may be administered with the o compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulaor, AdjuVax lOOa, QS-21 QS-18, CRL1005, Alununum salts, MF-59 and Virosomal adjuvant technology Vaccines that may be administered with the compositions of the mvention include, but are not limited to. vaccines directed toward IS protection against MMR (measles, mumps, rubella), polio, varncella, tetanus/diptheria, hepatitis A, hepatts B, haemophilus mfluenzae B, whooping cough, pneumonia, influenza, Lyme s Disease, rotavirus, cholera, yellow fever, Japanese encephalius, poliomyelits, rabies, typhoid fever, and pertussis, and/or PNEUMOVAX 23T1 Combinatons may be administered either concomiantly as an admixture, separately but simultaneously or concurrently- or sequentially This includes presentations in which the combined agents are administered together as a therapeutic nuxture, and also procedures in which the combined agents are administered separately but simultaneously as through separate intravenous lines into the same mdividual.

Administraton "in combmation further includes the separate administration of one of the compounds or agents given first, followed by the second.

In another specific embodiment, compositions of the invention arc used in combination with PNEUMOVAX 23 m to treat, prevent, and/or diagnose infection and/or any disease, disorder and/or condition associated therewith. In one emoodiment, compositions or me invention are usea in comoinaton win so PNEUMOVAX 23m to treat, prevent, and/or diagnose any Gram positive bactenal infection and/or any disease, disorder and/or condition associated therewith. In another embodiment, composmtions of the ivention are used in combination with PNEUMOVAX 23' to treat, prevent, and/or diagnose infection and/or any disease, COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 915 00 disorder, and/or condition associated with one or more members of the genus Enterococcus and/or the genus Streptococcus. In another embodiment, compostons of Sthe mvention are used in any combination with PNEUMOVAX 23T" to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condiuon associated with s one or more members of the Group B streptococci. In another embodiment, compositons of the invention are used m combinaton with PNEUMOVAX 23" to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated wtth Streptococcus pneumonas.

Cl The compositions of the invention may be admnistered alone or m 00 o 10 combination with other therapeutic agents, including but not limited to, Srchemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anumiflammatones, conventional immunotherapcuuc agents and cytokines. Combmauons may be admimstered either conconutantly as an admixture, separately but simultaneously or concurrently- or sequentially This includes presentatnons in which the combined agents are administered together as a therapeutc mixture, and also procedures in which the combined agents are administered separately but simultaneously as through separate intravenous lines into the same individual.

Administration "in combiation further ncludes the separate adminstration of one of the compounds or agents given first, followed by the second.

In one embodiment, the compositions of the invention are adrmnistered m combination with other members of the TNF family TNF TNF-related or TNF-like molecules that may be administered with the composiions of the mventuon include, but are not limited to, soluble forms of TNF-alpha, lymphotoxm-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotnmer LT-alpha2-beta).

OPGL, FasL. CD27L. CD30L, CD40L. 4-1 BBL, DcR3. OX40L. TNF-gamma (Internatonal Publication No. WO 96/14328), AIM-I (International Publication No.

WO 97/33899). AIM-II (Intermatonal Publication No. WO 97/34911), APRIL (J Exp.

Med. 188(6):1185-1190), endokme-alpha (Intemational Publication No. WO 983/7880). TRo (internaonal Puoiicanon NO. WO 9?/3u694). OPG, ana neucroinealpha (lnternational Publication No. WO 98/18921. OX40, and nerve growth factor (NGFI, and soluhle forms of Fas. CD30, CD27 CD40 and 4-IBB. TR2 (International Publication No. WO 96/34095). DR3 (International Publication No. WO 97/33904).

DR4 (International Publication No. WO 98/32856), TR5 (International Publication No.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 198 916 00 WO) 98/30693). TR6 (International Publication No. WO 98/30694), TR? (Inornanonal Publication No. WO 98141629), TRANK, TR9 (International Publication No. WO 98/56892), TRIO (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12.

s In a preferred embodiment, the compositions of the minvention are administered min combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g.

AVREND

T

biolozgacally active fragments, vanants, or denvauves of CD40L, anti- CD4OL antibodies agonstiuc or antagonisue antibodies), and/or anu-CD40 CI antibodies agonistic or antagonistic antibodies).

%to I certamin embodiments, compositions of the invention are administered in o combination with antiretroviral agents, nucleoside reverse transcrptase inhibitors, nonnucleoside reverse transcriptase minhibitors, and/or protease inhibitors. Nucleoside reverse transcriplase inhibitors that may be administered min combination with the compositions of the invention, include, but are not limited to, RETROVIRT is (zdovudine/AZT), VIDEXm (didanosme/ddl), HIVIDI (zalcitabinelddC), ZERT'" (stavndine/d4T), EPIVIRTS (lamvudine/3TC), and COMBIVIR' m (idovudinellamavudine). Non-nucleoside reverse trauscriptase minhibitors that may be administered min combination with the compositions of the invenution, include but are not limited to, VIRAMUNEm (nevirapine), RESCRIPTORI (delavirdine), and SUSTIVATM (efavirenz). Protease inhibitors that may be adminimstered min combmation with the compositions of the invention, include, but are not limited to, CRIXIVAN" (indinavir), NORVIRTm (ntonavir), INVIRASE'I (saqunavir), and VIRACEPr" (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse rtranscnptase inhibitors, non-nucleosde reverse transcnriptase inhibitors, and/or protease 2s minhibitors may be used in any combination with composinons of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent. and/or diagnose HIV infecuon.

In other embodiments, compositiuons of the minvention may be administered min combination waith ant-opportunistic infection agents. Anti-opportunistic agents that may be adminimstered in combination with the compositions of the invention, include, but are not linrted to, TRIMETHOPRIM-SULFAMETHOXAZOLENm DAPSONEN" PENTAMIDINE'" ATOVAQUONE

T

ISONIAZIDr" RIFAMPIN"h PYRAZINAMIDETP ETHAMBUTOL"' RIFABUTIN'" CLARITHROMYCINM COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/208 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 917 00 O 42.

AZITHROMYCIN'" GANCICLOVIRT FOSCARNETm CIDOFOVIR" FLUCONAZOLE"A ITRACONAZOLE'S KETOCONAZOLE" ACYCLOVIR

FAMCICOLVIR

T

M PYRIMETHAMINEr LEUCOVORINTU NEUPOENM (filgrasum/G-CSF), and LEUKINEI T (sargramosumrn/GM-CSF). In a specific embodiment, composmons of the invention are used in any combination wth TRIMETHOPRIM-SULFAMETHOXAZOLE DAPSONEW PENTAMIDINE

T

and/or ATOVAQUONEm to prophylactically treat, prevent, and/or diagnose an opportunistic Prannocyss carmii pncumomin mfecuon. In another specific embodiment. compositions of the Inveton are used in any combinabon with 00 oIo ISONIAZID" RIFAMPIN

T

PYRAZNAMIDETM and/or ETHAMBUTOLT to 0 prophylacially treat, prevent. and/ordiagnose an opportunistic Mvcobacterum awwn complex ifection. In another specific embodiment, compositions of the invention are used m any combination with RIFABUTIN

T

CLARITHROMYCINVm and/or AZITHROMYCrN"' to prophylactcally teat, prevent, and/or diagnose an is opportunistic Mycobacterum tuberculos infection. In another specific embodiment, composiations of the minvention are used in any combination with GANCICLOVIRT FOSCARNETrU and/or CIDOPOVIRT" to prophylactically treat, prevent, and/or diagnose an opportumstic cytomegalovirus infecon. In another specific embodiment, compositions of the minvention are used mn any combination with FLUCONAZOLE

T

ITRACONAZOLETM and/or KETOCONAZOLEN to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection. In another specific embodiment, compositions of the invention are used m any combination with ACYCLOVIRT'" and/or FAMCICOLVIRT to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment.

compositions of the invention are used in any combination with PYRIMETHAMINEM and/or LEUCOVORIN' to prophylactaecally treat, prevent, and/or diagnose an opportunistic Toxoplasma gondli infection. In another specific embodiment, compositions of the invention are used in any combination with LEUCOVORINTm and/or NEUPOGEN' to prophylactically treat, prevent, and/or diagnose an opportunisteic bactenal inmfection.

In a further embodiment, the comnposltons of the invention are administered n combination with an antiviral agent. Antiviral agents that may be adminimstered with COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 19/032008 16:39 BLAKE DAWSON WJALDRDN LAWYERS 4 062837999 NO. 198 P18 00 Cl the compositions of the invention include, but are not limited to, acyclovir ribavirnn, amantadine, and remantidine.

In a further embodiment, the compositions of the invention ame administered in combination with an antibiouc agent. Antibiotic agents that may be adinistered with s the compositions of the invention include, but are not limited to. amioxicillin, amuoglycosides. beta-lactam (glycopeptide), heta-lactainases, Clindamycin.

chlorAmphenicol, cephalospontus, ciprofloxacrn, ciprofloxacin, erythromycin, fhzoroqwinolones. macrolides, metronidazole, penucillins, qwinolones, nifamnpin.

0 strepitomycm, sulfonamide, tewracyclines timethopinm, znmetbopnrn- 00 to sulfamihoxazole, and vancomycmn.

o Conventional nonspecific immunosuppressive agents, that may be adrmrnstered in combination with the compositions of the invention include, but are not limited to, steroids, cyclosponne. cyclosponne analogs cyclophosphanude, cyclophosphamade IV methylprcdnisolonc, prednisolone. azathioprmne. FK 506, 1l5-deoityspergualin, and is other immunosuppressive agents that act by suppressing the function of responding T' cells.

In specific embodimnis, compositions of the invention are administered in combination with imnuosuppressaws. Lnmunoauppresaanrs preparations that may be admninistered wi ie compositions of the invention include, but ame not limited to, 2o ORTHOCLONEM (OKT3), SANDIMMUNETMJNEORALTM/SANCDYATM (cyclospona), PROGRAPm (cacrolimus). cELLCEPT T h (mycophenolate), Azatluopnne. glucoruicoswerotds, and RAPAMUNEM (sirolimus). In a specific embodiment, unmunosuppressants may be used to prevent rejection of organ or bone marrow transplantation. In a preferred embodiment, the compositons of the invention are administered in combination with steroid therapy Steroids that may be administered in combination with the compositions of the invention, include, but are not. limited to, oral comacoswerwds. prednisone, and mcethylpredzusolone, JV meihylprednisolone). In a %nmrflc embhodimnnt.compomuons of the ifV rltuf Ste .fUfMercu Ii CwnmnaaonUGF with prednisone. In a furthe specific embodiment, the compositions of the invention are administered in combination with prednasone and an nmnunosuppressive agent.

Immunosuppressive agents that may be administercd with the comupostons of the invention and prednisone are those described heren. and include. but are not limited COMS ID No: ARCS-i 83628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2009 19/032008 16:39 BLAKE DAWJSON WJALDRON LAWJYERS 4i 092937999 N.9 l NO. ige IP19 00 75431 Cl to, azathioprna, cylophosphamide, and cyclophosphazmde IV in a another specific Ct embodiment, compositions of the invention are administered in combination with mcthylpredwsolone. In a further specific embodiment, the composions of the invention are administered in combination with nieuhylpredwsolone anid an Simunfosuppmesive agent. Immunoisuppressive agents chat may be administered with the compositions of the invention and methylprednisolone are those described herein.

and include. but are nor limited to, azathioprine, cylophosphamide, and cyclophosphainide TV O In a preferred embodiment, the compositions of the inventiont are administered 00 )a in combination with an anuimalanal. Antunalanals that may be adinistered with the o compositions of the invention include, but are not lima ted to, hydroxychloroqwne, Cl cchioroqine. and/or qumnacnine.

In a preferred embodiment, the compositions of the invention are administered in combination with an NSAU) is In a nonexclusive embodiment, the compositions of the invention are admirusacred in combination with one, two, three. (our, f ive, ten, or more of the following drugs: NRD-1OI (Hoochst Marion Roussel), diclofenac (Dunetbaid), oxapironin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyamia), pemectrexed disodiurn (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), ehenac (Byk Gulden), caripath. AGM- 1470 (Takeda), CDIP 571 (Celitech Chiroscience), (CarboMed), NFL 3000 (Mcrckle). CB-2431 (KS Iliomnedix), CBF-BSZ (KS Bliomedlix), IL IRa gene therapy (Valentis). JTE-522 (Japan Tobacco), paclitaxel (Angiotech). DW 166HC (Doug Wha), darbufelone mesylate (Warner-Lainbedt), soluble TNF receptor I (synergen; Amngen), IPR-6001 (Instiute for Pbarmaceuuical Research), trocade (Hoffmnan-L-a Roche). EF-S (Scotia Pharmnaceuticals), BIlL 284 (Boehninger Ingelhetnfl. BIIF- 149 (Bochrnger Ingeiheimn), LeukoVax (Inflammatics). MK-66 (Merck), ST 1482 (Sigmav-Tau), and butixocort propionlate (Warnerl-ambent).

In a profenfed embodiment the rompositaons of the invent"'ar -ad'""'tered in combination with one, two, thre 7 four, five or more of the following drugs: ruethotrexate, sujfasalazmne. sodiumn aurothionialate, auranoifin, cyclosporine.

penicillamiace, azatbioprine. an antimalaral drug as described herein), COMS ID No: ARCS-i 83628 Received by IP Australia: Time (I-Pm) 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 198 00 02 N cyclophosphamide chlorambucil, gold, ENBREL M (Etanercept), anu-TNF antibody and prednasolone.

In a more preferred embodiment, the composmons of the invention are adnunastered m combination with an antimalaial, methotrexate, ant-TNF antibody s ENBRELTU andlar suflasalazime. In one embodiment, the compositions of the minvention are admianistered an combination with methoutrate. In another embodiment, the compositions of the invention are administered in combinmaon with ant-TNF Santibody In another embodiment, the compositions of the minvention are adnunistered in combination with mthotrexate and anti-TNF antibody In another embodiment, the 00 to compositions of the minvention are adminimstered min combination with suflasalazine. In another specific embodiment, the compositions of the invention are adminimstred in combmination with methotrexate, anti-TNF antibody and suflasalazine. In another embodiment, the compositions of the invention are administered min combination ENBRELM In another embodiment, the compositions of the invention are Is adrmastered in combination with ENBRELm and methotrexate. In another embodiment, the compositions of the minvention are administered in combination with ENBRELY methotrexate and suflasalazme. In another embodiment, the compositions of the invention are administered min combination with ENBREL'U methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one of the above-recited combinations. In a spectic embodiment, the compositions of the invention are admunistered in combination with an antmalarial(e.g., hydroxychloroquinme) ENBRELT" methotrexate and suflasalazine. In another specfic embodiment, the compositions of the minvention are admnanstered in combination with an antinalanal hydroxychloroquie), sulfasalazime, anu-TNF antibody and methotrexate.

In an additional embodiment, compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.

Intravenous immune globulin preparations that may be administered with the compositions of the minvention include, but not limited to, GAMMARTM IVEEGAM"r SANDOGLOBULINM GAMNIMAGARD SIPD" and GAMIMUNE' In a specific embodiment, compositions of the invention are administered min combination with COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 19/032002 16:39 BLAIKE DAWJSON WAJLDRON LAWJYERS 4 0921337999mis P2 NO. 19e U21 00 intravenous immaune globulin preparatioas in transplantation therapy bone ct imarrow transplant).

ligand (Ct)40L), a soluble form of CD40L AVRENDm"), biologically active fragments, variants, or derivatives of CD4OL. anri-CD4L antibodies agonisuc or antagonistic antibodies), and/or anui-CD4Q antibodies agonistic or anhagonistic antibodies).

In a additional embodiment, the compositions of the invention are administered alone or in combination With an anti-inflammiatoiy agent. Anti- 0 inflammatory agents that may be administered with the compositons of the invention o0 10 include, but are not limited to, glucocoruicoids and the nonsterwidal antiinflainmaxories, aminoiarylcarboxylic acid derivatives. arylacetic acid derivatives, arylbutrne acid derivatives, arylcarboxylic acids. aryipropionic acid derivatives, pyrazoles, pyrazolones. salicylic acid derivatives, thiazinecarboxamides, eacetumidocaproic acid. S-adenosylmerhianmne, 3-aminoA4-hydroxyburync acid, IS ainetrinc, bendazac. benzydamnc, bucolorm, difenpiramide, dimazo, ernorfazone, guazazulene, nabumetone, nintuide. orgotein. oxaceprol, pamayline, pensozat, pdfowne, proquazone, proxazole. and tenidap.

In another-embodimnent. compostions of the invention are administered in combmnation with a chcmrotherapeuts agent. Chemotherapeutic agents that may be adnrunistered with the compositions of the invention include, but arc not limited to, antibiotic derivatives doxomubicin, bleomycin, dauinozuibicin. and dacunomycin); amtiestrogens tamnoxifen): anuimetabolites fluorouracil, 5-PU. methotrexate, floxundine, interferon alpha-2b, gluzamic acid. plicamycin, mercaptopunne, and 6inoguanine); cytotoxic agents cannustine, BCNU lomustine. CCHU, cytosine arabmnosidc, cyclophosphanrude. estramushine, hydroxyuirea, procarhazine rmtomnycin.

busulfan, cis-plauin, and vinonisune sulfate): hormones medrozyprflgesterone.

esnmmusune phosphate sodium, ethinyl estradiol, esiradial, megeSITO acetate, methyltestosterone, diethyistilbestrol diphospbate. chloromrinmsene, and tesiolactone): nitrogen -musard dentes (a g rrncpakin, chorambacd, imliielw nuu tnrngcn mustard) and ihiorepa); steroids and combinations bethamrerhasone sodium phosphate); and others dicarbazine, asparaglnase, friotoane, vincnrstine sulfate.

vinbiastine sulfate, and elloposide).

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 P22 00 0 l In a specific embodiment, compositions of the inventon are admmnistered in ccombination with CHOP (cyclophosphamnde, doxorubici, vinnstme, and predmsone) or any combmauon of the components of CHOP In another embodiment, 0\ compositions of the ivention are adnminstered in combination with Rituxunab. In a further embodiment, compositions of the invention are adrmnstered with Rituxmab and CHOP or Rituxmab and any combmation of the components of CHOP In an additional embodiment, the compositions of the invention are administered in combination with cytokines. Cytokines that may be administered with o the compositions of the invention include, but are not limited to, GM-CSF G-CSF 00 to 1.2, IL3, IL4, IL5, IL6, 1L7 IL10, I112, IL13, IL15. ant-CD40, CD40L. IFN-alpha, o IFN-beta, IFN-gamma. TNF-alpha, and TNF-beta. In another embodiment, compositions of the invention may be administered with any interleukin, including, but not limited to, IL lalpha. IL Ibeta, IL 2, IL 3, IL-4, IL 5, IL-6, IL 7 IL-8. IL.9 IL IL 11 IL 12. IL 13 IL 14, IL 15. IL 16, IL 17 IL 18, IL 19 L 20, IL 2, and is IL 22. In preferred embodiments, the compositions of the invention are administered in combmation with ILA and IL10. Both IL4 and IL10 have been observed by the iventors to enhance Neutrokine-alpha mediated B cell proliferation.

In an additional embodiment, the compositions of the invention are administered with a chemokme. In another embodiment, the compostons of the invention are administered with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory proten-4. In a preferred embodiment, the compositions of the invention are administered with chemokine beta-8.

In an additional embodiment, the compositions of the invention are administered m combination with an IL-4 antagonist. IL-4 antagonists that may be administered with the compositons of the invention include, but are not liumed to: soluble IL-4 receptor polypeptdes, multimeric forms of soluble IL-4 receptor polypcptides; ani-IL-4 receptor antibodies that bind the IL-4 receptor without transducing the biological signal elicited by IL4, anti-IL4 antibodies that block binding of IL-4 to one or more IL- receptors, and muteims of IL-4 that bid I4 receptors bu do not transduce the biological signal elicited by IL-4. Preferably the antibodies employed according to this method are monoclonal antibodies (including antibody fragmenLs. such as. for example, those described herein).

