AU2003248191B2 - Novel PRO717 polypeptides and nucleic acids encoding the same - Google Patents

Description

H:\cintac\Keep\speci\2003248191 amendments.doc 07/04/05 SNOVEL PRO717 POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE SAME FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel DNA and to the 0 recombinant production of novel polypeptides encoded by that DNA.

BACKGROUND OF THE INVENTION All references, including any patents or patent application, cited in this specification are hereby 00 0 incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the C, references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications S are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.

Extracellular proteins play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment.

Secreted proteins have various industrial applications, including pharmaceuticals, diagnostics, biosensors and bioreactors. Most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines,-are secretory proteins. Their receptors, which are membrane proteins, also have potential as therapeutic or diagnostic agents. Efforts are being undertaken by both industry and academia to identify new, native secreted proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in the literature [see, for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Patent No. 5,536,637)].

Membrane-bound proteins and receptors can play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. Such membranebound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. For instance, transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also r H:\cintae\Kecp\speci\200324819 amendments.doc 07/04/05 Sact as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.

Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic O agents to block receptor-ligand interaction. The membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. Efforts are being undertaken by both industry and academia to identify new, native receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor 00 0 proteins.

C1 We herein describe the identification and characterization of novel secreted and transmembrane 0polypeptides and novel nucleic acids encoding those polypeptides.

PR0717 Efforts are being undertaken by both industry and academia to identify new, native transmembrane receptor proteins. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins. The results of such efforts are provided herein.

SUMMARY OF THE INVENTION PR0717 In a first aspect, the present invention provides an isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420).

Applicants have identified a cDNA clone that encodes a novel 12 transmembrane polypeptide, wherein the polypeptide is designated in the present application as "PRO717".

In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO717 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO717 polypeptide having amino acid residues 1 through 560 of Figure 169 (SEQ ID NO:420), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. The isolated nucleic acid sequence may comprise the cDNA insert of the vector deposited on April 28, 1998 with the ATCC as DNA50988-1326 which includes the nucleotide sequence encoding PRO717.

In another embodiment, the invention provides isolated PRO717 polypeptide. In particular, the invention provides isolated native sequence PRO717 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 through 560 of Figure 169 (SEQ ID NO:420). An additional embodiment of the present invention is directed to an isolated extracellular domain of a PRO717 polypeptide. Optionally, the PRO717 polypeptide is obtained or is obtainable by expressing the polypeptide encoded by the cDNA insert of the vector deposited on April 28, 1998 with the ATCC as DNA50988-1326.

In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the above or below described polypeptides. A host cell comprising any such vector is also provided. By H:\cintae\Keep\speci\2003248191 amendments.doc 07/04/05 O way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing any of the above or S below described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric molecules comprising any of the above or below O described polypeptides fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises any of the above or below described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.

SIn another embodiment, the invention provides an antibody which specifically binds to any of the above o0 or below described polypeptides. Optionally, the antibody is a monoclonal antibody.

In yet other embodiments, the invention provides oligonucleotide probes useful for isolating genomic and O cDNA nucleotide sequences, wherein those probes may be derived from any of the above or below described S nucleotide sequences.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a nucleotide sequence (SEQ ID NO:419) of a native sequence PRO717 cDNA, wherein SEQ ID NO:419 is a clone designated herein as AUNQ385" and/or ADNA50988-1326".

Figure 2 shows the amino acid sequence (SEQ ID NO:420) derived from the coding sequence of SEQ ID NO:419 shown in Figure 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions In the claims of this application and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation PRO/number) refers to specific polypeptide sequences as described herein. The terms "PRO/number polypeptide" and "PRO/number" as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.

A "native sequence PRO polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence PRO polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide an extracellular domain sequence), naturally-occurring variant forms alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In various embodiments of the invention, the native sequence PRO717 polypeptide is a mature or full-length native sequence PRO717 polypeptide comprising amino acids 1 through 560 19/09 200 FAX 61 3 92438333 GRIFFITH HACK IO010O of Figure 1 (SEQ ID NO:420).