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/20089 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 P23 00 In an additional embodiment, the compositions of the minvention are i admnistered in combination with heatopoteuc growth factors. Hematopoweue growth factors that may be adnisterd with the compositons of the Invention minclude, but are not linuted to, LEUKINE"h (SARGRAMOSTIM) and SNEUPOGENm (FILGRASTIM

M

In an additional embodiment, the compositions of the invention are adrnistered min combinbmation with fibroblast growth factors. Fibroblast growth factors that may be adnmnstred with the compositions of the invention include, but are nor limted to. FGF-1 FGF-2, FGF-3, FGF-4, FGF-5, FGF-6. FGF-7 FGF-8. FGF-9 FGF- 00 to 10, FGF-il, FGF-12, FGF-13., FGF-14, and Additionally the compositions of the invention may be administered alone or min combination with other therapeutic regimens, including but not limited to, radiation therapy Such combinatorial therapy may be administered sequentially andlor concomitantly IS Agntss andAntaganss Assays and folecules The Invention also provides a method of screening compounds to identify those which enhance or block the action of Neutrokme-alpha and/or Neutrokine-alphaSV polypeptide on cells, such as its interaction with Neutrokrne-alpha and/or Neuwokine-alphaSV binding molecules such as receptor molecules. An agonist is a compound which increases the natural biological functions of Neutrokmine-alpha and/or Neutrolune-alphaSV or which functions in a manner similar to Neutrokine-alpha and/or Neutrokne-alphaSV while antagonists decrease or eliminate such functions.

In another embodiment, the invention provides a method for identifying a receptor protein or other ligand-binding protein which binds specrifically to a Neutrokme-alpha and/or Neutrokine-alphaSV polypeptide. For example, a cellular compartment, such as a membrane or a preparation thereof. may be prepared from a cell that expresses a molecule that binds Neutrone-alpha and/or Neutrokine-alphaSV The Oreonaration s nrubated -ith labeled Neusrekie-aipha and/or a;ipbaS" and complexes of Neutrokine-alpha and/or Neutrokinc-alphaSV bound to the receptor or other binding protemin are isolated and charactmnzed according to routine methods known in the art. Alternatively the Neutrokmne-alpha and/or Neutrokzne-alphaSV polypeptide may be bound to a solid support so that bminding molecules solubilized from COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D24 00 0 Cl cells are bound to the column and then leuted and charactrinzd according to routine Smethods.

In the assay of the nvention for agoists or antagonists, a cellular compartment, Ssuch as a membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds Neutrokmnealpha and/or Neutrokine-alphaSV such as a molecule of a signaling or regulatory pathway modulated by Neutrolune-alpha and/or Neutrokine-alphaSV The preparation is incubated witb labeled Neutroklne-alpha and/or Neutrokme-alphaSV an the absence or the presence of a candidate molecule o which may be a Neutrolne-alpha and/or Neutroklne-alphaSV agonist or antagonist.

00 10 The ability of the candidate molecule to bind the binding molecule is reflected m o decreased binding of the labeled ligand. Molecules which bind gratuitously i.e., C" without inducmg the effects of Ncutrokne-alpha on binding the Neutrokine-alpha and/or Neutroklnc-alphaSV binding molecule, ae most likely to be good antagonists.

Molecules that bind well and elicit effects that are the same as or closely related to is Neutrolkne-alpha and/or Neutrokine-alphaSV are agonmsts.

Neutrokine-alpha- and/or Neutroknme-alphaSV like effects of potential agonists and antagonists may by measured, for instance, by determning activity of a second messenger system following interaction of the candidate molecule with a cell or appropnate cell preparation, and companng the effect with that of Neutrokine-alpha and/or Neutrolune-alphaSV or molecules that elicit the same effects as Neutrokine-alpha and/or Neutrokine-alphaSV Second messenger systems that may be useful m this regard include but are not limited to AMP guanylate cyclase. ion channel or phosphomositade hydrolysis second messenger systems.

Another example of an assay for Neutrokine-alpha and/or Neutrokne-alphaSV antagonists is a competinve assay that combines Neutroklne-alpha and/or Neutrokme-alphaSV and a potential antagonist with membrane-bound receptor molecules or recombinant Neutrokine-alpha and/or Neutroukne-alphaSV receptor molecules under appropriate conditions for a competitive inhibition assay Neutrokne-aloha and/or Nrutrokne-alphaSV can be labezd, such as by rajioactiv-ty such that the number of Neutrokine-alpha and/or Ncusrokane-alphaSV molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonist COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LlAWYERS 4 062837999 NO. 198 00 0 Potenual antagonists include small organic molecules, pepudes. polypeptades IL 13), and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish Its activity Potential antagonists also may be small organic molecules, a peptide, a polypeptde such as a closely related protein or antibody that bminds the same sites on a binding molecule, such as a receptor molecule, withour inducing Neutrokine-alpha and/or Neutrokne-alphaSV induced acuviues, thereby preventing the acton of Neutrokine-alpha and/or Neutrolne-alphaSV by excluding Neutrolne-alpha and/or Neutrokme-alphbaSV from binding.

0 Other potential antagonists include antisense molecules. Anusense technology 00 to can be used to control gene expression through antisense DNA or RNA or through o tnple-helix formation. Anuscase techniques are discussed, for example, in Okano, J.

Neurochem. 56: 560 (1991): -Oligodcoxynuclaotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton. FL (1988). Anusense technology can be used to control gene expression through anusense DNA or RNA, or through triple-helix formation. Antasense techniques are discussed for example, min Okano, Nenrochem.

56:560 (1991); Oligodeoxynucleotides as Anwnsense Inhibitors of Gene Expression.

CRC Press. Boca Raton, FL (1988). Triple helix formaton is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al.. Science 241 456 (1988); and Dervan et al., Since 251 1360(1991). The methods are based on bminding of a polynucleoude to a complementary DNA or RNA. For example, the coding portion of a polynucleode that encodes the extracellular domain of the polypeptide of the present mventon may be used to design an ansense RNA oligonucleonde of from about 10 to 40 base pars in length. A DNA oligonucleotde is Sdesigned to be complementary to a region of the gene involved in transenption thereby preventing transcription and the production of Neutrokine-alpha andlor Neutrokune-alphaSV The anusense RNA oligonucleoude hybridizes to the mniRNA us vivo and blocks translation of the mRNA molecule into Neutrokine-alpha and/or Neutrokine-alphaSV polypeptde. The oligonuclectides described above can also be delivered to cells such that the .nt:sese RNA or DNA ma-y bc expresscu an vivo Lu Inhibit producuon of Neutrolune-alpha and/or Neutrokne-alphaSV In one embodiment, the Neutrokine-alpha and/or Neutrokine-alphaSV antisense nucleic actd of the invention is produced inracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 926 00 8 3*1 producing an antssense nucleic acid (RNA) of the inventon. Such a vector would Scontain a sequence encoding the Neutrolone-alpha and/or Neurrokine-alphaSV anusense nucleic acid. Such a vector can remain epasomal or become chromosomally integrated, as long as It can be transcribed to produce the desired anusense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding Neutroklne-alpha and/or Neutrokine-alphaSV or fragments thereof, can be by any promoter know n m the N art to act in vertebrate, preferably human cells. Such promoters can be inducible or 00 o 1 consmutve. Such promoters rnclude, but are not limited to, the SV40 early promoter 0 region (Bernois and Chambon, Nature 29:304-310(1 981), the promoter contained in the 3' long termunal repeat of Rous sarcoma virus (Yamamoto et al.. Cell 22:787-797 (1980), the herpes thymrdine promoter (Wagner et al., Proc. Natl. Acad. Sca. U.S.A.

78.1441 1445 (1981), the regulatory sequences of the metallothionein gene (Bnnster, et al.. Nature 296:39-42 (1982)), etc.

The antlsense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a Neutroklne-alpha and/or Neutrokinc-alphaSV gene. However, absolute complementanty although preferred, is not required. A sequence "complementary to at least a portion of an RNA," referred to herein, means a sequence having sufficient complementarity to be able to hybndize with the RNA, forming a stable duplex; in the case of double stranded Neutrokme-alpha and/or Neutrokme-alphaSV anticense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementanty and the length of the antisense nucleic acid Generally the larger the hybrdizrng nucleic acid, the more base mismatches with a Neutrokne-alpha and/or Neutrokmne-alphaSV RNA it may contain and still form a stable duplex (or tnplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melt;ng point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the messago, the untranslated sequence up to and including the AUG iniiaton codon. should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences ofmRNAs have been shown to be effective at inhibiing COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062937999 NO. 198 927 00 0 cA translation of mRNAs as well. See generally Wagner, R, 1994, Nature 372:333-335.

Thus. oligonucleotides complementary to either the 5' or 3' non- translated, noncoding regions of Nentrokme-alpha and Neutroknce-alphaSV shown in Pigures IA-B and 5A-B, respectively could be used in an anusense approach to inhibit translation of endogenous Neutrokrne-alpha and/or Neutrokme-alphaSV mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Anusense oligonucleoudes complementary to mRNA coding regions are less efficient inhibitors of translaton but could be used in o accordance with the invention. Whether designed to hybridize to the 5' 3' or coding 00 1o region of Neutrokmne-alpha and/or Neutrokine-alphaSV mRNA, anusense nucleic acids should be at least six nucleodes min length, and are preferably oligonucleodes ranging "C from 6 to about 50 nucleoudes mn length. In specific aspects the oligonucleotide is at least 10 nuclcoudes, at least 17 nucleotides, at least 25 nucleotides or at least nucleodes.

is The palynucleotades of the invention can be DNA or RNA or chimenc mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety sugar moiety or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleobde may include other appended groups such as peptides for targetng host cell receptors n vivo), or agents facilitaung transport across the cell membrane (see, Letsinger et al., 1989 Proc. Nail. Acad. Set. U.S.A. 86:6553-6556; Lemaitre ct al., Proc. Natl. Acad. Sct. 84:648-652(1987); PCT Publication No. WO88/09810, published December IS, 1988) or the blood-brain barrier (see, PCT Publication No. W089/10134, published April 25. 1988). hybnrdization-tnggred cleavage agents.

(Sce, Krol ci al., BioTechniques 6:958-976 (1988)) or intercalating agents. (See.

Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleoude may be conjugated to another molecule, a peptide, hybnridization enriggered cross-linking agent, transponrt agent. hybridization-triggered cleavage agent. etc.

The antie'e eligonucleoude may comrpns at east one modifid ba= which as selected from the group including, but not limited to, 5-fluorouracil, bromouracil, 5-chlorouracil, -Sodouracil, hypoyanthine. xanune. 4-acetylcytosmae. (carboxyhydroxylmetrhyl) uracil, 5-carboxymehylamnomehyl-2-thaouridine, carboxymethylaminomethyluracil, dihydrouracil, bera-D-galacrosylqucosine. anosine.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 19/032009 1:38 BLAKE DAWJSON WAPLDRON LAWJYERS 4 082937999 .19 P8 No. i9e P28 00 0 '105 N6.isopentenylaenine. I -merbylguanine, I -methylinosine, 2,2-dimerhylguane. 2 Ct rmethyladenine, 2-methylguamne, 3-methylcytosine. 5-methylcytosine. N6-ademine, 7mcthylgnmne. 5-methylamwnomethyluracil, 5-ineihoxymannomethyl-2-tlnouracil, beta-D-mannosylqueosznc, 5-methoxycaztxymerhyluracil, 5-methoxyuracii, 2s rmthylthuo-N6-usopeutenyladen. uracil-5-oxyacetc acid wybuzoxosmne, pseUdotlratiI. qusosmne. 2-thiocyroszne. S-mechyl42-chzounacil. 2-duouracil, 4-thiouracil, uracil-5-oxyaccic acid merhylesrer. uracil-5-oxyaccuc acid netyl-2-ihiourcil. 3-(3-anuno-3-N-2-cmarhxypropyl) untcil, (acp3)w and 2.6- 0 diaminopunne.

0i 0 2 Theorarabiosens oligonucicouide may also comrise a eione modified sugar moiety selected frmtegopincluding, u o iuedt,2aioe 2-loorbnsxylulose, and hexose.( In yet another embodiment, the antasense, oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a is phosphorotioate. a phosphorodiioate. a phosphoranudotluoare, a pbosphoranudate. a phosphordianuclate. a niethylphosphonare. an alkyl phosphornester. and a formace~tal or analog thereof.

In yet another embodiment, the antisense oligonucleoude is an aipha-anomeric oligonucleotide. An aipha-anomene oligcnrncleotidle forms specific double-stranded hybrids with complementary RNA in which, contrar to the usual beta-units, the strands run parallel to each other (Gautier et al., Nod. Acids Res. Ib:6625-664I (1987)). The oligonucleotide is a 2-O-merhyllribonucleozde (Inoue et al., Nuci. Acids lRes. 15:6131-6148 (1987)), or a cliene RNA-DNA analogue (Inoue et al., FEBS Lent. 215:327-330 (1997)).( Polynuicleotides, of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizcr (such as ame conunercialy available from Biosearch, Applied B~iosystmrs, etc.). As examples, phosphorothtoatc. oligonucleou~des may be synthesized by the method of Stein et al.

(Nui, i. ju Rcs i o:3209 ti19SS)), methyipnospnonaiec oiigonuczeouaes can De prepared by use of controlled pore glass polymer supports (San a ct al., Proc. Natil.

Arad. Sm. U.S.A. 85-7448-7451 (198W~ etc- COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO.198 D29 3o' c While antsense nucleotides complementary to the Neutrokiue-alpha and/or cNeutokine-alphaSV coding region sequence could be used, those complementary to Sthe transcribed untranslated region are most preferred.

SPotential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, PCT International Publication WO 90/ 11364, published October 4, 1990; Sarver et al, Science 247-1222 1225 (1990). While ribozymes that cleave mRNA at site specific recognmon sequences can be used to destroy Neutrokine-alpha andlor Neutrokme-alphaSV mRNAs, the use of hammerhead ribozymes is preferred.

C Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that 0 0 10 form complementary base pairs with the target mRNA. The sole requirement is that 0 the target mRNA have the following sequence of two bases: 5'-UG-3' The C construction and production of hammerhead ribozymes is well known m the an and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites withm the nucleoide is sequence of Neutrokine-alpha and Neutrokine-alphaSV (Figures IA-B and SA-B, respectively). Preferably the ribozyme is engineered so that the cleavage recogmuon site is located near the 5' end of the Neutrokne-alpha and/or Neutrokne-alphaSV mRNA, to increase efficiency and minmize the intraccllular accumulation of nonfunctional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleoudes for improved stability targeing, etc.) and should be delivered to cells which express Neutrokne-alpha and/or NeutrokJne-alphaSV in wvo DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of anusense encoding DNA. A preferred method of delivery ivolves using a DNA construct encoding the ribozyme under the control of a strong constitutive promoter, such as, for example, pol Ill or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous Neutrokine-alpha and/or Neutrokne-alphaSV messages and innibit itrnstiauun. Since ribuzymues unlike anusense mouecuies. arc caialyuc, a iuWel intracellular concentration is required for efficiency Endogenous gene expression can also be reduced by inactivating or "knocking out" the Neutrokine-alpha and/or Neutrokme-alphaSV gene and/or its promoter using targeted homologous recombination. see SmtIhIes et al., Nature 317:230-234 COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 L_ 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.19e 00 C (1985); Thomas Capecchi, Cell 51:503-512 (1987); Thompson ct al., Cell 5:313-321 t (1989); each of which is incorporated by reference herein mn is entirety). For example, a mutant, nonfunnetonal polynucleotde of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleoude s sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypcptides of the invention nr vivo. In another embodiment, lechniques known in the art are used to generate knockouts m cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous 0 0 10 recombinaton, results in macuvauon of the targeted gene. Such approaches are 0 particularly suited in research and agrcultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene see Thomas Capecchi 1987 and Thompson 1989 supra). However this approach can be routinely adapted for use m humans provided the recombinant DNA constructs Is are directly administered or targeted to the required site in wvv using approprate viral vectors that will be apparent to those of skill in the ar. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety In other embodiments, antagonists according to the present invention include soluble forms of Neutrokne-alpha and/or Neutrokine-alphaSV fragments of Ncutrokne-alpha shown in Figures A-B that include the ligand binding domain, TNF conserved domain, and/or extracellular domain of Neutrokme-alpha and/or Neutrokine-alphaSV and fragments of Neotrokine-alphaSV shown in Figures that include the ligand binding domain. TNF conserved domain, and/or extracellular domain of Neutrokme-alpha and/or Neutrokme-alphaSV). Such soluble forms of the Neutrokne-alpha and/or Neutrokmne-alphaSV which may be naturally occurring or synthetic. antagonize Neutrokine-alpha and/or Neutrokme-alphaSV mediated signaling by competing with native Neutrokme-alpha and/or Neutrokme-alphaSV for binding to Neutrokme-alpha and/or Neutrokme-alphaSV receptors DR5 (See, International Publicatton 1N. WO 98!4'629), ML 0 (See, lntemnatiuia Puolicanon na. WO 98/54202), 312C2 (See, Internatonal Publicaton No. WO 98/06842), and TR 11 TRI 1SVI and TRI ISV2 (See, U.S. Application Senal No. 09/176.200)). and/or by forming a nultimer that may or may not be capable of binding the receptor, but which is incapable of inducing signal transduction. Preferably these antagonists inhibit COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 19/032228 16:39 BLAKE DAWJSON WAJLDRON LAWJYERS 4 062837999 lJ~9 3 NO. Ige P31 00 Neutrolane-alph-A and/or Neutrotzue-alphaSV mediated stimulation of lymphocyte Ct(e.g.. B-cell) prolifranon. differentisuon, and/or activation. Antagonists of the present invention also include antibodies specific for ThE-family ligands CD3O) and ONNeutrokme-alpha-Fc and/or Neutrokaie-alpbaSV-Fc fusion proteins.

s By a "TNF-family ligand" is intended naturally occumnng. recombujani, and synthetic ligands that are capable of banding toa member of the TN? receptor famnily and inducing anid/or blocking the ligand/recepror signaling pathway Members of the TNF ligand (wmily include, but are not limited 10, TNF-alpha. lymphozoxin-alpha (LT o alpha. also known as ThF-beta), LT-beta (found in complex hererotnner LT-alpha2- 00 to0 beta), FasL, CD4OL. (ThlF-ganuna (International Publication No- WO 9(V/14328), O AIM-I (International Publication No. WO 97/33899), AIM-Il (International Publication C']No. W0 97/3491). APRIL Exp. Mod. 188(6)-1185-1190), endobune-alpha (International Publication No. WO 98/07880), neutrolcinc-alpha (international Publication No. WO 98118921). CD27L.CD3OLI 4-IBBL. OX4OL, CD27 (2130.4- 1BB. 0X40. and nerve growth factor (NOD. In preferred embodimentsi the Neutrokine-alpha anid/or Neurroksne-alphaSV TNF-farnily ligands of the invention arm DRS (See International Publication No. WO 98141629). TRiO0 (See, International Publication No. WO 9815420M), 31' XC2 (See, International Publication No. WO 98/06842), and TRi 11, TR I iSYI1. and TR iISV2 (See. U.S. Application Senal No.

09/176,2OO).

Antagonists of the present invention also include antibodies spcific for TNF famnily receptors or the Neutrokine-alpha and/or Neutrokine-alpbaSV polypepudes, of the invention. Antibodies according to the present invention may be prepared by any C of a variety of standard methods using Neutrokine-alphm and/or 2$ iminunogens of the present invention. As indicated, such Neutrokine-aipha and/or Neutrokine-alphaSV iinmunogens, include the complete Neutrokine-alpha and Neutrokine-alphaSV polypeptides depicted in Figures l A-8 (SE ID) NO;.2) and Figures SA-3 (SEQ ID N0-19), respectively (which may or may not include the leader sequcnicej anu icutruuuic-aapuia aiOor Neuurokinc-ailphaS" poiypcpu-dc 'tAMn-s compnszng, for example, the ligand binding domain, TNIF-conserved domain, extracellular domain. crausmemnbrane domain, and/or intracellular domain, ar any combination thereof.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D32 0 0 Polyclonal and monoclonal antibody agomsts or antagonists according to the preset invention can be raised according to the methods disclosed m Tataglia and Goeddel, J. Bio. Chem. 267(7):4304-4307(1992)); Tartaglia et al., Cell73.213-216 (1993)). and PCT Application WO 94/09137 and are preferably specific to hind s uniquely to polypeptides of the mvention having the amino acid sequence of SEQ ID NO:2. The term antibody" (Ab) or monoclonal antibody" (mAb) as used herein is meant to include itact molecules as well as fragments thereof (such as, for example, Fab and F(ab) fragments) which are capable of binding an antigen. Fab, Fab and o F(ab') fragments lack the Fc fragment intact antibody clear more rapidly from the circulation, and may have less non-specific ussue bmding of an intact antibody (Wahl 0 et al.. J. Nuc. Med., 24.316-325 (1983)).

C In a preferred method, antibodies according to the present mvention are mAbs. Such mAbs can be prepared using hybndoma technology (Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4.376.110. Harlow et al., Antibodies. A Laboratory Manual, Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, NY 1988; Monoclonal Antibodies and Hvbrdomas: A New Dimenson n Biological Analyses, Plenum Press, New York, NY 1980; Campbell, "Monoclonal Antibody Technology In: Laboratory Techniques M Biochemistry md Molecular Biology, Volume 13 (Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds which bind the Neutrokine-alpha and/or Neuirokine-alphaSV domains are also candidate agomsts and antagonists according to the present invention. Such binding compounds can be captured" using the yeast twohybnd system (Fields and Song, Nature 340:245-246 (1989)). A modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuns, Cell 75:791-803 (1993); Zervos et al, Cell 72:223-232 (1993)). Preferably the yeast two-hybrd system is used according to the presem invenuon to capture compounds which bind to the ligand binding domain, extracellular, ntracellular, ransmembrane, and death domain of the Neutrokme-alpha and/or Neutroknc-alphaSV Such compounds ar good candidate agsoncts n.d 2.annn.ts of the present enton.

For example, using the two-hybrid assay described above, the extracellular or intracellular domain of the Neutrokme-alpha and/or Neutrokme-alphaSV receptor, or a portion thereof, may be used to identify cellular proteins which interact with Neutrokine-alpha and/or Neutrokine-alphaSV the receptor Mn vivo. Such an assay may COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 P33 00 o 0 C also be used to identify ligands with potenual agonmsuc or antagonistc acuvlty of SNeutrokine-alpha and/or Neutrolkne-alphaSV receptor function. This screning assay t has previously been used to identify protein which interact with the cytoplasmic domain of the munne TNF-RII and led to the identificanon of two receptor associated S proteins. Rothe et Cell 78:681 (1994). Such proteins and amnno acid sequences winch bind to the cytoplasnuc domain of the Neutrokme-alpha and/or SNeutrokne-alphaSV receptors are good candidate agonist and antagonist of the present invention.

o Other screenig techniques include the use of cells which express the 00 to polypepude of the present invention (for example, transfected CHO cells) in a system

O

Swhich measures extracellular pH changes caused by receptor activation, for example, as IC described m Science, 246; :81 296 (1989). In another example, potential agonists or antagonrsts may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, signal transduction may be measured to determine whether the potential antagonist or agonist is effective.

Agonists according to the present invention include naturally occurring and synthetic compounds such as, for example, TNF family ligand peptide fragments, transformming growth factor, neurotransmitters (such as glutamate, dopammine, N-mehyl- D-aspartate). tumor suppressors (p53), cytolytlc T cells and animetabolites. Preferred agonists include chemotherapeuuc drugs such as, for example, cisplain, doxorubicin, bleomycin, cytosne arabmoside, nitrogen mustard, methotrexatc and vncristme.

Others include ethanol and -amylold peptde. (Science 267-1457 1458 (1995)).

Preferred agomsts are fragments of Neutrokme-alpha and/or C Neutrokme-alphaSV polypeptides of the mvention which stimulate lymphocyte B cell) proliferation, differentiation and/or activation. Further preferred agonists include polyclonal and monoclonal antibodies raised against the Ncutrokine-alpha and/or Neutrokine-alphaSV polypeptdes of the invention, or a fragment thereof. Such agonist antibodies raised against a TNF-family receptor are disclosed in Tanaglia et al., Proc.

atl. Arad. Vc-_ ISA RS8: 9 29 2 .Q296 1 Q and T rag!ia et J. Bot Ch.,n.

267-4304- 4307(1992). See. also, PCT Application WO 94/09137 In an additional embodiment, immunoregulatory molecules such as, for example. IL2. IL3. IL4, IL5 IL6. IL7 IL10. ILl2, IL13, IL11, anti-CD40, IFN-gamma and TNF-alpha, may be used as agoumss of Neutrokne-alpha and/or COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D34 00 0 -03, C Neutrokmne-alphaSV polypepltdes of the nventon which stimulate lymphocyte B ccell) proliferation, differentiation and/or activation. In a specific embodiment, IL4 and/or IL10 are used to enhance the Neutroine-alpha- and/or Neutrokine-alphaSV ON mediated proliferation of B cells.

s In further embodiments of the invention, cells that are genetically engineered to express the polypeptades of the invention, or alternatively thai are genetically engineered not to express the polypeptdes of the invention knockouts) are administered to a patient m vwvo. Such cells may be obtained from the patient O ammal, including human) or an MHC compatible donor and can include, but are not 00 lo linmted to fibroblasts, bone marrow cells, blood cells lymphocytes), adipocytes, 0 muscle cells, endothelial cells etc. The cells are genetically engineered in vtro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypcptides of the invention, e.g., is by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to. the use of plasmds, cosmids, YACs, naked DNA, electroporation, liposomes, etc- The coding sequence of the polypeptdes of the invention can be placed under the control of a strong constitutive or mducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically in the circulation, or intrapenoncally Alternatively the cells can be incorporated into a matrix and implanted in the body genetically engineered fibroblasts can be implanted as part of a skin graft: genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349, and Mulligan Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the ce!ls to be adm.instered ar non- auologous or non-M.C compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 00 0 c of components with the immediate extracellular environment, does not allow the Sintroduced cells to be recognized by the host immune system.

In yet another embodiment of the invention, the activity of Neutroklne-alpha and/or Neutrokine-alphaSV polypeptide can be reduced using a dominant negative.

To this end, constructs which encode defective Neutrokme-alpha and/or Neutrokne-alphaSV polypepude, such as, for example, mutants lacking all or a portion of the TNF-conserved domain, can be used m genc therapy approaches to dirmntsh the activity of Neutrokine-alpha and/or Neurokine-alphaSV on appropnate target cells.

O For example, nucleotide sequences that direct host cell expression of Neutrokine-alpha and/or Neuirokme-alphaSV polypeptde in which all or a portion of the TNF-conserved Sdomain is altered or missing can be introduced into monocytic cells or other cells or c tissues (either by in vivo or ex vive gene therapy methods described herein or otherwise known in the art). Alternatively targeted homologous recombination can be utilized to introduce such deletions or mutations into the subject's endogenous Neutrokme-alpha and/or Neutrokine-alphaSV gene in monocytes. The engineered cells will express non-funcional Neutrokme-alpha and/or Neutrokme-alphaSV polypeplides a ligand mulumer) that may be capable of binding, but which is incapable of inducing signal transduction).

Chromosome Assays The nucleic acid molecules of the present invention are also valuable for chromosome identfication. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphusms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

In certam nreferrerd embdiments nm this regard. the cDN.A andler polynuclcotides herein disclosed is used to clone genomic DNA of a Neutrokne-alpha and/or Neutrokne-alphaSV gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially The COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2088 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 P36 00 0 c" genomic DNA then is used for m saru chromosome mapping using well known techniques for thus purpose.

SIn addition, in some cases, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon m the genomic DNA. thus complicating the amplification process.

These pnmers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Fluorescence tn sru hybridization ("FISH") of a oc DNA clone to a metaphase chromosomal spread can be used to provide a precise 00 10 chromosoral location m one step. This technique can be used with probes from the ScDNA as short as 50 or 60 bp. For a review of this technique. see Venna er Human C"1 Chromosomes: A Manual Of Basc Techniques. Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, m V McKusick, Mendelian Inhertance In Man, available on-line through Johns Hopkms University Welch Medical Library The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysts (coinhntance of physically adjacent genes).

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

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to achromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes I megabase mapping resolution and one gene per 20 kb).

Utilizng the techmques described above, the chromosomal location of Netlrmknr-alpha and Neurokmc-alphaSV ,.3s determined w::h hizh confid ce using a combination of somatic cell hybrids and radiation hybrids to chromosome position 13q34.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWISON WALDRON LAWYERS 4 062837999 N0.198 P37 00 Having generally described the invention, the same will be more readily Sunderstood by reference to the following examples, winch are provided by way of illustration and are not intended as lirtmng. Many of the following examples are set s forth referring specifically to Neutrokme-alpha polynucleoudes and polypeptdes of the invention. Each example may also be practiced to generate and/or examne Neutrokinc-alphaSV polynucleotudes and/or polypepndes of the invention. One of ordinary skill m the art would easily be able to direct the following examples to Neutrokine-alphaSV 00 o Example la: Expresson and Puriicafion of "His-tagged" Neutrokme-alpha r m E. col The bactenal expression vector pQE9 (pD10) is used for bactenal expression in this example. (QIAGEN, Inc., supra), pQE9 encodes ampicillin antibiotic resistance s ("Ampr") and contains a bactenal orngm of replication an IPTG inducible promoter, a ribosome binding site six codons encoding histidine residues that allow affinmty puification using nickel-ntrilo-n-acec acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra, and suitable single restnction enzyme cleavage sites.

These elements are arranged such that an inserted DNA fragment encoding a polypeptide expresses that polypeptide with the six His residues a "6 X His tag") covalently linked to the ammo terminus of that polypepide.

The DNA sequence encoding the desired portion of the Neutroknmealpha protein comprsmg the extracellular domn sequence is amplified from the deposited cDNA clone using PCR oligonucleotide prmers which anneal to the amino terminal sequences of the desired portion of the protein and to sequences in the deposted construct 3' to the cDNA coding sequence. Additional nucleotides containing restincion sites to facilitate cloning in the pQE9 vector are added to the 5' and 3' primer sequences. resoectivelv For cloning the extracellular domain of the protein, the 5' pnmer has the sequence 5'-GTG GGA TCCAGC CTC CGG GCA GAG CTG-3' (SEQ ID contining the underlined Bam HI restncnon site followed by 18 nucleotides of the COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062937999 N. 198 D38 00 c ammo terminal coding sequence of the extracellular domain of the sequence in Figures Ct IA and IB. One of ordinary skill m the art would appreciate, of course, that the point in the protein coding sequence where the 5' primer begins may be vaned to amplify a DNA segment encoding any desired porton of the complete Neutrolkne a protein s shorter or longer than the extracellular domain of the form. The 3' prmer has the sequence 5'-GTO AAG TTTA TTAA CAG CAG TTT CAA TGC ACC 3 (SEQ ID NO' 11) containing the underlined Hind III restriction site followed by two stop codons and 18 nucleoudes complementary to the 3' end of the coding sequence of the DNA C sequence m Figures IA and IB.

00 The amplified DNA fragment and the vector pQE9 are digested with Bam HI and Hind III and the digested DNAs are then ligated together. Insertion of the DNA ito the restricted pQE9 vector places the protein coding region downstream from the IPTG-nducible promoter and in-frame with an matung AUG and the six htstidine codons.

Is The ligauon mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Fd., Cold Spnng Harbor Laboratory Press, Cold Spnng Harbor, NY (1989). E. coli stran M15/rep4, containing multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance is used in carrying out the illustratve example described herein. This strain, which is only one of many that are suitable for expressing protein, as available commercially from QIAGEN, Inc., supra. Transformants are identified by their ability to grow on LB plates m the presence of ampicillin and kanamycn. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restrition analysis, S PCR and DNA sequencing. Clones containing the desired constructs are grown overnght in liquid culture in LB media supplemented with both ampicillin (100 jg/ml) and kanamycin (25 pg/nll). The OIN culture is used to inoculate a large culture, at a dilution of approximately 1.25 to 1.250. The cells are grown to an opucal density at 600 nm ("OD600") of between 0.4 and 0.6.

Isopropyl-beta-D*thiogalactopyranostae ("IPTG") is then added to a final concentrauun of I mM to induce transcription from the lac repressor sensitive promoter, by COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 939 00 C' inactivating the lac repressor Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centnfuganon.

SThe cells are then strred for 3-4 hours at 4 C in 6M guanidine-HCl, pH 8. The cell debns is removed by centnfugaton, and the supematant contaimng the is loaded Son to a nickel-nitrilo-tn-acetc acid ("Ni-NTA") affinity resin column (available from QIAGEN, Inc.. supra). Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be punfied in a simple one-step procedure (for details see: The QlAexpressiomst. 1995, QIAGEN, Inc.. stupra). Briefly the supernatant is loaded on to 0 the column in 6 M guawdine-HCl. pH 8, the column is first washed with 10 volumes of o o 6 M guanldine-HC. pH 8, then washed with 10 volumes of 6 M guanidine-HCI pH 6.

o and finally the Neutrolkne-alpha and/or Neutrokme-alphaSV polypeptide is eluted with C 6 M guamdine-HCl, pH The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaC. Alternauvely the iS protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M- I M urea gradient in 500 mM NaCI, 20% glycerol, 20 mM Tns/HCI pH 7 4. containing protease mhibitors. The renaturauon should be performed over a period of 1.5 hours or more.

After renaturation the proteins can.be eluted by the addition of 250 mM immidazole.

Imimdazole is removed by a final dialyzmg step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaC. The punfied protein is stored at 4*C or frozen at -80 g

C.

C.

Example lb: Expresston ad Pur(Wcation of Neutrokune.alpha an E. coli The bacterial expression vector pQE60 is used for bacterial expression n this example. (QlAGEN, Inc., 9259 Eton Avenue, Chaisworth, CA, 91311). encodes ampicillin antibiouc resistance ("Ampr") and contains a bactenal origin of replication an IPTG inducible promoter, a ribosome binding ste six codons encoding hisudine residues that allow affinity purification using nckel-nmrilo-in-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., supra.

and suitable single restriction enzyme cleavage stes. These elements are arranged such COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/0F^3/2008 1 4 1 1ae III1 s 1 0. unrM Ar -wMUIN WHLJUKUN LHWYEh 4 0628337999 NO.198 940 00 C that a DNA fragment encoding a polypeptde may be iserted in such as way as to produce that polypepude with the six His residues a "6 X His tag") covalently Slinked to the carboxyl terminus of that polypeptzde. However, m this example, the polypeptide coding sequence is inserted such that translanon of the six His codons is S prevented and, therefore, the polypeptide is produced with no 6 X His tag.

The DNA sequence encoding the desired portion of the protein compnsing the extracellular domain sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers which anneal to the amino termnal sequences of the desired N portion of the protein and to sequences in the deposited construct 3 to the cDNA coding sequence. Additional nucleoudes conmaing resnction sites to facilitate m cloning in the pQE60 vector are added to the 5' and 3' sequences, respecuvely For cloning the extracellular domain of the protein, the 5' pnmer has the sequence 5'-GTG IA TGA GCC TCC GGG CAG AGC TG-3' (SEQ ID NO: 12) containing the underlined Bsp HI restricon site followed by 17 nucleotides of the s amino ternmnal coding sequence of the extracellular domain of the sequence in Figures IA and IB. One of ordinary skill in the art would appreciate, of course, that the point t the protein coding sequence where the 5' pnmer begins may be varied to amplify a desired poruon of the complete protein shorter or longer than the extracellular domain of the form. The 3' pimer has the sequence 5'-GTG AAG CTT TTA TTA CAG CAG TTT CAA TGC ACC 3' (SEQ ID NO: 13) containing the underlined Hind UI restriction site followed by two stop codons and 18 nucleotdes complementary to the 3' end of the coding sequence in the DNA sequence m Figures IA and IB.

The amplified DNA fragments and the vector pQE60 are digested with Bsp HI and Hind Ill and the digested DNAs are then ligated together. Insertion of the DNA nro the restricted pQE60 vector places the protein coding region including its associated stop codon downstream from the IPTO-inducible promoter and m-frame with an iniatnmg AUG. The associated stop codon prevents translation of the six lustidine codons downstream of the insertion point.

The ligaton mixture is transformed into competent E. coli cells using standard procedures such as those described in Sambrook et al., Molecular Cloning: a Laborarory Manual 2nd Ed., Cold Spring Harbor Laboratory Press. Cold Spnng COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 41 00 Cl Harbor, NY (1989). E coli strain M 5/rep4, containing multple copies of the plasrmd c pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kanr"), Sis used ns carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expressing protein, as available commercally Sfrom QIAGEN. Inc., supra. Transformants are identified by their ability to grow on SLB plates in the presence of ampicillin and kanamycm, Plasnmd DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by resmcton analysis, PCR and DNA sequencing.

C One of ordinary skill in the an recognizes that any of a number of bacterial 00 O0 O 10 expression vectors may be useful n place of pQE9 and pQE60 in the expression Cl- protocols presented in this example. For example, the novel pHE4 series of bacterial expression vectors, i particular, the pHE4-5 vector may be used for bacterial expression in this example (ATCC Accession No. 209311 and variations thereof). The plasimd DNA designated pHE4-5/MPIFD23 in ATCC Deposit No. 209311 is vector is plasmld DNA which contains an insert which encodes another ORF The construct was deposited with the American Type Culture Collection, 10801 University Boulevard.

Manassas, Virginia 20110-2209 on September 30, 1997 Using the Nde I and Asp 718 restnction sites flanking the irrelevant MPIF ORF insert, one of ordinary skill in the art could easily use current molecular biological techniques to replace the irrelevant ORF im the pHE4-5 vector with the Neutrokne-alpha ORF of the present invenuon.

The pHE4-5 bacterial expression vector includes a neomycin phosphotransferase gene for selection, an E. coli origin of replication, a T5 phage promoter sequence, two lac operator sequences, a Shine-Delgaro sequence, and the lactose operon repressor gene (laclq). These elements are arranged such that an inserted DNA fragment encoding a polypeptude expresses that polypepude with the six His residues a "6 X His tag") covalently linked to the amino terminus of that polypepude. The promoter and operator sequences of the pHE4-5 vector were made synthetically Synxhene production of nucleic acid sequences is well known m the art (CLONETECH 95/96 Catalog. pages 215-216, CLONETECH. 1020 East Meadow Circle, Palo alto, CA 94303).

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 P42 00 0, 0 Clones contaung the desired Neutrokme-alpha constructs are grown overnight O("OIN") in liquid culture m LB media supplemented with both ampicillin (100 pg/ml) and kanamycin (25 pg/mi). The 01N culture z used to inoculate a large culture, at a dilution of approximately 1.25 to 1.250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. asopropyl-beta-D-thdogalactopyranossde (cIPT) is then added to a final concentration of I mM to mnduce tanscnpton from the lac repressor sensitive promoter, by inmavating the lad repressor, Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centnfugatzon.

00 o The cells are then stirred for 3-4 hours at 4C min 6M guanidine-HCI, pH 8. The O call debris is removed by centnrfugation and the supernatant containimg the Neutroknme a is dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with 200 mM NaC.

Alternauvely the protein can be successfully refolded by dialyzing it against 500 mM NaCI, 20% glycerol, 25 mM Tns/HCI pH 7 4, containing protease inhibitors. After ranaturaton the protein can be punrified by son exchange, hydrophobic interaction and size exclusion chromatography Alternatively an affinity chromatography step such as an antibody column can be used to obtain pure protein. The purified protein is stored at 4'C or frozen at In crtamI embodiments, a ist preferred to generate expression construcls as detailed m this Example to mutate one or more of the three cysteme residues in the Neutrobn6-alpba polypeptide sequence. The cystene residues in the Neutrokzne-alpha polypeptide sequence are located at positions 147 232, and 245 as shown in SEQ ID NO:2 and at positions 213 and 226 of the Neutrokine-alpha polypepude sequence as shown in SEQ ID NO' 19 (there as no cystemne mn the Neutrokine-alphaSV polypeptide 2 sequence which corresponds to Cys-147 in the Neutrolane-alpha polypepude sequence because amino acid residues 143-160 of the Neutrokme-alpha polypepude sequence are not present in the Neutrokune-alphaSV polypepude sequence).

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 43 00 o cN Example 2: Cloning, Expresson, and Purification of Neuroklan-alpha Pr lotein a Ba yvarus Expresson System In this illustrative example. the plasmid shuttle vector pA?2P is used to insert the cloned DNA encoding the extracellular domain of the protein, lacking its naturally associated intracellular and transmembrane sequences, into a baculovirus to express the extracellular domain of the Neutrokine-alpha protein, using a baculovirus leader and standard methods as described in Summers et al., A Manual ofMerhods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agrcultural Expermental Station 0 Bulletin No. 1555 (1987). This expression vector contains the strong polyhednn 0 0o promoter of the Aurographa califorica nuclear polyhedrosis virus (AcMNPV) o followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as Barn HI. Xba 1 and Asp 71 The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of recombimant vims, the plasnmd contains the beta-galactosidase gene from E coli under control of a weak Drosophila promoter in the same oncntauon, followed by the polyadenylation signal of the polyhednn gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombnation with wild-type viral DNA to generate viable virus that expresses the cloned polynucleotide.

Many other baculovirus vectors could be used m place of the vector above, such as pAc373, pVL941 and pAcIMI as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for ranscrnption, translation, secretion and the like, including a signal pepude and an m-frame AUG as required. Such vectors arc described, for instance, in Luckow et al., Virology 170;31 39(1989).