The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PR3 polypeptide whi h is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a PRO polyp ptide ECD will ha e less than 1% of such u anmarmnbrane and/or cytoplasmic domains and preferably, will have s than 0.5% of su h domains. It will be understood that any transmembrane domais identified for the PRO poly ptides of the presmen invention arc identified pursuant to criteria routinely employed in the art for identifying ith type of hydrophot i domain. The exact boundaries of a tmrnsmembrane domain may vary but most likely by no more than about 5 ami 1o acids at either end of the domain as initially identified. Optionally, therefore, an extracelllar domain of a PRO polypeptide may contain from about 5 or fewer amino acids on either or the transmembraa domain as initia ly identified.

"PRO polyprptide variant" means an active PRO polypeptido as defined above or below having at le st about 80% amino acid sequence identity with the full-length native sequence PRO polypeptide sequence as disclosd herein. Such PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid kequonce. Ordinari y, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, more p cierably at least abcut amino acid sequrenc identity, and even more preferably at least about 90% amino acid equence identity, Cv n more preferably at least about 91% amino acid sequence identity, even more preferably at east about 92% amilo acid sequence identity, even more preferably at least about 93% amino acid sequence idcnit, even more prefra ly at least about 94% arinn acid sequence identity, even more preferably at least about 959 amino acid sequen c identity, yet more preferably at least about 96% amino acid sequence identity, yet more preferably at least about 97 amino acid sequence identity, yet more preferably at least about 98% amino acid sequefc identity and most preferably at least about 99% amino acid sequence identity with the amino acid sequence of the full-length nati e amino acid sequence as disclosed herein.

"Percent amino acid sequence identity" with respect to the PRO polypeptidb sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that arc iJentical with the amio acid residues in the :pecific PRO polypeptide sequence, after aligning the sequences ard introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutio as part of the sequence identity. Alignment for purposes of determining percent amino aciq sequence identity m be achieved in various ways that are within the skill in the art, for instance, using publi y available compulcr software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. The pref Ted software alignu nt program is BLAST. Those skilled in the art can determine appropriate parameters for measuring alignmet, including any algorithms needed to achieve maximal alignment over the full length of the seqeces being compa d.

The identity values used herein have been generated using the WU-BLAST-2 computer irogram (Altschul et I., Methods in Enzvmonu 266:460-480 (1996); http://blast.wust/edu/blast/README.htnl). Most of the W BLAST-2 search parameters were set to the default values. The adjustable parameters er set with the followi g values: overlap span= overlap fraction 0125, word theshold I and scoring matix BLOSUM62. T HSP S and HSP S2 parameters, which are dynamic values used by BLAST-2, are establish d by the program its lf depending upon the composition of the sequence of interest and composition of the dauhtase against which t 4 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:22 FAX 61 3 92438333 GRIFFITH HACK 001oii sequence is being searched. However, the values may he adjusted to increase sensitivity. sequence identit value Is determined by the fraction of matching identical residues divided by the total number of residues in 8 aligned region.

"Percent nucleic acid sequence identity" with respect to PRO-encoding nucleic acid sequcncss identified herein is defined as the percentage of nucleotides in a candidate sequence thai are identical with die nucleotides in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximumi percent sequence identity. Alignment for purposes of determining prcnt nucleic acid sequence identity can be achieved in various ways that are within the skill in the it, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software Those skilled in the aic can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve max nal alignment over the full lngth of the sequences being compared- The identity values us d herein were generated by the BLASTN module ofWU-BLAST-2 set to the default parametrs, with overlap sp and overlap fraction st to 1 and 0.125, respectively.

The term "positives", in the context of sequence comparison performed as dosctibed above, includes residues in the sequernes compared that are not identical but have similar properties as result of consrvative substiutions). The value of positives is determined by the fraction of residues scoring 1 positive value in he BLOSUM 62 matrix divided by the total number of residues in the aligned region, as defined above.