The cDNA sequence encoding an N-terminally deleted form of the extracellular domain of the Neutrokine-alpha protein in the deposited clone, lacking the AUG initiation codon, the naturally associated intracellular and transmembrane domain sequences, and ammno acids Gin-73 through Leu-79 shown in Figures IA and IB (SEQ ID NO:2), is amplified using PCR oligonucleotade primers corresponding to the 5' and 3' sequences of the gene. The 5' primer has thb sequence 5'-GTG G A ICC CCG GGC AGA GCT GCA GGG C 3' (SEQ ID NO'14) containing the underlined Bam HI COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D44 00 03-1 Srestriction enzyme site followed by 18 nucleotides of the sequence of the extracellular domain of the Ncutrokie-alpha protein shown in Figures IA and 1 B, beginning with Sthe indicated N-enmanus of the exracellular domain of the protein. The 3' pnmer has the sequence 5'-GTG GGA TCC TTA TTA CAG CAG TTT CAA TGC ACC 3' (SEQ ID NO: 15) containing the underlined Barn HI restnction site followed by two stop codons and 18 nucleotidcs complementary to the 3' coding sequence m Figures IA and

^IB.

In certain other embodiments, constrmcts designed to express the entire C predicted extracellular domain of the Neutroklne-alpha amino acid residues 0 0 to Gln-73 through Leu-285) are preferred. One of skill in the art would be able to use the o polynuclecotde and polypeptide sequences provided as SEQ ID NO I and SEQ ID NO:2, respectively to design polynucleotade primers to generate such a clone.) In a further preferred embodiment, a pA2GP expression construct encodes amlno acid residues Leu-1 12 through Leu-285 of the Neutrokmne-alpha polypeptide sequence shown as SEQ ID NO:2.

In another preferred embodiment, a pA2GP expression construct encodes ammo acid residues Ser-78 through Leu-285 of the Neutrokine-alpha polypeptide sequence shown as SEQ ID NO:2.

The amplified fragment is isolated from a 1% agarose gel usmg a commercially available kit ("Geneclean, BIO 101 Inc., La Jolla, The fragment then is digested with Barn HI and again s purified on a 1% agarose gel. This fragment is designated herein Fl.

The plasmid is digested with the restriction enzymes Barn HI and optonally can be dcphosphorylared using calf intestnal phosphatase, using routine procedures known in the art The DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean BIO 101 Inc., La Jolla, This vector DNA is designated herein "V Fragment Fl and the deohosohorvlated olasmid VI are ligated together wih T4 DNA ligase. E. coli HBI 01 or ther suitable E. coli hosts such as XL 1 Blue (Statagene Clonng Systems, La Jolla, CA) cells are transformed with the ligaion mixture and spread on culture plates. Bacteria are dentified that contain the plasrmd with the human gene by digesting DNA from individual colonies using Bam HI and COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 19e 00 0 S2o O0 C then analyzing the digestion product by gel electrophoresis. The sequence of the cloned t fragment is confirmed by DNA sequencing. This plasmad is designated herein SpA2GP-Neutrokne-alpha.

O Five micrograms of the plasrmd pA2GP-Ncutrokine-alpha is co-trnsfected with 1.0 microgram of a conmmercally available lineanzed biculovirus DNA ("BaculoGold r baculovirus DNA Pharnungen, San Diego, CA), using the lipofecton method described by Feigner et al., Proc. Natl. Acad. Set. USA 84' 7413-7417 (1987). One pg of BaculoGold' m vrus DNA and 5 nucrograms of the C plasrud pA2GP Neutrokmen-alpha are nuned in a sterile well of a mncrouter plate 1 0 contanmng 50 rmcroliters of serum-free Grace's medium (Life Technologies Inc., O Ganhersburg, MD). Afterwards, 10 microliters Lapofecun plus 90 rmcroliters Grace's L( medium are added, rmxed and incubated for 15 mmnutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27'C. The transfection solution is then removed from the plate and I ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27'C for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Rockville, Maryland) is used to allow easy identification and isolaton of gal-expressing clones, which produce blue-stamed plaques. (A detailed descripton of a "plaque assay" of this type can also be found in the user's guide for Sinsect cell culture and baculovirology distributed by Life Technologies Inc., Rockville, MD page 9-10). After appropriate incubation, blue stained plaques are picked with the up of a mncropipettor Eppendorf). The agar containing the recombinant viruses is then resuspended n a nucrocentnfuge tube containing 200 microliters of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4*C. The recombinant virus is called V-Neutrokine-alpha.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 198 P46 00 o 32 O0 c-l To verify the expression of the Neutrokme-alpha gene Sf9 cells are grown mn C Grace s medium supplemented with 10% heat-nactuvated FBS. The cells are infected Swith the recombinant baculovirus V-Neutrokne-alphaa aa multiplicity of mfection of about 2. If radiolabeled proteins are desired, 6 hours later the medium as Sremoved and is replaced with SF900 II medium minus methionne and cysteme (available from Life Technologies Inc., Rockville, MD). After 42 hours, 5 microcunes of "S-methiomne and 5 micmrounes "S-cysteine (available from Amersbam) are added.

The cells are further incubated for 16 hours and then are harvested by centrfugation.

C'I The proteins in the supernatant as well as the intracellular proteins are analyzed by 00 StO SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencmg of the amino acid sequence of the amino temmnus of punfied protein may be used to determine the armno terminal sequence of the extracellulardomain of the protein and thus the cleavage point and length of the secretory signal peptide.

In a specific expenmental example, recombinant Neutrokine-alpha was punfied from baculovnrus infected Sf9 cell supematants as follows. The msect cells were grown in EXCEL4A0 medium (JRH Scientific) with I fetal bovine serum. At 92 hours post-mfection, the harvested supematant was clarified by centnfugation at 18,000 x g followed by 0.45 m depth filtration. A de-lipid filtration step might be also used to remove the lipid contaminants and in turn to improve initial capturing of the Neutrokme-alpha protein.

The supernatant was loaded on to a set of Poros HS-50/HQ-50 in tandem mode.

As alternatves, Toyopearl QAF, Toyopearl Super Q (Tosohass), Q-Sepharose (Pharmacia) and equivalent resins might be used. This step as used as a negauve punfication step to remove strong anion binding contaminants. The HS/HQ flow through material was adjusted to pH 7.5 with I M Tns-HCI pH 8, diluted with equal volume of 50 mM Tns-HCI pH 8, and loaded onto a poros PI-20 or P1-50 column. The PI column was washed first with 4 column volumes of 75 mM sodium chloride in mM Tns-HCI at pH 7.5. then eluted using 3 to 5 column volumes of a stepwise gradient of 300 mM, 750 mM, 1500 mM sodium chlonde in 50 mM Tns-HCI pH Neutrokne-alpha protein appears as a 17 KD band on reduced SDS-PAGE and is present in the 0.75 M to 1.5M Sodium chloride fractions.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 D47 0 C" The PI fraction was further purfied through a Sephacryl S100 HR (Pharmacia) Ssize exclusion column equilibrated with 0.15 M sodium chlonde, 50 mM sodium Sacetate at pH 6. The S200 fractions were mixed with sodium chloride to a final concentrauon of 3 M and loaded onto a Toyopearl Hexyl 650C (Tosohass) column.

The Hexyl column was eluted with a linear gradient from 3 M to 0.05 M sodium chloride m 50 mM Sodium acetate pH 6 m 5 to 15 column volumes. The sodium chlonde gradient can also be replaced by ammomum sulfate gradient of IM to 0 M in mM sodium acetate pH 6 in the Hexyl chromatographic step. Fractions contarinng O pnnfied Neutrokme-alpha as analyzed through SDS-PAGE were combined and 00 10 dialyzed against a buffer containing 150 mM Sodium chlonde, 50 mM Sodium acetate.

SpH 6 The final purified Neutrokne-alpha protein expressed in a baculovirus system as explained herein has an N-termmus sequence which begins with amino acid residue Ala- 134 of SEQ ID NO:2. RP-HPLC analysts shows a single peak of greater than ls purity Endotoxin level was below the detecuon limit in LAL assay In another example, recombinant Ncucrokanc-alpha was purified from baculovirus infected 5f9 cell supernatants containing 0.25% bovine serum as follows.

The Sf9 supernatant was harvested by cenmfuganon at 18.000 x g The supernatant was then treated with 10 mM calcium chloride an slightly alkaline conditions for 10-15 minutes followed by centnfuganon and then 0.22 micrometer depth filtration. The resulting Sf-9 cell supernatant was then diluted 2-fold and loaded on to a Poros PI-SO column (available from PE Biosystems). The column was equilibrated with 50 mM Trls (pH=7 The PI-50 column was washed with I CV of 3 50 mM Tns (pH= 7 4) and then eluted with 1.5 M NaCI in 50 mM NaOAc (pH=6) over 2 3 CV The PI fracenon was loaded on to a Sephacryl 5200 column equilibrated with mM NaOAc 125 mM NaCI. The 5200 fraction was mixed with salts to final concentrations of 0.7 M ammonium sulfate and 0.6 M NaCI and loaded on to a Toyopearl Hexyl 650C column (available from Toso Haas) that had been equilibrated buff-r containing 0.6 M NCI M 'amrnmunim sulfate m 50 rrmM NaOAc (pH= 6 The column was then washed with 2 CV of the same buffer. Recombinant Neurokne-alpha was then eluted stepwise with 3 CV of 50 mM NaOAc (pH=6) followed by 2 CV of 20% ethanol wash. The recombmant Neutrokine-alpha protein was then eluted at the end of the ammonium sulfate (0.3 to 0 M salt) gradient. The COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 062837999 NO. 198 Q48 00 0 3j3 appropriate fractions were pooled and dialyzed against a buffer containing 50 mM SNaOAc and then passed through a Poros 50 HQ column. The HQ flow- G through was dilted to 4 ms and loaded on to a Toyopearl DEAD 650M column and then eluted with 25 mM NaCitrate, 125 mM NaCI.

In another example, recombnant Neutrokme-alpha was expressed and purified using a baculoviral vector system in Sf+ insect cells.

First, a polynucleoude encoding amnno acid residues Ser-78 through Leu-285 of the Neutrokne-alpha polypeptide sequence shown in Figures IA and IB (which is 0 exactly identical to anuno acid residues Scr-78 through Leu-285 of the Neurokme 00 to alpha polypepude sequence shown as SEQ ID N0:2) was subeloned into the O baculovarus transfer construct PSC to generate a baculovirus expression plasnud. The

C"

1 pA2GP transfer vector, denved from pVL941, contains the gp 6 7 signal peptide, a i) modified multiple cloning site, and the lac Z gene cloned downstream of the Drosophila hear-shock promoter for selection of blue plaques. Using the sequence of is Neutrokne-alpha (SEQ ID NO:2) and the sequence of the pA2GP vector, a cloning strategy was designed for scamlessly fusing the PSC signal peptde coding sequence to the Neutrokne-alpha coding sequence at Ala-134 (SEQ ID NO:2 and Figures IA and IB) and msering it into a PSC baculovirus transfer plasmad. The strategy involved the use of a two-stage polymierase chain reacuon (PCR) procedure. First, pnmers were designed for amplifying the Neutrokne-alpha sequences. The 5' primer consisted of the sequence encoding Ala-134 and following residues (5'-GGT COC CGT TTC TAA CGC GGC CGT TCA GGG TCC AGA AG-3'- SEQ ID NO:31), preceded by the sequence encoding the PSC signal peptide C-temunus. The 3' pnmer (5'-CTG GTT CCG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTA GAT C 2s SEQ ID NO:32) consisted of the reverse complement of the pA2GP vector sequence immediately downstream from the Neurokine-alpha coding sequence, preceded by a Kpn I restrieton endonuclease sue and a spacer sequence (for increased cutting efficiency by Kpn PCR was performed with the pA2GP containing Neutrokne alnha nlasrnud t mplate and pnmers O-i 887 and O- 'S8S, and the resulting CR product was purified using standard techniques.

An additional PCR reacton was performed using the PSC baculovirus transfer plasmnd pMGS 12 as a template. The pMGS 12 plasmnd consists of the AcNPV EcoRI fragment inserted into pUC8. with the polyhedrm coding sequences after the ATG COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 349 00 Cl start codon replaced with the PSC signal peptide and a polylinker site. The PCR C reacton used pMGS12 as a template, a 5' pnmer (5'-CTG GTA GTT CTT CGG AGT SGTG-3'- SEQ ID NO:33) whtch annealed in AcNPV ORF603 upstream of the unique NgoM IV and EcoR V sites, and a 3' primer (5'-CGC GTT AGA AAC GGC GAC C 3' SEQ ID NO:34) which annealed to the 3' end of the sequence encoding the PSC signal peptide.

To generate a PCR product in which the PSC signal peptide was seamlessly Sfused to the Ala-134 of the Neuirokme-alpha coding sequence, the PCR product was o combined with the PSC signal peptde-polybedrm upstream region PCR product and 00 10 subjected to an additional round of PCR. Because the 3' end of the PSC signal peptide

O

0 PCR product (pMGS 12 0-959 0-1044) overlapped the 5' end of the Neutrokine c' alpha PCR product prepared with primers 0-1887 0.1888, the two PCR products were combined and overlap-extended by PCR using primers 0-959 and 0-1888.

The resultng overlap-extended PCR product containing the PSC signal pepude IS fused to the Neutrokmne-alpha sequence subsequently was inserted into baculovius transfer plasmid pMGS12. The PCR product was digested with NgoM IV and Kpn 1, and the fragment was purified and ligated into NgoM IV-Kpn I-cut pMGS 12. After transformation of competent E coli DH5alpha cells with the ligation mix, colonies were picked and plasmid DNA mini-preps were prepared. Several positive clones from each ligation were identified by restrncton digestion analysis of the plasnud DNA, and three clones (pAcC9669 pAcC9671 and pAcC9672) were selected for large scale plasmid punfication. The resulting plasmid DNA was subjected to DNA sequence analysis to confirm and sequence the Neutrokine-alpha insert.

The following steps describe the recovery and punfication process of recombinant Neutrokine-alpha from Sf+ insect cells. Unless stated otherwise, the process is conducted at 2-8'C.

Recovery Step I CaCI, Treatment Sf-- cell supernatant was harvested by c.ntnfugaison at 8,000 x g. Recovery buffer-I (I M CaCI) was added to the supernatant so that the final concentration of CaCI, was 10 mM. (In a further preferred embodiment, IM ZnCI, is used in place of 1M CaCI,.) The pH of the solution was adjusted to 7 7 with Recovery buffer-2 (1M COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 9 00 o us C" Tns pH 8 (t The solution was incubated for 15 minutes and then centrifuged at 8,000 x g.

Purificanon Step 1 Chromaiography on Poros P.I50 Column Sf+ cell suprnatant was loaded on to a Poros PI-50 column (PE Biosystem).

The column was equilibrated m PI-i buffer (50 rnM Tris. 50 mM NaCI, pH 7 4 The PI-50 column was washed with 1-2 CV of P- 1 buffer and then eluted with PI-2 buffer (50 mM Na Citrate pH 6 over 3 CV linear gradient. The elutnon o was monitored by ultraviolet (UV) absorbance at 280 nm. Fractions were collected 00 to across the eluate peak and analyzed by SDS page. Appropriate fractions were pooled.

0 Slep 2. Chromalography on Toypearl Hexyl 650C Column C' The PI pool was mixed with salts to final concentrations of 0.7M (NH),SO, and loaded on to a Toyopearl Hexyl 650C (Toso Haas) column equilibrated in HIC 1 buffer (50 mM NaOAc, 0.6M NaC, 0.7M pH 6 The column was then washed with 2 CV of HIC I buffer. Subsequently recombinant Neutrokine-alpha was then eluted stepwise with 3-5 CV of HIC 2 buffer (50mM NaOAc pH 6.0 0.2)) followed by a 2 CV 20% ethanol wash. The elution was monitored by UV absorbance at 280 nm and conductivity Fractions were collected across the eluate peak and analyzed by SDS-PAGE. The approprate fractions were then pooled.

Step 3. Chromatography on SP sepharose FF The Hexyl fraction was dialyzed and ajusted to pH 4.5 with SP 1 buffer mM sodium acetate pH 4.5 diluted to 4 ms and loaded through a SP sepharose (cation exchanger, Pharmacia) column equilibrated wih SP I buffer (50 mM sodium acetate pH 4.5 Recombinant Neutrokine-alpha protein was then eluted from the SP column with SP 2 buffer (50 mM sodium acetate pH 5.5 at pH 5.5 The eluton was then monitored by ultraviolet (UV) absorbance at 280 nm. Fractions were collected across the eluate peak and analyzed by SDS page. Appropriate fractons were pooled.

Step v Dialysis of R1.ioriibumn NeAtr6okmei-alpnu The SP fractions were placed into a 6-8 kd cutoff membrane device and then dialyzed or diafilered into Dialysis Buffer (10 mM sodium citrate, 140 mM sodium chloride pH 6 (2 overnight.

Step 5. Filtramlon and Fill COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N0.198 D51 00 The protein concentration of the recombinan Neutrokmne-alpha soluton from t Step 6 was determined by bicmchonimei acid (BCA) protein assay Recombinant SNeutrokane-alpha formulation was adjusted to the final protein concentraton with the appropriate buffer and filtered under controlled conditions. The filtrate (bulk s substance) was stored m suitable sterilized containers below In a specific embodiment, Neutrokine-alpha protein of the invention produced as described Infra was adjusted to a final protein concentration of I to 5 mg/ml and buffered in 10 mM sodium citrate, 140 mM sodium chloride, pH 6.0 t an C< stored at or below 20'C in Type I glass vials.

During chromatography runs. the processes are monitered by UV absorbance at 0 280 nm. When applicable, in-process chromatography intermediates are tested for conductivity pH, and monitored by SDS and/or RP-HPLC.

Columns and purificauon equipment are cleaned and sanitized with 0.2 or M NaOH followed by deomnzed water and then 0.1 or 0.5 M acetic acid. The column is and purification equipment are nnsed with deionized water and, if necessary stored m the appropriate storage solution. Prior to use, the equipment is equilibrated with appropriate buffers (as described herein or as is well known in the art).

In a further preferred embodiment, IM ZnCi, is used in place of IM CaCI, i Step 1 of the Recovery section described above. Also, in this embodiment, a combmaton of ZnC1, and CaCl, may be used. Many combinations of 0.1 M ZnCI, and 0.9 M CaCI,. may be used in the Recovery process of recombinant Neutrokine-alpha protein such as, for example, but not limited to, a combination of O.1 M ZnCl, and 0.9 M CaCI, 0.2 M ZnCI, and 0.8 M CaC,, 0.3 M ZnCI, and 0.7 M CaCl,, 0.4 M ZnCI, and 0.6 M CaCI., 0.5 M ZnCI, and 0.5 M CaCI,, 0.6 M ZnC., and 0.4 M CaC,, 0.7 M ZnCI, and 0.3 M CaCl,, 0.8 M ZnCI, and 0.2 M CaCI,, 0.9 M ZnCh1 and 0.1 M CaCI,.

and others. However, the presence of EDTA will inhibit the recovery process.

Moreover, the presence of ZnCI, and/or CaC, in Recovery Buffer-I will induce the formation of larger amounts of lugher molecular weight (or molecular mass) Neutrolinc-aipha nriuii wrs.

Example 3. Cloning and Expression of Neutrokme-alpha in Mammalian Cells COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 P52 00 1 A typical mammalian expression vector contains the promoter clement, which t mediates the miiataon of transcrpuon of mRNA, the proein coding sequence, and signals requred for the termnation of transenpton and polyadenylaion of the transcnpL Additional elements include enhancers. Kozak sequences and tervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient Stranscripuon can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses. RSV HTLVI, HIVI and the early promoter of the cytomegalovmrus (CMV). However, cellular elements can also be used C the human actn promoter). Suitable expression vectors for use in practicing the 00 o to present inventon include, for example, vectors such as pSVL and pMSG (Pharmacia, SUppsala, Sweden). pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC 12MI (ATCC 67109). Mammalian host cells that could be used include, human HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells. Cos 1, Cos 7 and CVI, quail QC1 3 cells, mouse L cells, Chinese hamster ovary (CHO) cells, and HEK 293 cells.

s Alternatively the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycmn allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of mterest.

Another useful selection marker is the enzyme glutarmne synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington ec al., Binfechnology 10-169-175 (1992)). Using these markers, the mammalian cells arc grown m selcctive medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

The expression vectors pCI and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen ct al., Molecular and Cellular Biology 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart er al., Cell 41:521 530 (1985)).

Multiple cloning sites, with the restriction enzyme cleavage sites Barn HI, Xba I COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 353 00 0 ,1 and Asp 718, facilitate the cloning of the gene of interest. The vectors contain m t addition the 3' mtron, the polyadenylaion and ternnnaton signal of the rat prepromisulin gene.

s Example Clonmg and Expression w COS Cells 4 The expression plasmid, pNeulrokin-alpha-HA, is made by cloning a portion of the deposited eDNA encoding the extracellular domain of the protein into the O expression vector pcDNAl/Amp or pcDNAIII (which can be obtained from Invitrogen, 00 Inc.). To produce a soluble, secreted form of the polypeptide, the extracellular domain to is fused to the secretory leader sequence of the human IL-6 gene.

The expression vector pcDNAl/amp contains: an E. coli ongm of replication effective for propagation m E. coli and other prokaryotic cells; an ampicillin resistance gene for selection of plasmsd-containmg prokaryotic cells: an origin of replicaton for propagation in eukaryouc cells; a CMV promoter a polylinker, an SV40 intron; several codons encoding a hemagglutinm fragment an "HA" tag to facilitate punficaton) followed by a termination codon and polyadenylanon signal arranged so that a eDNA can be conveniently placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites n the polylinker. The HA tag corresponds to an epitope derived from the influenza hemaggluun protein described by Wilson et al., Cell 37- 767 (1984). The fusion of the HA tag to the target protein allows easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope. pcDNAIll contains, in addition, the selectable neomycin marker.

A DNA fragment encoding the extracellular domain of the Netrokine-alpha polypeptide is cloned into the polylinker region of the vector so that recombinant orotein exoression is directed by the CMV oromoter. The vlasmid construction strategy is as follows. The Neutrokine-alpha cDNA of the deposited clone is amplified using primers that contain convenient restnction sites, much as described above for construcuon of vectors for express on of Neutrokine-aipha in E. col. Suitable primers include the following, which are used in this example. The primer, containing the COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DFAWSON WALDRON LAWYERS 4 062837999 NO.198 D54 00 Cl underlined Bar HI site, a Kozak sequence, an AUG stan codon, a sequence encoding c the sccretory leader pcptde from the human IL-6 gene, and 18 nucleotides of the coding region of the extracellular domain of Ncutroklne-alpha protein, has the O followmg sequence: 5'-GCG GGA CC GCC ACC ATG AAC TCC TTC TCC ACA s AGC C TTC GGT CCA GTT GCC TTC TCC CTG GGG CTM CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTI GTG AGA CAA GGG GAC CTG GCC AGC 3' (SEQ ID NO: 16). The 3' pruner, containing the underlined Barn HI Srestriction site and 18 of nuclcotdes complementary to the 3" coding sequence

O

00 :g immediately before the stop codon, has the following sequence: 5'-GTG GGATCC O 1i TTA CAG CAG TTT CAA TGC ACC 3' (SEQ ID NO: 17).

The PCR amplified DNA fragment and the vector, pcDNAl/Amp, are digested with Bam HI and then ligated. The ligatlon mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037). and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colones. Plasmid DNA is isolated from resistant colonies and examnued by restriction analysts or other means for the presence of the fragment encoding the Ncutrokine-alpha extracellular domain.

For expression of recombinant Neutrokine-alpha, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Clonng: a Laboratory Manual, Cold Spring Laboratory Press, Cold Sprng Harbor, New York (1989). Cells are incubated under conditions for expression of Neutrokme-alpha by the vector. Expression of the Neutroklne-alpha-HA fusion protein is detected by radiolabeling and immunopreciptation, using methods described in, for example Harlow et al.. Antibodies: A Laboratory Manual, 2nd Ed., Cold Spnng Harbor Laboraiory Press, Cold Spnng Harbor, New York (1988). To this end. two days after transfection, the cells are labeled by incubation m media containing "S-cysteme for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer- 150 mM NaCI, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM TRIS. pH 7.5, as described by Wilson et al. cited above.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 198 00 0 Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody The precipitated proteins then are analyzed by SSDS-PAGE and autoradiography An expression product of the expected size is seen mi the cell lysate, which is not seen in negative controls.

Example Clonmg and Expression m CHO Cells The vector pC4 is used for the expression of Neutrokne-alpha protein. Plasmad pC4 is a derivative of the plasnud pSV2-dhfr (ATCC Accession No. 37146). To C produce a soluble, secreted form of the Neutrokme-alpha polypeptide, the portion of Sgo the deposited cDNA encoding the extracellular domain is fused to the secretory leader Ssequence of the human IL-6 gene. The vector plasnud contans the mouse DHFR gene under control of the SV40 early promoter- Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfectcd wih these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM. Life Technologies) supplemented with the chemotherapeutc agent methotrexate. The amplification of the DHFR genes in cells resistant to methorexate (MTX) has been well documented (see, Alr. F Kellems. R. Beruno, J- and Schimke. R. 1978, J. Biol.

Chem. 253,1357-1370, Harmlin, J. L. and Ma, C. 1990, Blochem, et Biophys. Aca.

1097-107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9.64-68). Cells 32 grown m increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it as usually co-amplified and over-expressed. It is known in the art that this approach may be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently when the methotrexate is withdrawn, cell lines are obtained which contain the amplified gene integrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terrmnal repeat (LTR) of the Rouse Sarcoma Virus (Cullen. et al., Molecular and Cellular Biology, March 1985:438-447) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Bushan ct al., Cell 41:521 530 (1985)). Downstream of the promoter are the following single restriction COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS e 062837999 NO.198 956 00 l enzyme cleavage sites that allow the Integraton of the genes: BamHI, Xba I, and t Asp718. Behind these cloning sites the plasmid contains the 3 mtron and polyadenylauon site of the rat prepromsulin gene. Other high efficiency promoters can Salso be used for the expression, the human beta-actn promoter, the SV40 early or late promoters or the long termnal repeats from other retroviruses, HIV and HTLVI. Clontech s Tet-Off and Tet-On gene expression systems and similar systems can be used to express the Neutrokine-alpha in a regulated way in mammalian cells (Gossen. Bujard, H. 1992. Proc. NaIL Acad. Sca. USA 89' 5547-5551). For the polyadenylanon of the mRNA other signals, from the human growth hormone or O 10 globm genes can be used as well. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycan. It is advantageous to use more than one selectable marker in the beginning, G418 plus methotrexate.

The plasnud pC4 is digested with the restriction enzymes Ban HI and then iS dephosphorylated using calf intestinal phosphates by procedures known in the an. The vector is then isolated from a agarose gel.

The DNA sequence encoding the extracellular domain of the Neutrokine-alpha protein is amplified using PCR oligonucleoude primers corresponding to the 5' and 3' sequences of the gene. The 5' primer, containing the underlined Bar HI site, a Kozak sequence, an AUG star codon. a sequence encoding the secretory leader peptade from the human IL-6 gene, and 18 nuclcotdes of the 5' coding region of the extracellular domain of Neutrokine-alpha protein, has the followmg 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). The 3' pnmer containng the underlined Ban HI and 18 of nucleoudes complementary to the 3' coding sequence immediately before the stop codon, has the following sequence: GGA TCC TTA CAG CAG TTT CAA TGC ACC 3' (SEQ ID NO:17).

The amplified fragment is digested with the endonuclease Barn HI and then purified again on a 1% agaros gel. The isolated fragment and the dephosphorysatic vector are then ligated with T4 DNA ligase. E. coli HB 101 or XL I Blue cells are then COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 P57 00 o 32 0 N transformed and bacteria are identified that contain the fragment inserted into plasmid tpC4 using, for instance, restrcton enzyme analysts.

SChinese hamster ovary cells lacking an actve DHFR gene are used for transfection. Five pg of the expression plasmnd pC4 is cotransfected with 0.5 pg of the s plasrmd pSVneo using lipofecun (Feigner et al., supra). The plasmid pSV2.neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotcs including G418. The cells are seeded in alpha minus MEM supplemented with I mg/hl G418. After 2 days, the cells arc

O

C-I trypsmized and seeded in hybndoma cloning plates (Greanr, Germany) in alpha minus 00 01o MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus I mg/ml G418.

0 After about 10-14 days single clones are trypsmzed and then seeded m 6-well petn C dishes or 10 ml flasks using different concentrations of methotrexace (50 nM, 100 nM, 200 nM. 400 nM. 800 nM). Clones growing at the highest concentratons of methotrexate are then transferred to new 6-well plates containing even higher concentrations of imehotrexare (1 pM, 2 pM, 5 pM, 10 pM. 20 pM). The sane procedure is repeated until clones are obtained which grow at a concentration of 100-200 pM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

At least six Neutrokne-alpha expression constructs have been generated by the inventors herein to facilitate the production of Neutrokmne-alpha and/or Neutrolune-alphaSV polypepudes of several sizes and in several systems. The expression constructs are as follows: pNa.A71-L285 (expresses ammo acid residues Ala-71 through Leu-285), pNa.A81-L285 (expresse anuno acid residues Ala-81 through Leu-285), pNa.L 12-L285 (expresses amino acid residues Leu-112 through Leu-285), pNa.A 134-L285 (expresses amino acid residues Ala-134 through Leu-285), pNa.L147-L285 (expresses amino acid residues Lcu-147 through Leu-285). and pNa.G161-L285 (expresses anuno acid residues Gly-161 through Lean-2RA).

In preferred embodiments, the expression constructs are used to express various Neutrokme-alpha muteins from bacteal, baculoviral. and mammalian systems.

In certain additonal preferred embodiments, the constructs express a Neutrokme-alpha polypeptde fragment fused at the N- and/or C-terminus to a COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N.198 D58 00 0 533 C hetcrologous polypeptid, the signal pepude from human IL-6. the signal pepude t from CK-beta8 (amino acids 21 to 1 of the CK-beta8 sequence disclosed n published SPCT application PCT/US95/09058), or the human IgG Fe region. Other sequences 0could be used which are known to those of skill m the an.

Example 4: Tissue distribution of Neutrokme-alpha mRNA expression Northern blot analysis is camed out to examie Neutrokine-alpha gene expression in human tssues, using methods described by among others, Sambrook'; Sal., cited above. A cDNA probe contaimng the entire nucleotde sequence of the 00 10 Neutrokmne-alpha protein (SEQ ID NO-1) is labeled with "P using the redipnmerm o DNA labeling system (Amersham Life Science), according to manufacturer's Sinstrucuons. After labeling, the probe s punfied usig a CHROMA SPIN-100 column (Clontech Laboratores, Inc.), according to manufacturer's protocol number PT1200-1 The purified labeled probe is then used to examine various human tssues for Neutrokine-alpha and/or Neutrokme-alpha mRNA.

Multiple Tissue Northern (MTN) blots conaining various human tissues or human immune system tissues (IM) are obtained from Clontech and are examined with the labeled probe using ExpressHybTM hybrdization solution (Clontech) according to manufacturer's protocol number PT 190-1 Following hybridization and washing, the blots are mounted and exposed to film at 70° C overnght, and films developed according to standard procedures.

To determine the pattern of Neutrokine-alpha and/or Neutrokme-alpha expression a panel of multiple tissue Northern blots were probed. This revealed predominant expression of single 2.6 kb mRNA in peripheral blood leukocytes, spleen, 2s lymph node and bone marrow and detectable expression in placenta, heart, lung, fetal liver, thymus and pancreas. Analysis of a panel of cell lines demonstrated high expression of Neutrokne-alpha and/or Nutrokmne-alpha in HL60 cells. detectable expression an K562, but no expression m Raji, HeLa, or MOLT-4 cells. Overall at appcirS thm aiC euuounc-a ap anwtor ieuuroKine-alpna mRiNA expression is enncnea in the immune system.

Example 5. Gene Therapy Using Endogenous Neutrokine-atpha Gene COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 198 959 00 0 33Z7 c Another method of gene therapy according to the present invenuon involves c operably associating the endogenous Neutrokine-alpha sequence with a promoter via G homologous recombnmaon as described, for example, m U.S. Patent No. 5,641,670, issued June 24. 1997- International Publication No. WO 96/29411, published s September 26, 1996; International Publicaton No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra ct al., Nature 342:435-438 (1989). This method involves the activation of a gene which is present m the target cells, but which is not expressed in the cells, or is expressed at a 0 lower level than desired. Polynucleotide constructs are made which contain a 00 o1 promoter and targeung sequences, which are homologous to the 5' non-coding Ssequence of endogenous Neutrokme-alpha, flanking the promoter The targeting sequence will be sufficiently near the 5' end of Neutrokme-alpha so the promoter will be operably linked 1t the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR Preferably the amplified promoter contains distinct restrction enzyme stes on the 5' and 3' ends.

Preferably the 3' end of the first targeting sequence contains the same restriction enzyme sie as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restncuon site as the 3' end of the amplified promoter.

The amplified promoter and the amplified targetinmg sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatasc. The digested promoter and digested targeting sequences are added together an the presence ofT4 DNA ligase. The resulting mixture is maintained under conditions appropriae for ligation of the two fragments. The construct is size fractionated on an agarose gel then purified by phenol extraction and elhanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynuclcotide constructs may also be admi;stered with transfcction-facilitatng agents, such as liposoame, viral sequences, viral particles, precipiating agents, etc. Such methods of delivery are known m the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous Neutrokmne-alpha COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAUWSON WALDRON LAWYERS 4 062837999 N0.198 00 0 c sequence. This results i the expression of Neutroklnc-alpha in the cell. Expression may be detected by immunological staining, or any other method known m the art.

Fibroblasts are obtained from a subject by skin biopsy The resulting tissue is placed n DMEM 10% fetal calf serum. Exponentially growing or early stationary s phase fibroblasts am trypstuzed and nased from the plastic surface with nutnent medium. An aliquot of the cell suspension is removed for counting, and the remaining Scells are subjected to centrfugauon. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporaton buffer (20 mM HEPES pH 7.3, 137 mM NaCI, O mM ,KCI, 0.7 mM Na2 HP04. 6 mM dextrose). The cells are recentrnfuged, the 00 It supernatant aspirated, and the cells resuspcnded in clectroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The rmal cell suspensioon cotains c approximately 3X 10 cells/ml. Electroporaton should be performed imnediately i following rcsuspension.

Plasmid DNA is prepared according to standard techniques. For example, to Is construct a plasmid for targeting to the Neutrokmi-alpha locus, plasmid pUCl 8 (MBI Fermentas, Amherst. NY) is digested with HindllI. The CMV promoter is amplified by PCR with an Xbal sie on the 5' end and a BamHI site on the 3'end. Two Neutroklne-alpha non-coding sequences are amplified via PCR: one Neutrokinc-alpha non-coding sequence (Ncutroune-alpha fragment i) is amplified with a HindlI site at the 5' end and an Xba site at the 3'end; the other Neutrokine-alpha non-coding sequence (Neutrokne-alpha fragment 2) is amplified with a BamHI site at the 5'end and a Hind]I site at the 3'end. The CMV promoter and Neutrokmne-alpha fragments are digested with the appropnate enzymes (CMV promoter Xbal and BamHI; Neutrokine-alpha fragment 1 Xbal; Neutrokme-alpha fragment 2 BamHI) and ligated together. The resulting ligation product is digested with Hindll, and ligated with the HindIII-digested pUCIS plasmid.

Plasrmd DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio- Rad). The final DNA concenranon is generally at least 120 pg/ml. 0.5 ml of the cell suspension tconaiimang approx niazily .5.X 106 cells, is then added to th civcc, and the cell suspension and DNA solutions are gently muxed. Electroporation is performed with a Gene-Pulser apparatus (Bto-Rad). Capacitance and voltage are set at 960 pF and 250-300 V respectively As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 361 00 0 Cl genome increases dramatically Given these parameters, a pulse time of approximately t 14-20 mSec should be observed.

Electroporated cells are maintained at room temperaure for approximately r mm., and the contents of the cuvette are then gently removed with a sterile transfer s pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37"C. The followmg day the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

O The engineered fibroblasts are then injected into the host, either alone or after 00 to having been grown to confluence on cytodex 3 nucrocarrier beads. The fibroblasts o now produce the protein product. The fibroblasts can then be introduced into a patient N as described above.

Example 6. Neurokume-alpha, a Novel Member of the Tumor Necrosis IS Factor Ltgand Family that Functions as a B Lymphocyte Stimulator A 285 amino acid protein was identified in a human neutrophil/monocytederived cDNA library that shared significant homology within its predicted extracellular receptor-ligand binding domain to APRIL (Hahne, et al., J.Exp.Med. 188,1185-90 (1998)), TNF-alpha (Penmca, et al., Nature 312,724-729 (1984)) and LT-alpha (Gray Nature 312,721 724 (1984)) (Figure 7A). We have designated this cyokine Neutrokne-alpha (we have also designated this molecule as 8 Lyjmphocyte Stimulator (BLyS) based on its biological activiy). Hydrophobiclty analyses of the the Neutrokme-alpha protein sequence have revealed a potential transmembrane spanning domain between amino acid residues 47 2S and 73 which as preceded by non-hydrophobic ammno acids suggesting that Neutrokine-alpha. like other members of the TNF ligand family is a type II membrane bound protein (Cosman, D Szem.Cells. 12:440-55 (1994)). Expression of this cDNA m mammalian cells (HEK 293 and Chinese Hamster Ovary) and Sf9 insect cells latcnufiea a i52 anUno acmi suiubie iWlil with uil -L IIuliIal squcilc begirir ug with the alanine residue at ammno acid 134 (arrow m Figure 7A). Reconstruction of the mass to charge ratio defined a mass for Neutrokne-alpha of 17,038 Daltons. a value in consistent with thai predicted for this 152 armno acid protein with a single disulfide bond (17037.5 Daltons).

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062937999 N. 198 U62 00 o 339 SUsing human/hamster somatc cell hybrids and a radiaton-hybnd mapping panel, the gene encoding Neutrolune-alpha was found linked to marker SHGC 36171 which maps to human chromosome 13q34, a region not previously associated with any other member of the TNF superfamily of genes (Cosman, D. Stem.Cells. 12:440-55 s (1994)).

The expression profile of Neutrokne.alpha was assessed by Northem blot (Figure 7B) and flow cytomemc analyses (Table V and Figure Neutrokine-alpha is encoded by a single 2.6kb mRNA found at high levels in peripheral blood ]cukocytes, 0 spleen, lymph node and bone marrow Lower expression levels were detected in 00 10 placenta, heart, lung, fetal liver, thymus and pancreas. Among a panel of cell lines, Neutrokine-alpha mRNA was detected m HL-60 and KS62, but not in Raji, HeLa, or l MOLT-4 cells. These results were confirmed by flow cytometnc analyses using the Neutrokne-alpha-specific mAb 2E5. As shown in Table V Neutrokmne-alpha expression as not detected on T or B lineage cells but rather restricted to cells within is the myeloid origin. Further analyses of normal blood cell types demonstrated significant expression on resting monocyres that was upregulated approximately 4fold following exposure of cells to IFN-gamma (100 U/mL) for three days (Figure 8A). A concomitant increase in Neutrokme-alpha-spectfic imRNA was also detected (Figure 8B). By contrast, Neutrokine-alpha was not expressed on freshly isolated peripheral blood granulocytes. T cells, B cells, or NK cells.

Purified recombmant Neutrokne-alpha ("rNeutrokine-alpha") was assessed for its ability to induce activaton, proliferation, differentanon or death in numerous cell based assays involving B cells, T cells, monocytes, NK cells, hematopoietic progenitors, and a variety of cell types of endothelial and epithelial origin. Among these assays, Neutrokine-alpha was specifically found to increase B cell proliferation in a standard co-stmulatory assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized ant-human IgM as priming agents (Sieckmann. D et al, J.ExpMed.

:47.8tS-29t i978); Ruigucn, at .,Scuura..himunu 6. L 59-69 19 r1), shown in Figure 9A, recombinant Neutrokne-alpha induced a dose-dependent proliferation of tonsillar B cells. This response was similar to that of rlL2 over the dose range from 0. 1 to 10,000 ng/mL. Neutrokne-alpha also induces B cell proliferation when cultured with cells co-stimulated with immobilized anti-lgM COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D63 00 o 33 CN (Figure 9B). A dose-dependent response as readily observed as the amount of Scrosslinking agent increases in the presence of a fixed concentration of either IL2 or SrNeurokine-alpha.

SIn an attempt to correlate the specific biological activity on B cells with s receptor expression, punfied Neutrokine-alpha was biotnylated. The resultant bioun- Neutrokmne-alpha protein retained biological function m the standard B cell proliferation assays. Lineage-specific analyses of whole human peripheral blood cells indicated that binding of biotinylated Neutrokine-alpha was undetectable on T cells, Smonocytes, NK cells and granulocytes as assessed by CD3, CD 14, CD56, and CD66b Cl 00 10 respectively (Figure 10A). In contrast, biotnylated Neutrokine-alpha bound peripheral B cells. Receptor-expression was also detected on the B cell tumor lines REH, SCl ARH-77 Raji, Namalwa. RPMI 8226, and IM-9 but not any of the myeloid-derved lines tested including THP I HL-60, K 562, and U-937 Representative flow cytometnc profiles for the myeloma cell line IM-9 and the hstiocytac line U-937 are shown in Figure 10B. Similar results were also obtained using a biologically active FLAG-tagged Neutrokine-alpha protein instead of the chemically modified biotin- Neutrokine-alpha. Taken together, these results confirm that Neutrokine-alpha displays a clear B cell tropism in both its receptor distribution and biological activity It remains to be shown whether cellular activation may induce expression of Neutrokine-alpha receptors on peripheral blood cells, other normal cell types or established cell lines.

To examine the species specificity of Neutrokine-alpha, mouse splenic B cells were cultured in the presence of human Neutrokme-alpha and SAC. Results demonstrate that rNeutrokme-alpha induced m vtro proliferation of murne splenic B cells and bound to a cell surface receptor on these cells. Interestingly immature surface Ig negative B cell precursors isolated from mouse bone marrow did not proliferate m response to Neutrokine-alpha nor did they bind the ligand.

To assess the n vvo activity of rNeutroane-alpha. BALB/c mnce (3/group) were injected wice per day with buffer only or 0.08 mg/kg, 0.8 npgkg. 2 rg/kg or 8 mg/kg of rNeutrokine-alpha. Mice received this treatment for 4 consecutive days at which ume they were sacrificed and vanous tissues and serum collected for analyses. in an altemative embodiment, BALB/c mice may be injected twice per day with any amount of rNeutrokane-alpha in a range of 0.01 to 10 mg/kg. In a COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008&03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 D64 00 o c- preferred embodiment. BALBIc rmce are mjected twice per day with any amount of rNcutrokane-alpha in a range of 0.01 to 3 mg/kg (specific preferred exemplary Sdosages i this embodiment include, but are not limited to, 0.01 m/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 mng/kg. 1.n mg/kg. 1 mg/kg, 1.2 mg/kg. 13 me/kg. 1 4 mg/kg, mg/kg. 1.6 mg/kg 1.6 mg/kg. I 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 O mg/kg, and 3.0 mg/kg). In an additional preferred embodiment, BALB/c mice are 00 1 injected twice per day with any amount of rNeutrokme-alpha in a range of 0.02 to S2 mg/kg (specific preferred exemplary dosages in this embodiment include, but are not limited to. 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 mglkg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, is 1.4 mg/kg, 1-5 mg/kg, mg/kg, 1.6 mg/kg 1.7 mg/kg, 1.8 mg/kg. 1.9 mg/kg, and mg/kg).

Microscopically the effects ofNeutrokine-apha administration were clearly evdent in sections of spleen stained with rounne hematoxylin and eosin and immunohtstochemically with a mAb specific for CD45R(B220) (Figure 11 Normal splenmc architecture was altered by a dramatic expansion of the white pulp marginal zone and a distinct increase in cellularty of the red pulp (Figure I IA). Marginal zone expansion appeared to be the result of increased numbers of lymphocytes expressing the B cell marker CD45R(B220). In addition, the T cell dense penartenolar lymphoid sheath (PALS) areas were also ifiltrated by moderate numbers of CD45R(B220) positive cells. This suggests the white pulp changes were due to increased numbers of B cells. The densely packed cell population that frequently filled red pulps spaces did not stain with CD45R(B220). Additional experiments will be required to characterize all the cell types involved and further define the mechanism by which Neutrokme alpha alters splenic architecture.

Flow cytometnc analyses of the spleens from mice treated with 2 ng/kg Neutrokine-alpha-treated indicated that Neutrokne-alpha increased the proportion of mature (CD45R(B220)" ThBh") B cells approximately 10-fold over that observed in control mice (Figure 11B). Further analyses performed in which mice were treated COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 00 0 3 o c with buffer. 0.08 mg/kg. 0.8 mg/kg, 2 mg/kg, or 8 mg/kg Neutrolkne-alpha indicated t that 0.08 mg/kg. 0.8 mg/kg, and 2 mg/kg each increased the proporton of mature S(CD45R(B220)" ThB"* B cells approximately 10-fold over that observed in control mice, whereas buffer and 8 mg/kg produced approximately equal proportions S of mature B cells. See, Table IV Table IV FACS Analyss of Mouse Spleen B cell Population.

Neutrokne-alpha O to a10 Mature B Cells (R2) CD4R-posnve (RI) SControl (buffer) 1.26 52.17 S0.08 mg/kg 16.15 56.53 C 0.8 mg/kg 18.54 57.56 (C 15 2 mg/kg 16.54 57.55 8 g/kg 1.24 6142 A potential consequence of increased mature B cell representation m vo is a relative increase in serum Ig titers. Accordingly serum IgA, IgG and IgM levels were compared between buffer and Neutrokmne-alpha-treated mice (Figure 1 IC).

Neutrokme-alpha administration resulted in a 2 and 5-fold increase m IgA and IgM serum levels respectively Interestingly circulating levels of IgG did not increase.

Moreover, a dose-dependent response was observed in serum IgA titers m mice treated with various amounts of Neutrokine-apha over a period of four days, whereas no apparent dose-dependancy was observed by administration of the same amounts of Neutrokine-alpha over a perod of two days. In the case of admmistrauon over four days, administration of 8, 2,0.8. 0.08, and 0 mg/kg Neutrokne-alpha resulted in serum IgA tlters of approximately 800 micrograms/ml, 700 micrograms/ml, 400 mrcrograms/ml, 200 micrograms/ml and 200 micrograms/ml. That is. administraton of 8, 2, 0.8, and 0.08 mg/kg Neutrokme-alpha over four days resulted m approximately 4-fold, 3.75-fold, 2-fold, and minimal-fold, respectively increases in IgA serum levels over background or basal levels observed by adrmnistration of buffer only In an alternative embodiment, these experiments may be performed with any amount of rNeutrokine-alpha in a range of 0.01 to 10 mg/kg. In a preferred embodiment.

Neutrokine-alpha is administered in a range of O.O0 to 3 mg/kg (specific preferred exemplary dosages in this embodiment include, but are not limited to, 0.01 mg/kg, COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAW~SON WJALDRON LAWJYERS 4 0929237999 mO.198 P66 00 (N 0.02 mg/kg. 0.03 mg/kg, 0.04 mng/kg. 0.05 mg/kg. 0.06 mg/kgS. 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 mglikg, 0.6 .mg/kg, 0.7 mg/kg, 0.8 mg/kg. 0.9 mg/kg. 1.0mg/kg, I1.1 mg/kg. 112mg&kg. 1.3mg/kg.

14 mg/kg. 1.5 mg/kg. 1.6 mg/kg, 1.6 mg/kg, 17 mg/kg. 1.8 mg/kg, 1.9 mg/kg. 2.1 mg/kg, 2.2 mg/kg. 2.3 mng/kg. 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/k, 2.8 mg/k, 2.9 mig/kg, amd 3.0 mg/g). In an additional preferred embodiment, Neutrokine-alpha is adnrunisterd in a range of 0.0210o2 mg/kg (specific preferred exemplary dosages in tins embodimtent include, but are not limited to, 0.02 mg/kg, 0 0.03 mg/kg. 0.04 mg/kg, 0.05 mg/kg, 0.06 mng/kg, 0.07 mg/kg, 0.08 mg/kg 0.09 00 lo mg/kg. 0. 1 mg/kg, 0.2 mg/kg, 0.3 mg/kg 0.4 mng/kg. 0.5 mg/kg. 0.6 mg/kg. 0.7 mg/kg, o0.8 mg/kg,.0.9mrrg/kg, 1.0 mg/g, I11mg/kg, 1.2 mg/kg, 1.3 mg/kg, 14mg/kg.I mg/kg. 1.6 mg/kg. 1.6 mg/kg.!t 7 mgg 1.8 mg/kg, 1.9 mg/kg, and 2.0 mg/kg).

The dam presented hereto define Neutrokane-alpha, as a novel member of the TNP-ligand superfamily that induces both in vivo and ini varo B cell proliferation and IN differentiation. Ncwtrokmne-alpba. L% distimguished from other B cell growth and differentiation factors such as 1L2 (M~etzger. D) W. et at. Res.limmunot 146:499-505 (1995)), ILA4 (Axnuage. LIJ., eral., Adv. Exp.Med.Swt. 292:121 30 (199 Yokota, T., r nat.. Proc.NOI-Acad.ScL U.SA 83:5894-98 (1986)). [U5 (Takatsu, ca., Proc. N&IAcad.Sci. US.A 4:4234-38 (1987); Bertol i et atI, Eur.J.Iminwot 23:398-402- (1993)), 1L6 (Poupart, etaL, EMBO J 6:1219-24 (1987); Hirano, ea at. Nature 324:73-76 (1986)) 1L7 (Goodwin, at al.. Proc. Nat Acad. Set. U.S.A.

86:302-06 C1989)- Namnen, eral., Nature 333:571 73 (198R)), 1L13 (Punnonen.

I1.. et at., Allergy. 49:576-86 (1994)), IL15 (Armartage, Iii., cr al., J.lrnmunat. 154-.493- (1995)), CD4OL (Armihtage. RI., et at., Nature 357.80-82 (1992); Van Kooxen, C, 23 and Banchereau, J1. lnrArch.Allergy.Immunol. 113.393-99 (1997)) or CD27L (CD7O) (Oshima, ci at., hn.mmwnoL 10:5 17-26 (1998); Lens, ei at., Semnlniwot 10:491-99 (1998)) by uts monocyte-specafic gene/protein expression pattern and its specifi receptor distribution and biological activity on B lymphocytes. Taken logeiatt thosa data gpst Eh 6 1 Nlab 11 rk 1 *e-alpha is Iikely ,nvolvcd in the exchnge of 3o signals between B cells and monocyzes or their differentiated progeny Although all B cells may utilize this mode of signaling, te restricted expression patterns and Igsecretion suggest a role for Neuurokmnc-alpha irn the acivation of CD5 t or "unconventional" A3 cell responses. These B cells provide a critical component to the COMS ID No: ARCS-i 83628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 N. 198 367 00 rc innate immune system and provide protection from environmental pathogens through Stheir secretion of polyreactive IgM and IgA antibodies (Pennell, et aL.

t Eur.J.lmmunl. 19 1289-95 (1989); Hayakawa, el al.. Proc.NatAcad.ScjU.S.A.

81.2494-98 (1984)). Alternatively Neutrokme-alpha may function as a regulator ofT s cell independent responses in a manner analogous to that of CD40 and CD40L in T cell dependent antgen actvation (van den Eertwegh, AJ., e al., J.Exp.Med.

178:1555-65 (1993); Grabstemn, KH., atl., Jlmmunl. 150:3141-47 (1993)). As such, Neutrokine-alpha. as receptor or related antagonists have utility in the treatment 0 of B cell disorders associated with autoimmuny neoplasia and/or immunodeficient 00 10 syndromes.

SMethods SMice. BALB/cAnNCR (6-8 weeks) were purchased from Charles River Laboratones, Inc. and maintained according to recommended standards (National Research Council, Guide for the care and se of laboratory animals (1999)) in is mnucmosolator cages with recycled paper bedding (Harlan Sprague Dawley Inc., Indianapolis, IN) and provided with pelleted rodent diet (Harlan Sprague Dawley Inc) and bottled drinking water on an ad libitum basis. The animal protocols used in this study were reviewed and approved by the HGS Institutonal Animal Care and Use Committee.

Isolation of full length Neutrokme-alpha eDNA. The BLAST algorithm was used to search the Human Genome Sciences Inc. expressed sequence tag (EST) database for sequences with homology to the receptor-binding domain of the TNP family A full length Neutrokine-alpha clone was identified, sequenced and submitted to GenBank (Accession number AF132600). The Neutrokme-alpha open reading frame was PCR amplified utilizing a 5' primer (5'-CAG ACT GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3') annealing at the predicted start codon and a 3' primer (5'-CAG ACT GGT ACC GTC CTG CGT GCA CTA CAT GGC 3') designed to anneal at the predicted downstream stop codon. The resulting amplicon was tailed wsth Ban HI and 4-p restction sites; ad subc oned :a 3mammralian expression vector Neutrokne-alpha was also expressed in p-CMV I (Sigma Chemicals).

Purification of recombinant human Neutrokme-alpha. The full length cDNA encoding Neutrokne-alpha was subcloned into the baculovirus expression COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRDN LAWYERS 4 062837999 NO.198 G6E 00 c- vector pA2 and transfected Into 59 msect cells (Patel er aL. J.Exp.Med.

185:1163-72 (1997)). Recomnbmant Neutokine-alpha was purified from cell supcmarans at 92 h post-tftection using a combination of anion-exchange, size exclusion, and hydrophobie interaction columns. The purified protein was formulated S in a buffer containmmg 0.15 M NaCI, 50 mM NaOAc at pH 6, sterile filtered and stored at 4'C until needed. Both SDS-PAGE and RP-HPLC analyses ndicate that SrNutrokme-alpha is greater than 95% pure. Endotoxin levels were below the detection limit mn the LAL assay (Associates of Cape Cod. Falmnouth, MA). The final purfied Neutrokine-alpha proletn has an N-termnuns sequence of Ala-Val-Gin-Gly- OO 10 Pro. This corresponds identically to the sequence of soluble Neutrokine-alpha derived 0 from CHO0 cell lines stably rtransfected with the full length Neutroklune-alpha gene.

S~Monoclonal antibody generation. BALBcAnNCR mnuce were immunized with 50 rmicrograms of HisTag-Neutrokine-alpha suspended in complete Frund's adjuvant followed by 2 challenges in meincomplete Freund's adjuvant. Hybndomas and monoclonal antibodies were prepared as described (Gefter, er aL. Samanc.Cell Gene:. 3.231 36 (1977): Akerstrom, Et al., J.lmmunol. 135:2589-92 (1985)).

Cell lines. All human cell lines were purchased fromn ATCC (Amenrican Type Culture Collection, Manassas, VA).

FACS analyst Neutrolkne-alpha expression was assessed on human cell lines, freshly isolated normal penpheral blood nucleated cells, and in vLtro cultured monocytes, a mouse anti-human Neutrokine-alpha mAb 2E5 (IgGl1) followed by PEconjugated F(ab)2 goat antibody to mouse IgG (CALTAG Laboratonries. Burlingamet, CA). Cells were analyzed usming a FACSan (Becton Dickinson Immunocytometry Systems. San Jose, CA) with propidium iodide to exclude dead cells. Neutrokne alpha binding was assessed using rNeutroklone-alpha botinylated with a Nhydroxysuccnunidobsoin reagent (Pierce, Rockford, 1L) followed by PE-conjugated streptavidin (Dako Corp, Glostrup, Denmark).

Chromosomal mapping. To determine the chromosomal location of the Neurokine-alpha gene, a panel of monochromosomal somatic cell hybrids tQuantum Biotechnology Canada) retaining individual chromosomes was screened by PCR using Neutrokine-alpha specific primers 5' pnmer 5' TGG TGT CIT TCT ACC AGG TGG-3' and 3' primer 5' TT CTT CIG GAC CCT GAA CGG-3'). The predicted 233 bp PCR product was only detected in human chromosome 13 hybnds.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2001 16:39 BLAKE DPASON WAJLDRDN LAWJYERS 4> 062937999 N0. 1993 P69 00 1 c-I Using a panel of R3 radiation hybrids (Research Genetics, St. Louis, MO) and the Stanford Human Genome Center Database.

(htzp:Ilwww.shgc.stanford.edu.RIHrbserver). Neutrolune-alpha was found l inked to the SHGC 36171 marker on chromosome 13, Superposition of this map with the s cytogencuc moap, of human chromosome 13 allowed the assignment of human Neutrokmec-alpha to chmoiosoinal band 13q34, B lymphocyte proliferation eay. Human tonsillar B cells were purifled by magnetic bead (MACS) depletion of CD3-posiuivc cells. The resgulting cell population o was routinely greater than 95% B cells as assessed by expresion of 019 and CD2O.

00 1o Various dilutions or human rNcutrobne-alphba or the control protein recombinant o human 1L2 were placed into individual wells of a 96-well plate to which was added I C? ClU cells suspended mn culture medium (RPM! 1640 containing 10% PBIS, 5 X I10 5

M

2ME. 100U/mnl penicillin, 100 nrucrogram/mI streptomycin, and IOr'dilution of Pansorbmn (SAC) or anti-lgM) in a total volume of 150 icroliters. Proliferation was 1s quantitated by a 20h pulse (I microCiweDl) of '11-rhymuidine (6.7 Cl/mM) beginning 72h post factor addition.

Histological analyses. Spleen were fixed in 10% neutral buffered forinalin, embedded in paraffin, secilionted at 5 micrometer,, mounted on glass slides and stained with hematoxylin and cosin or by enzyme-labeled indirect method 2o ziinunohiswocherwsrry for CD4SR(B220) (Hilbeit, et Eur.J.lmmunoL.

23:2412 18 (1993)).

Table V Nensrolane-alpha cell surface uxpnession 25 Neutrokmne-alpha cell Cell line Cellular Morpholog surface expression Monocytic lineage U-937 Lymphomna, histaocyuic/macrolphuge Leukemia, acurcprcwnyelocytic K 562 Leukemia, chronlcmyelogenous THP I Leukemia, acutemonocynec T-lineage COMS ID No: ARCS-i 83628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO.198 o Jurkat Leukemia. T lymphocyti SUP T13 Leukemia, T lymphoblastic MOLT-4 Leukerna. Tlymphoblastc 0 B-lineage Daudi Burktt's, lymphoblastic Namalwa Burktt's, lymphocyte to Raji Burlutt's, lymphocyte Reh Leukemna, lymphocytc 'ARH-77 Lukerma, plasma cell O IM9 Mycloma RPMI 8226 Myeloma 00 IS 0 Example 7 Assays to detec stimulation or inhibition ofB cell proliferation and 1 qdifferentiadon i Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their mncroenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stmulus that instructs the cell to arrest its current developmental pathway To date. numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL 2. IL-4, ILS. L6, IL 7 IL 13. IL14 and IL15. Interestmngly these signals are by themselves weak effectors but can, in combination with various co-suimulatory proteins, induce activation, proliferation, differentiation, honnng, tolerance and death among B cell populations. One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily Within this family CD40. CD27 and CD30 along wnth their respective ligands CDI54, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detecuon and/or observation of the proliferation and differentiauon of these B-cell populations and their precursors are valuable tools in determing the effects vanous proteins may have on these B-cell populations in terms of proliferation and differentiaton. Listed below are two assays designed to allow for the detection of the differentiaton, proliferation, or inhibition of B-cell populations and their precursors.

In Vitro assay- Punned Neutrokne-alpha and/or Neutrokine-alphaSV protein, or truncated forms thereof, is assessed for its ability to induce activation, proliferation.

COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/2008 16:39 BLAKE DAWSON WALDRON LAWYERS 4 062837999 NO. 199 71 00 C1 differentation or ihibition and/or death m B-cell populations and their precursors. The c activity of Neutrokme-alpha and/or Neutroknc-alphaSV protein on purfied human Stonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, SIs assessed m a standard B-lymphocyte co-sumulaton assay in which purfied tonsillar S B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I fSAC) or immobilized anti-human IeM antibody as the primin aent. Second signals such as IL 2 and IL 15 synergaze with SAC and IgM crosslinkmng to elicit B cell proliferation as measured by tnuated-thymdine incorporatton. Novel synergizmg 0 agents can be readily identified using thus assay The assay involves isolating human 00 to tonsillar B cells by magnetic bead (MACS) depletion of CD3-posmve cells. The Sresulting cell population is greater than 95% B cells as assessed by expression of C CD45R(B220). Various dilutions of each sample are placed nto individual wells of a 96.well plate to which are added 10 s B-cells suspended n culture medium (RPMI 1640 containing 10% FBS, 5 X 10'M 2ME, 100U/ml penicillin, 10 ug/ml streptomycin, and 10' 5 dilution of SAC) in a total volume of 150ul, Proliferation or ihibition is quantitated by a 20h pulse (luCi/well) with "H-thymidine (6.7 Ci/mM) beginning 72h post factor addition. The positive and negative controls are IL2 and medium respectively Agonists (including Neutrokne-alpha and/or Neutrokme-alphaSV polypeptide fragments) demonstrate an increased B cell proliferation when compared to that observed when the same number of B cells is contacted with the same concentration of pnnung agent. Antagonists according to the invention exhibit a decreased B cell proliferation when compared to controls containing the same number of B cells, the C same concentration of prmnng agent, and the same concentration of a soluble form of Neutrokme-alpha that elicits an increase in B cell proliferative actvity 71 285, 81-285, 112 285 or 134-285 of the Neutrokmne-alpha polypeptide shown m SEQ ID NO:2) in the absence the antagonist.

In Vivo assay- BALBc rmce are injected twice per day with buffer only or 2 mng/Kg or reurrorne-aspna anwaor reutroKlne-apnaSV proticu, ur truntalrcu forms thereof. Mice receive tins treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal and Neutrokine-alpha and/or Neutrokine-alphaSV protcenteated spleens identify the results of the actviny of Neutrolune-alpha and/or COMS ID No: ARCS-183628 Received by IP Australia: Time 17:02 Date 2008-03-19 19/03/200e 19/03/009 1:37 61 2 9259G999 4 062837999 N.2 0 NO. 72? Poll FAX TRANSMISSION No of pages (including this sheet) lo Level 35, Grosvenor Place 225 George street Sydney NSW 2000 Australia n olake Dawson PATENT ATTORNEYS To The Commissioner of Patents P Australia F 02 6283 7999 H-uman Genome Sciences, Inc New Australian divisional patent application Title! Neutrokine-aipha and neutroklne-nlpha splice variant PART 6 OF 6 T 61 2925a88000 IF 6 12 92588999 OX M55 Sydney Looked Bag No 8 Grosvenor Place Sydney NSW 2000 Australia www.blakedawson.coni 19 March 2008 Our reference 000 SJ 02 1430 4452 Partner David Clark T7612 9259 699 davidolark *blakedawson.com Please check that you have received this document in full. If not. please telephone the sender or call 61 2 9256 wool.

Confidentiality This documnent Is confidential and may contain legally privileged inforniadon. if you are not a named or authorisad recipientd you must not read, copy, distribute or act in reliance on it, If you have eceived this document in error, please telephone our operator immedlaeL-y onl 21 a8258 8000 and return the document by mall.

Sydney Melbourne Brisbane Canberra 203937378.1 1 COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 925986999 4 062837999 NO.727 P02 00 o 34i 7 Neutrokme-alphaSV protein on spleen cells, such as the diffusion of pen-arteral lymphatic sheaths, andlor significant mereases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochenucal studies using a B cell marker, antis CD45R(B220), are used to determine whether any physiological changes to splenc cells. such as snleme disorgatuzaton. are due to increased B-ccil roresentauon within loosely defined B-eell zones that infiltrate established T-cell regions.

Flow cytometreic analyses of the spleens from Neutrokme-alpha and/or 0 Neutroklone-alphaSV proten-treated rmce as used to indicate whether Neutroktme-alpha to and/or Neutrokme-alphaSV protein specifically increases the proportion of ThB+ O CD45R(8220)dull B cells over that which as observed an control mace.

Likewe, a predicted consequence of increased mature B-cell representation in ivo is a relative increase in serum Ig tters. Accordingly serum IgM and IgA levels are compared between buffer and Neutrokme-alpha andlor Neutrokme-alphaSV Is proten-treated mice Example 8: Effect of Neutrokmne-alpha and rs agonsts in treating graftversur-host disease asscrwated lymphoid atrophy and hypoplasa in ace An analysis of the use of Neutrokincm-alpha to treat, prevent. and/or diagnose graft-versus-host disease (GVHD)-associated lymphold hypoplasisalatrophy is performed through the use of a CS7BL/6 parent into (BALB/c X C57BIJ6) Fi (CBFl) mouse model. This parent into F1 mouse model as a well-charactnzed and reproducible anunmal model of GVHD an bone marrow transplant panents, wich is well know to one of ordinary skill in the art (see, Gleichemann, e al., bnmunoL Today 5:324. 1984). Soluble Neutrokne-alpha is expected to minduced the proliferation and differentiation of B lymphocyte, and correct the lymphold hypoplasia and atrophy observed in this animal model of GVHD (Piguet, ct al. J Exp. Med. 166:1280 (1987); Hation, e al., Blood 90:42 (1997)).

initiation ot the GVHD condition is mIoucco Dy me inmtavenous injeeton oi approximately 1 5 x 11Y spleen cells from C57BL/6 mice into (BALB/c X CS7BL/6) Fi muce (both are available from Jacksnn La, Bar Harbor Maine. Groups of6 to 8 mance receive daily either 0.1 to 5.0 mg/kg of Neutrokine-alpha or buffer control mntrapentoneally intrarascullarly or Intraderrmally starting from the days when COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 N0.727 D03 00 o 7 0 c lymphoid hypoplasia and atrophy are mild (-day moderate (-day 12) or severe t (-day 20) following the parental cell injection. The effect of Neutroklne-alpha on lymphoid hypoplasa and atrophy of spleen is analyzed by FACS and histopathology at C muluplc time points between day 10-30. Briefly splenocytes are prepared from s normal CBFI, GVHD or Neutrokine-alpha-treated mice, and stained with fluorescem phycoerythnn-conjugated ants- H-2Kb, biotn-conjugated anti- H-2Kd, and FITC conjugated antnCD4, anu-CDS, or anti-B220, followed by a CyChrome-conjugated Savidin. All of these conjugated antibodies can be purchased from PharMingen (San o Diego, CA). Cells are then analysis on a FACScan (Becton Dickmson, San Jose. CA).

00 10 Recipient and donor lymphocytes are identified as H-2Kb+ Kd+ and H-2Kb+ Kd- 0 cells, respectively Cell numbers of CD4+T CD8+ T and B220+ B cells of recipient or donor orgin are calculated from the total numbers of splenocytes recovered and the percentages of each subpopulation are determined by the three color analysis.

Histological evaluation of the relative degree of tssue damage in other GVHDis associated organs (liver, skin and intestine) may be conducted after sacrificing the animals.

Finally Neutrokine-alpha and buffer-treated animals undergo a clinical evaluation every other day to assess cachexia, body weight and lethality Neutrokme-alpha agotmss and antagonists may also be examed in this acute GVHD murme model.

Example 9. Isolation of antibody fragments directed agastst Netrokane-alpha polyp eptides from a library of scFvs.

Naturally occurnng V-genes isolated from human PBLs are constructed into a large library of antibody fragments which contain ractivties against Neutrokinealpha and/or Neutrokine-alphaSV to which the donor may or may not have been exposed (see U.S. Patent 5,885,793 incorporated heren in its entirety by reference), Resce of the library A library of scFvs is constructed from the RNA of human PBLs as described in W09201047 (which is hereby incorporated by reference in its entirety). To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2x TY containmg 1% glucose and 100 COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 is /03/2009 16:3? 61 2 92596999 4 062937999 NO. 727 U04 wcroms/m ofampjiiln. (ZxTY-AMP-GLU) =nd grpwn to an O.1) of 0.81 with shaking. Five -ml of this cultur Is used to inoculat S50 of QxTY -AMP-GL.U 2 x TU. of delta gene 3 heLper (4 13delta gene HI, -see W092/01047) are added *and the culture mincued at.-37'C fr 45:minutes without shaIng and- tn at 3V*C for s nunuics. with. shaking. Ther eure is. cetrifuged at 4000-r.p~m -for 10 nun-; and the pelk: -rcsui~en4ed n2 litersof 2x TV containing tOOimcroigram/mi amPteillin and' nucrograms/ni kannnyomi and. grown, overnight. Phage amw prepared as described inW09Mf047* MN13 delta gene Ifl is prepared as foJ)iijis: MI 3 delta gene IUT helper phage meuis !azir vivditof biningto:3mten. nfiaMl3 deCt6 gaec M.

paftOwt;r umadeby growp h hlm pbagz~jweells harbonnt a 1C9' dirtvatOiv spplying the il W W& tyek TI protenn durn phagernrhngenesis.

Theeuhwn i infcubated for 1 hour at 31T-without shaking; and tn for a- further i~hour at 37*C wit shaking. Cells Were spu down (IEC-Cega &.4000 revstrm for mrlink; resuspended ini 300 m) 2xTY. broth contaizuing 100.tmierograins ampzcillini and 25 mcrogrims kanarnycmtlml (Uz TY AMP-KCAN) and grown overnight, 4$alarg at 37'C. Phig purtic1ks are purified and concentrated fromn the cu -V mdu wqPttptuu-in (etoo ciij a 90Y Mresvende In 0 2nfl PBS idpsc thirough a 045rmicromecterf Ite (Min,'art N4ML, Sartonuis) to; give A fina conccnwraulon of approximately 10ll transducing units/nd (amicilinbunin~tbes (Nuac) arm coatead ovcnntut n PBS with. 4 mit of eitr' 100 microgramm or 10a mwnrdgtamnslml of a polypepntde. of the present ,nventiohi.

Tuhes are blocked wiOth 2%1'4arvel-PBS for.2-hoiurs at 3*C- and then washed 3 tims in aMS. Approxiiatgly 1-0 TU of' phage is applied to thre tube and incubated for 30 iniuce -at. room temperture tiblingon an over and under. turntable andI Tween-20 and 10 tznm with PB8S. Pliage awe eluted by adding I rut of 100 ruM trethylamnine and rowaing 15 minutes on an under and over turntable after which the solution is immnediately neutrraittd with 0.5 ml of ILOM.Tns-HCl. pH 7 4. Phage are then used to Infect 10 ml! of mid-log S. coli TOJI by incubating eluted phiage with *9) COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 N0.727 085 00 CN bactena for 30 mnutes at 37'C. The E. coli are then plated on TYE plates t containig 1% glucose and 100 merogranmsml ampicillin. The resulting bactenal Slibrary is then rescued with delta gene 3 helper phage as described above to prepare 0 phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity punfication with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders.

T Eluted phage from the third and fourth rounds of selection are used to infect E.

0 coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies 00 o1 for assay ELISAs are performed with microtler plates coated with either o ptcograms/ml of the polypeptide of the present invention in 50 mM bicarbonate pH C' 9.6. Clones positive in ELISA are further characterized by PCR fingerpnntmg (see W092/01047) and then by sequencung.

s1 Example 10. Neutralization of Neutroklne-apha/Neutrokae-alpha Receptor Ineraction with an and-Neutrokne-alpha Monoclonal Antody.

Monoclonal antibodies were generated against Neutrokine-alpha protein according to the following method. Bnefly mice were given a subcutaneous injection (front part of the dorsum) of 50 micrograms of His-tagged Neutrokmtealpha protein produced by the method of Example 2 in 100 microliters of PBS emulsified in 100 microliters of complete Freunds adjuvant. Three additional subcutaneous injectons of 25 mncrograms of Neurokine-alpha m incomplete Freunds adjuvant were given at 2-week intervals. The animals were rested for a C mounth before they received the final mtrapenioneal boost of 25 micrograms of 2s Neutrokne-alpha in PBS. Four days later mice were sacnficed and splenocytes taken for fusion.

The process of "Fusion was accomplished by fusing splenocytes from one spleen were with 2x10E7 P3X63Ag8.653 plasmacytoma cells using PEG 1500 (Boehnnger Mannheim), according to the ranufacturc,~ mdifications ui an ctalie described method. (See, Gefter, M.L. er at Somatic Cell Genet 3.231 36 (1977); Boehnnger Mannhcem, PEG 1500 (Cat.No. 783641), product descrption.) After fusion, the cells were resuspended in 400 ml of HAT medium supplemented with 20% FBS and 4% Hybndoma Supplement (Boehnnger COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 D06 00 8D 551 C Mannheim) and distributed to 96 well plates at a density of 200 microliters per well.

c At day 7 post-fusion, 100 mncroliters of medium was aspirated and replaced with 100 nucroliters of fresh medium. At day 14 post-fusion. the hybndomas were Sscreened for antibody production.

S Hybndoma supematants were screened by ELISA for binding to Neutroknealpha protein mmobilized on plates. Plates were coated with Neutrokmne-alpha by overnght incubation of 100 nucroliters per well of Neutrokmc-alpha in PBS at a concentration of 2 mncrograms per ml. Hybridoma supematants were diluted O with PBS were placed in individual wells of Neutrokmin-alpha-coated plates and 00 o0 incubated overnight al 4C. On the following day the plates were washed 3 times Swith PBS containing 0.1 Tween-20 and developed using the anti-mouse IgG ABC cl system (Vector Laboratories). The color development reaction was stopped with the addition of 25 ml/well of 2M HLSO 4 The plates were then read at 450 nm.

Hybridoma supernatants were checked for Ig isotype using Isostrips.

Cloning was done by the method of limiting dilutions on HT medium. About 3xl0E6 cells in 0.9 ml of HBSS were injected m pristane-pnmed mnce. After 7-9 days, ascinc flud was collected using a 19 g needle. All antibodies were punfied by protem G affinity chromatography using the Acta FPLC system (Pharmacia).

After primary and two consecutive subcutaneous injections, all three mice developed a strong Immune response; the serum tiler was 10E-7 as assessed by EUSA on Neutrokine-alpha-coated plates.

In one expernment, using the splenocytes from the positive mouse more than 1000 primary hybridomas were generated. 917 of them were screened for producing anti-Neutrokine-alpha antibody Screening was performed using I I diluted supernatants in order to detect all positive clones. Of 917 hybndomas screened, 76 were found to be positive and 17 of those were found to be IgG producers. After affinity testing and cloning, 9 of them were chosen for further expansion and punfication.

All punfied monoclonal antibudicb were absai wu uin different tornms o Neutrokine-alpha (including His-tagged and protein produced from a baculoviral system (sec Example in both Western blot analysis and ELISA. Six of nine clones were also able to bind Neutrokme-alpha on the surface of THP 1 cells.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 ?07 00 Cl However, none of the antibodies tested were able to capture Neutrokine-alpha from solution.

G High affinity antt-Neutrolune-alpha monoclonal antibodies were generated that recognize Neutrokine-alpha expressed on the cell surface but not i solution can be used for neutralization studies in vivo and in monocyte and B cell assays in vitro.

These antibodies are also useful for sensitive detection of Neutrokine-alpha on Western blots.

In an independent experiment, using the splnocytes from the positive O mouse, more than 1000 primary hybndomas were generated. 729 of the primary 00 o1 hybridomas were then screned for the production of an anu-Neutrokme-alpha Santibody Screening was performed under stngent conditons using 1.10 diluted "c suprnatants in order to pick up only clones of higher affinity Of 729 hybndomas screened. 23 were positive, including 16 IgM and 7 IgG producers (among the latter.

4 gave a strong IgM background). In this expenment, the isotype distribution of IgG is antibodies was biased towards the IgG2 subclasses. Three of seven IgG hybndomas produced antibodies of IgG2a subclass and two produced an antibody of IgG2b subclass, while the remaining two were IgGI producers.

Supernatants from all positive hybndomas generated in the second experiment were tested for the ability to inhibit Neutrokine-alpha-medialed proliferation of B cells. In the first screening experiment, two hybrtdomas producing IgG-neutralizing antibodies were detected (these are antibodies 16C9 and 12C5). In additional experiments, the IgG-neutralizmg activity of the hybndomas 16C9 and 12C5) were confirmed and two additional strongly neutralizing supernatants from hybridomas 15C10 and 4A6 were indentified.

Three clones were subsequently expanded a vio (a single clone, 15C10, was also expanded in a hollow fiber system), and the antibody purified by affinity chromatography All three of the clones were able to bind Neutrokmne-alpha on the surface of THP 1 cells and were also able to bind capture") Neutrokine-alpha from solut.on.

Specifically experiments were performed using the anti-Ncutrokine-alpha monoclonal antibodies described in the second experiment above to determine whether the antibodies neutralize Neurokine-alpha/Neutrokine-alpha Receptor binding. Bnefly Ncutrokine-alpha protein was biotinylated using the EZ-link T COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 D08 00 355 0 0 NHS-biotin reagent (Pierce, Rockford, IL). Biounylated Neutrokme-alpha was then used to idcntify cell surface protems that bind Neutrokne-alpha. Prelinunary Sexperiments demonstrated tha Neutrokme-alpha binds to a receptor on B lymphoid Scells.

s The inclusion of anu-Ncutrokme-alpha antibodies generated m the second expenment described above neutralized binding of Neutrokme-alpha to a Neutrokmie-alpha receptor. In a specific embodiment, ant-Neutrokme-alpha antibody 15C10 neutralizes binding of Neutrokine-alpha to a Neutrokine-alpha o Receptor, 0 10 Thus, the ant-Neutroknc-alpha monoclonal antibodies generated in the second expenment described above (in partcular, antibody 15C10).rccognze and Sbind to both membrane-bound and soluble Neutrokine-alpha protein and neutralize Neutrokune-alpha/Neutrakinmealpha Receptor binding m vitro.

Is It will be clear that the inventuon may be practiced otherwise than as particularly described m the foregoing descrnpon and examples. Numerous modifications and vanatons of the present invention are possible m light of the above teachings and, therefore, are within the scope of the appended clams.

The enure disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.

Further, the Sequence Lsting submitted herewth, and the Sequence Lstings submtued in copending application Serial Nos. 09/005,874, filed January 12, 1998, US60/036,100. filed January 14,1997 and PCT/US96/17957 filed October 25, 1996, m both computer and paper fpnns mI ea ca.s, are hbrby ncorporated by reference m ther entireties.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/o3/2ooe 19/032009 16:37 61 2 925936999 4 062837999 0 NO.72? P09 0 ci 00 0 0 CAr ApluntO Oes lt PF343PCT2 nwhn pkmo unassigned INDICATINS RELATING TO A DEPOSITE MICROORGANISM (PCT tult I fLu) conivale 21 3.IENTIFICATIONOPPPOSIr Nvmwfdupooiavirnwmin AMeric Type Culture koecton Addnhss of depomlan WsiltutOO fuChAdin posit a* and CROMr W001 University Boukevard Mimas, Virginia 20110G-2209 United States or Amer"c 22 Octobe 1998 9778 r- ADDITIONAL INLDICATrIQNSikavW Mmkifnwqphobfr) ThsuformauolcDn uursdoA ddiiOhciil DM DESIGNATED STATES FOR WHICH INDICATINS ARE MADE 1eti wumamc-wctaddtireiStaia Europe In respect to those designations in which a European Paten is souht a sample of ft. deposited nuicroorgantsm will be made availabe until t publication of the mention of ithe gat of te European patent or until the date on whwi applicatio hasp been refused or withrawn or is deemeod to be withdrawn. only by mhe issue of sud' a sample to an expert nominated by the person requesting the sample (Rule 28 EPC).

E. SEPA.IATFtURNISHINCO~tNDCATIONS~eebYbkifhswt&kat Tha rnieuow liad bala will he mm' to the IrnmMauonl Bau Iar0,4.rSf/euIt4'e Manic Of fbpswfl Farrftceiving~flitmwcoty Foam P1rctRo/l34 (July 1 i2) for mirnmil Bureau mainly COMS ID No: ARCS-183622 Received by IP Australia: Time (1-Pm) 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 00 0 355 Ct ATCC Deposit No. 97768

CANADA

The applicant requests that, until either a Canadian patent has been issued on the basis of an application or the application has been refused, or is abandoned and no longer subject to reinstatement, or is withdrawn, the Commissioner of Patents only authonzes the furnishing of Sa sample of the deposited biological material referred to in the application to an independent expert nominated by the Commissioner, the applicant must, by a wntten statement, iform O the International Bureau accordingly before completion of technical preparations for 0 publication of the international application.

0 0

NORWAY

The applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnislung of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act If such a request has been filed by the applicant, any request made by a third party for the fumnshing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recogrzed experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the mdividual case.

AUSTRALIA

The applicant hereby gives notice that the fumishmg of a sample of a microorganism shall only be effected pnor to the grant of a patent, or prnor to the lapsing, refusal or withdrawal of the applicaion, to a person who is a skilled addressee without an nterest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).

FINLAND

The applicant hereby requests that, until the application has been laid open to public inspection (by the National Board of Patents and Regulations), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.

UNiTED KINGDOM The applicant hereby requests that the furnislung of a sample ofa microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant with the Intemational Bureau before the complenon of the technical preparations for the international publication of the application.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2009 16:37 61 2 92596999 4 06293?999 N0.727 0 I 00 0 o 356 ATCC Deposit No. 97768

DENMARK

The applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the fummhling of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made o available to the public trder Sections 22 and 33(3) of the Damsh Patents Act. If such a ci request has been filed by the applicant, any request made by a third party for the fiumishing of 00 a sample shall indicate the expert to be used. That expert may be any person entered on a list oof recognized experts drawn up by the Danish Patent Office or anyperson by the applicant in othe individual case.

SWEDEN

The applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expet in the art. The request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the pnonty date (preferably on the Form PCTIRO/1 34 reproduced in annex Z of Volume I of the PCT Applicant s Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.

NETHERLANDS

The applicant hereby requests that until the date ofi grant of a Netherlands patent or until the -date on which the application is refused or withdrawn or lapsed, the microorgantsm shall be made available as provided in the 31 F( 1) of the Patent Rules only by the issue of a sample to an expert. The request to this effect must be furnished by the applicant with the Netherlands Industal Property Office before the date on which [he application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever of the two dates occurs earlier.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 19/032009 16:37 61 2 92596999 4 062937999 .2 D1 NO. 727 P12 00 0 357 ClApplcaffl's or almes ile PF34SPCr2 twmmioeiaupplwcailoeNP.

.enevAnmbur!Ot ,W INDICATIONS RELATING TO A DEPOSIED MICROORGANIM icr Rtule l3bu) *tThe ndwctnsffpdt hklwvwI~ii@AticiflaiirttmriiudtatitLhrnpuac onpage -i IL wplrMCxKNoFl#DroSlT V.unhapomaidcnfedwiasad4;enasI E Cl ecrddqosatwy pwzva Ainericsn Typ Cultur Collection 00 o Address ctf dqiosiwy insktio w3 un nmAadqpna cb ad COMArr ClI 0801 University Boulevard Manassas, Virginia 20110-2M0 Uldtd States of Ameia 1ac~cpt Ia0December 1990 203S1 C ADDIflONAL IT4DICATIOKa rkwwo bal ffnoiw appkblu This mfammiorunuoda anaddionalshec DL DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE fdAemhnrwsaniarfdgasfws Europe hn.renpect to thoe desinallion ,nvich a European Patent is nought a sample of the deposited mltcrorgammwill be made avalabletuntil fth pubcAmlion ofhs niention of the grant of"th European patent or untilt the date on which application hm been) refused or withdawn armi deemed to be Wmthain. only by(3 the maw of such a sample to aun expert ncwnmptuc by the purscon requestinig the sample (Rule 28 VPC).

Th suhw wasre lved lwwldbueusuiuiaanSt thple cw m 5uca Thaswm vdbthlcufaaJB ac: Aut- of Dofiqraa Tens PCT17IUOI34 (iuf 1992) COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 D13 00 0 S358 c ATCC Deposit No. 203518

CANADA

The applicant requests that. until either a Canadian patent has been issued on the basis of an application or the application has been refused, or is abandoned and no longer subiect to remistatement, or is withdrawn, the Commissioner of Patents only authorizes the fumrishing of a sample of the deposited biological matenal referred to in the application to an independent expert nominated by the Commissioner, the applicant must, by a written statement, inform Sthe International Bureau accordingly before completion of technical preparations for Cq publication of the international application.

00

NORWAY

The applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open mspection, the firmshing of a sample shall only be effected to an expert m the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the funushing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recogntied experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the mdividual case.

AUSTRALIA

The applicant hereby gives notice that the furnshmg of a sample of a microorganism shall only be effected prior to the grant of a patent, or pnor to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an mterest m the invention (Regulation 3.25(3) of the Australian Patents Regulations).

FINLAND

The applicant hereby requests that, until the application has been laid open to public inspection (by the National Board of Patents and Regulations), or has been finally decded upon by the National Board of Patents and Registration without having been laid open to public inspection, the fnirmshing of a sample shall only be effected to an expert in the an.

UNITED KINUUM The applicant hereby requests that the furnshmg of a sample of a microorgamnsm shall only be made available to an expert. The request to this effect must be filed by the applicant with the International Bureau before the completion of the technical preparations for the nternational publication of the application.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 P14 00 0 359 C ATCC Deposit No. 203518

SDENMARK

The applicant hereby requests that, until the application has been laid open to public inspection (by the Dansh Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public mspection, the furnishing of a sample shall only be effected to an expert m the art. The request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made O available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a Cl request has been filed by the applicant, any request made by a third party for the furmshing of 00 a sample shall indicate the expert to be used. That expert may be any person entered on a list 0 of recognied experts drawn up by the Danish Patent Office or any person by the applicant m 0< the individual case.

SWEDEN

The applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without havmg been laid open to public mspecton, the furnshmg of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Intenational Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/i 34 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recogmzed experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.

NETHERLANDS

The applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided m the 3 IF(1) of the Patent Rules only by the issue of a sample to an expert. The request to this effect must be furmshed by the applicant with the Netherlands Industnal Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands. whichever of the two dates occurs earlier.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2098 16:37 -61 2 92566999 4 062937999 J.2 P1 NO. 72? 00 0t 0 Awplieantswor flsRI PF3I3PCT2lmmwlapfhqk INWCA1"OMS RELAhC; TO A DEPOSITD NDCROORCANIM (CTa ItOe 130's) MW229.)n 3rOtITICATONOFDEPOSIWu raaufrdlfrf~~dI~illf ?&,ncafdupnsiaty nsuiuflon AmeOa~n TYPe Culture Colletion Adfets i daimmirv isuwuoc f~btungpannl cad, ud csrn) 10501 LkuvOAtdy Boulevard Masses, Virgina 20110-2209 Un~ited SlatS of AnCIecaI DatunftepamAccenon~umber 07 January 2000 IPTA-I 158 12- AD~t)IONAL TNDUC^flOPNSdaVwltifuxAwip&Gtt* Thismfnnwionmsconwcdcnuanuihhcct Mt DZ$IGNATED STATES VOR WHICHi INDICATIONS ARE NIADE- itfhndieanutmfrdnuweD Europe Irv respect to tos desagnilion in wtucha European Patentsa sought a sample of the deposited mmoorgunism wil be made av-ailabl until the publiaion ofithe mention af tin grant ot "b Europen patent or unti the date on wtwdi applicaton bas been refused at witimwnmu riseoed wn be withdrawn, only by the issue of such a sampte to an expert nominated by the parson requesting the samrple (Rule 2B8(4) EPC).

L SEPARATE flRNISHINOOf INDICATIONSkwbatjmeppabW The sndieaamo lined below will be submuuad 26 On ldaulwmf l Bfupamu tne q*uqmiwrqea lsa.

NWmRW4DAvmn7 X Fatmcpngofieen ly Tlwsbesawurccovedwihmdanenmnplicauloq Fern PCaitORI34 (July 1992) ForIntrnakbnal Bureausmanly Auihanedcffict COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 [16 00 0 0 361 ATCC Deposit No. PTA-1I58 0 CANADA The applicant requests that, until either a Canadian patent has been issued on the basis of an application or the application has been refused, or is abandoned and no longer subject to Sreinstaement, or is wIthdrawn, the Lommissioner of Parents only authorizes the funushing of a sample of the deposited biological material referred to in the application to an independent expert nominated by the Commissioner, the applicant must, by a written statement, mform o the International Bureau accordingly before completion of technical preparations for l publication of the international application.

00 O NORWAY The applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open mspection, the furnishing of a sample shall only be effected to an expert in the art The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishng of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.

AUSTRALIA

The applicant hereby gives notice that the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or pnor to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an interest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).

FINLAND

The applicant hereby requests that, until the application has been laid open to public nspecton (by the National Board of Patents and Regulations), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert m the art.

UNrED KINGDOM The applicant hereby requests that the furmshmg of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the apolicant with the International Bureau before the completion of the technical preparations for the international publication of the application.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 N0.727 017 00 0 0 362 t ATCC Deposit No. PTA-1158

DENMARK

The applicant hereby requests that, until the application has been laid open to public ispection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert m the art. The request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made o available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a Cl request has been filed by the applicant, any request made by a third party for the furnishing of 00 a sample shall indicate the expert to be used. That expert may be any person entered on a list o ofrecogmzed experts drawn up by the Danish Patent Office or any person by the applicant in Cl the individual case.

SWEDEN

The applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public mspectton, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnshing of a sample shall mdicate the expert to be used. That expert may be any person entered on a list ofrecognized experts drawn up by the Swedish Patent Office or any person approved by a applicant m the mdividual case.

NETHERLANDS

The applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31 F(1) of the Patent Rules only by the issue of a sample to an expert. The request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands, whichever of the two dates occurs earlier.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/200e 16:37 61 2 92596999 4 662837999 hC.727 pie 00 o 363 AppLcaits nt ams 11it: PFS.4SPCT2 lnnmmnuuob -4 INDVICATIONS RELATiNG TO A DEPOSITE NUCROORCANMI (PCT Rule M3ali A. The mdicatInSsffladtbCkflhbttOtChOncoWIWnsiflftntdbIin twflnpt*Of an pect Z2 line Il. IDETTWICArIONOVDKPOSIT Fwuherdepeuuremtifluioanunddiitalushen f Naueofdepsijayn~nuhjom AMflhCe Typ Culture Cotection Atkkes of' depwi4a aSINumnW mrac1dn FOND! cdc and mv) lanai Universit Boutevsod.

Manassas. Viginia 20110-2209 United Status of America Dsao~pnt AersNmenber 017 January 2000 PTA-1159 c- ADDITIONAL IN4DICATtONSdnrbmhijwAj~pka&j Tm~mn~cmsccad*nthc TL.UDESIGNATED STATES FOR WHICH INDICATIONS ARE MADE frccautweodpdSi Europe In respect to those designations inwhmdi a European Patent is sought a sample of the deposited muorgansm will be made available until the pu~blication oft.e mention of the grant of the European patent or unti the dte on which applIcalion has be" refused or withdruwn or ms damned to be withdawnl. only by the issue of such a Sample to an expert nominated by the person requesting the sample (Rule 28 EPC).

SEPARATE FUiRNISHINGOFNDCATON"#mwbm~kaaPan&f The rndicw1o 1sd balow wilt bq nubnnnedutoaft IsemnUca Bureau Iater opt qrcji wmf~ saulaq MYu.06 *f Dqowl

(N)

C

z Tcrtaceavsu~f~cisscoaly lThsslucwamsvcdwnbtcmiananlapicsian form PaJWmi3S (Juy IM9) Fednflu.Omel Duweauiseenly 5 mis dwnwuncnvr4bydwlnfernhfoniflunaa AiflhenrdolWciuq COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 019 00 0 364 ^c ATCC Deposit No. PTA- 159 0 CANADA The applicant requests that, until either a Canadian patent has been issued on the basis of an auDlicauon or the aoolication has been refused or is abandoned and no longer subject to reinstatement, or is withdrawn, the Commissioner of Patents only authorizes the furnishing of a sample of the deposted biological materal referred to in the application to an independent expert nominated by the Commissoner, the applicant must, by a wntten statement, inform 0 the Internatonal Bureau accordingly before completion of techncal preparatons for Cl publication of the internatonal application.

00 0 NORWAY The applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert m the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the finmshing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.

AUSTRALIA

The applicant hereby gives notice that the fuirnshing of a sample of a microorganism shall only be effected pnor to the grant of a patent, or prior to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an interest in the mvention (Regulation 3.25(3) of the Australian Patents Regulations).

FINLAND

The applicant hereby requests that, until the application has been laid open to public inspection (by the National Board of Patents and Regulatons), or has been fmally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishmg of a sample shall only be effected to an expert in the art.

UNITED KINGDOM The applicant hereby requests that the frmshing of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant with the Intematonal Bureau before the completion of the technical preparations for the international publication of the application.

COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92596999 4 062837999 NO.72? 00 0 o 365 ATCC Deposit No. PTA-I 159

DENMARK

The applicant hereby requests thai. until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspction, the furnishing of a sample.

shall only be effected to an expertrn the art. The request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made o available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any requet made by a third party for the furnishing of 00 a sample shall indicate the expert to be used. That expert may be any person entered on a list Oof recogruzed experts drawn up by the Danish Patent Office or any person by the applicant in Othe individual case.

SWEDEN

The applicant hereby requests that, until the application has been laid open to public ispection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this cffect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I of the PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third patty for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up-by the Swedish Patent Office or any person approved by a applicant in the individual case.

NETHERLANDS

The applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31F(l) of the Patent Rules only by the issue of a sample to an expert. The request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 of the Patents Act of the Kingdom of the Netherlands. whwhcver of the two dates occurs earlier, COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19

Claims (20)

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: a nucleotide sequence encoding the Neutrokine-alpha polypeptide o having the complete amino acid sequence in Figures 1A and 1B (SEQ ID NO:2); OO a nucleotide sequence encoding the Neutrokine-alpha polypeptide Shaving the complete amino acid sequence encoded by the cDNA clone contained in the C" 10 deposit having ATCC accession number 97768; a nucleotide sequence encoding the Neutrokine-alpha polypeptide extracellular domain; a nucleotide sequence encoding the Neutrokine-alpha polypeptide transmembrane domain; a nucleotide sequence encoding the Neutrokine-alpha polypeptide intracellular domain; a nucleotide sequence encoding a soluble Neutrokine-alpha polypeptide comprising the extracellular and intracellular domains but lacking the transmembrane domain; and a nucleotide sequence complementary to any of the nucleotide sequences in or above.

2. The nucleic acid molecule of claim I wherein said polynucleotide has the complete nucleotide sequence in Figures 1A and IB (SEQ ID NO:1).

3. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence in Figures IA and 1B (SEQ ID NO: 1) encoding the Neutrokine-alpha polypeptide having the complete amino acid sequence in Figures 1A and 1B (SEQ ID NO:2). COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 ig/o3/2ooe0 16:37 61 2 92586999 4 062837999 NO.727 U22 00 WO 00/50597 PCT/US00/04336 S367 Cl 4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a soluble Neutrokine-alpha polypeptide comprising the extracellular domain shown in Figures IA and 1B (SEQ ID NO:2).

5. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: a nucleotide sequence encoding a polypeptide having the amino acid C sequence consisting of residues n-285 of SEQ ID NO:2, where n is an integer in the 00 0 10 range of 2-190 0, a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues 1-m of SEQ ID NO:2, where m is an integer in the range of 274-284; a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues n-m of SEQ ID NO:2, where n and m are integers as defined respectively in and above; and a nucleotide sequence encoding a polypeptide consisting of a portion of the complete Neutrokine-alpha amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC accession number 97768, wherein said portion excludes from 1 to 190 amino acids from the amino terminus and from 1 to 11 amino acids from the C-terminus of said complete amino acid sequence.

6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in the deposit having ATCC accession number 97768.

7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding the Neutrokine-alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC accession number 97768.

8. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence encoding a soluble Neutrokine-alpha polypeptide comprising COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 N0.727 D23 00 WO 00150597 PCTUSOO/04336 0 368 Cl the extracellular domain encoded by the cDNA clone contained in the deposit having ATCC accession number 97768, S9. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in or of claim 1 wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence C, consisting of only A residues or of only T residues. 00 oo An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Neutrokine-alpha polypeptide having an amino acid sequence in or of claim 1.

11. The isolated nucleic acid molecule of claim 10, which encodes an epitope-bearing portion of a Neutrokine-alpha polypeptide selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe-115 to about Leu-147 (SEQ ID N0:2); a polypeptide comprising amino acid residues from about Ile-150 to about Tyr-163 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-171 to about Phe-194 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Glu-223 to about Tyr-246 (SEQ ID NO:2); and a polypeptide comprising amino acid residues from about Ser-271 to about Phe-278 (SEQ ID NO:2).

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

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

14. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 13 into a host cell. A recombinant host cel produced by the method of claim 14. COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 D24 00 WO 00/5097 PCT/US00/04336 0369 S 16. A recombinant method for producing a Neutrokine-alpha polypeptide, Scomprising culturing the recombinant host cell of claim 15 under conditions such that said polypeptide is expressed and recovering said polypeptide.

17. An isolated Neutrokine-alpha polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: the amino acid sequence of the Neutrokine-alpha polypeptide having the complete amino acid sequence in Figures IA and IB (SEQ ID NO:2); 00 0 10 the amino acid sequence of the Neutrokine-alpha polypeptide having the 0 complete amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC accession number 97768; the amino acid sequence of the Neutrokine-alpha polypeptide extracellular domain; the amino acid sequence of the Neutrokine-alpha polypeptide transmembrane domain; the amino acid sequence of the Neutrokine-alpha polypeptide intracellular domain; the amino acid sequence of a soluble Neutrokine-alpha polypeptide comprising the domain; and the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of or

18. An isolated polypeptide of claim 17 comprising an epitope-bearing portion of the Neutrokine-alpha protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe-115 to about Leu-147 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ile-150 to about Tyr-163 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-171 to about Phe-194 (SEQ ID NO:2); a polypeptide comprising amino acid residues from about Glu-223 to about Tyr-246(SEQ ID NO:2); a polypeptide comprising amino acid residues from about Ser-271 to about Phe-278 (SEQ ID NO:2). COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 025 00 WO 00/50597 PCTUS01/04336 370 Cl 19. An isolated antibody that binds specifically to a Neutrokine-alpha c polypeptide of claim 17. A pharmaceutical composition comprising a polypeptide of claim 17 and a pharmaceutically acceptable carrier.

21. An isolated polynucleotide encoding a modified Neutrokine-alpha protein, wherein, except for at least one conservative amino acid substitution, said C modified peptide has an amino acid sequence that is identical to a member selected 00 o 10 from the group consisting of: 0 amino acids 1 to 285 of SEQ ID NO:2; amino acids 2 to 285 of SEQ ID NO:2; amino acids 1 to 46 of SEQ ID NO:2; amino acids 47 to 72 of SEQ ID NO:2; and amino acids 73 to 286 of SEQ ID NO:2.

22. A modified Neutrokine-alpha polypeptide molecule, wherein, except for at least one conservative amino acid substitution, said modified peptide has an amino acid sequence that is identical to a member selected from the group consisting of: amino acids 1 to 285 of SEQ ID NO:2; amino acids 2 to 285 of SEQ ID NO:2; amino acids 1 to 46 of SEQ ID NO:2; amino acids 47 to 72 of SEQ ID NO:2; and amino acids 73 to 286 of SEQ ID NO:2.

23. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected selected from the group consisting of: the nucleotide sequence of SEQ ID NO:7; the nucleotide sequence of SEQ ID NO:8; the nucleotide sequence of SEQ ID NO:9; the nucleotide sequence of a portion of the sequence shown in Figures 1A and 1B (SEQ ID NO:1) wherein said portion comprises at least 30 contiguous COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 NO.727 P26 00 WO O/5057 PCT/USOO/04336 S371 Cl nucleotides from nucleotide 1 to nucleotide 2442, excluding the sequence from C nucleotide 1387 to 1421, the sequence from nucleotide 9 to 382, the sequence from Snucleotide 1674 to 1996, the sequence from nucleotide 1401 to 1784, the sequence from nucleotide 900 to 1237. and any fragments located within these sequences; and s a nucleotide sequence complementary to any of the nucleotide Ssequences in or above.

24. An isolated nucleic acid molecule comprising a polynucleotide having a C' nucleotide sequence at least 95% identical to a sequence selected from the group OO 0 10 consisting of: 0 a nucleotide sequence encoding the Neutrokine-alphaSV polypeptide having the complete amino acid sequence in Figures 5A and 5B (SEQ ID NO: 19); a nucleotide sequence encoding the Neutrokine-alphaSV polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC accession number 203518; a nucleotide sequence encoding the Neutrokine-alphaSV polypeptide extracellular domain; a nucleotide sequence encoding the Neutrokine-alphaSV polypeptide transmembrane domain; a nucleotide sequence encoding the Neutrokine-alphaSV polypeptide intracellular domain; a nucleotide sequence encoding a soluble Neutrokine-alphaSV polypeptide comprising the extracellular and intracellular domains but lacking the transmembrane domain; and a nucleotide sequence complementary to any of the nucleotide sequences in or above. The isolated antibody of claim 19 wherein said isolated antibody inhibits binding of the protein of SEQ ID NO:2 to a Neutrokine-alpha receptor. COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19 19/03/2008 16:37 61 2 92586999 4 062837999 N0.727 [27 372 00 o 26. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide O sequence at least 95% identical to a sequence substantially as described with reference to and as illustrated in the accompanying figures.

27. An isolated Neutrokine-alpha polypeptide comprising an amino acid sequence at least a, 5 identical to a sequence substantially as described with reference to and as illustrated in the accompanying figures.

28. An isolated polynucleotide encoding a modified Neutrokine-alpha protein, wherein, except for at least one conservative amino acid substitution, said modified peptide has an amino acid sequence that is identical to a member substantially as described with reference to and as 0 10 illustrated in the accompanying figures. 0 0 29. A modified Neutrokine-alpha polypeptide molecule, wherein, except for at least one 0 conservative amino acid substitution, said modified peptide has an amino acid sequence that is C N identical to a member substantially as described with reference to and as illustrated in the accompanying figures.

30. An isolated nucleic acid molecule comprising a polynucleotide having a sequence of at least 95% identical to a sequence substantially as described with reference to and as illustrated in the accompanying figures. COMS ID No: ARCS-183622 Received by IP Australia: Time 16:56 Date 2008-03-19