The term "cpitope tagged" where used herein refers to a chimeric polypcptidB comprising a O polypeptidc, or domain sequence thereof, fused to a "tag polypcptide". The tag polypeptidb has enough residues to provide an epitope against which an antibody may be made, or which can be Identified by some other agent, yet is short enough such that it does not interfere with the activity of the PRO polypepdde of interest. The a polypeptide preferably is also fairly unique so that the antibody does not substantially cross-reict with other epitop.

Suitable tag polypeplidcs genrally have at least six amino acid residues and usually bctwen about 8 to about 0 amino acid residues (preferably, between about 10 to about 20 residues).

"Isolated," when used to describe the various polypeptides disclosed herein, meas polypeptide that Ias been identified and cparated and/or recovered from a component of its natural environment. Contammi components of its natural environment are materials that would typically interfere with diagnstic or therapeutic ut s for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaccous soluts.

In preferred embodiments, the polypeptide will be purified to a degree sufficient to obtin at least 15 resid es of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to[homogeneity by S 3- PAGE under non-reducing or reducing conditions using Coonassie blue or, preferably, silver stain. Isolad polypeptide includes polypeptide in situ within rccombinant cells, since at least one c6mponent of the P O polypeptide natural environment will not be present Ordinarily, however, isolated polypeptde will be prepared by at least one purification step.

An "isolated" PRO polypoptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natu al source of the PRO polypeptide nucleic acid. An isolated PRO polypeptide nucleic acid mlaecule is other than in the form or setting in which it is found in nature. Isolated PRO polypeptide nucleic add moleculcs therefore e ar COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:22 FAX 61 3 92438333 GRIFFITH HACK a012 distinguished from the specific PRO polypeptide nucleic acid molecule as it exists in natur 4 cells. However, mi isolated PRO polypeptide nucleic acid molecule includes PRO polypeptide nucleic acid moco ldes contained in ce ls that ordinarily express the PRO polypeptide where, for example, the nucleic acid molecule is in a chromosoial location different from that of natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a p;rticular host organism. The control sequences that are suitable for pro aryotes, for exanp include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryo ie cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when itis placed into a functional relationship with another nucleic acid sequence. For oxamplt, DNA for a presequence or secretory leader is operably linked to D A for a polypeptide if it is expressed as a preprocin that participates in the secretion of the polypeptide; a pro moter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribpsome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linke1" means that the DNA sequences being linked ar contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction 'ites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers ar used in accordance with conventional practice.

The term "antibody" is used in the broadest sense and specifically covers single nti-PRO polypeptide monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-PRO polypeptide antibody compositions with polyepitopic specificity. The term "monoclonal antibody" as ted herein refers to i antibody obtained from a population of substantially homogeneous antibodies, i.e, the individual antibodis comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. i "Active" or "activity" for the purposes herein refers to form(s) of PRO polypep4de which retain the biologic and/or immunologic activities of the specific native or naturally-occurring PRO polyppidde.

"Trealnmnt" r "treating" refers to both therapeutic treatment and prophylactic or p ventative measm Those in need of treatment include those already with the disorder as well as those prone t have the disorder if those in which the discorder is to be prevented.

"Mammal" for purposes of treatment refers to any animal classified as a mamnm. including bumans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, hoars, cais, cows, and the like.

Preferably, the mamnral herein is a human.

"Carriers" as used herein include pharmaccutically acceptable carriers, excipintms, o stabilizers which a e nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations mployed. Often t e physiologically acceptable carrier Is an aqueous pH buffered solution. Examples of phys ologically acceptabe carriers include buffbrs such as phosphate, citrate, and other organic acids; antioxidants incl ng ascorbic acid; lw molecular weight (lecs than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, r immunoglobulins; hydrophilic polymers such as polyvinylpyrrulidone; amino acids such as glycine, glutamini.

asparagine, arginine or lysin; monosaccharides, disaccharides, and other carbohydrates inclu ng glucose, anno 6 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:23 FAX 61 3 92438333 GRIFFITH HACK Q013 or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; sa 1 -forming counteriOS such as sodium; and/o nonionic surfacLants such as TWEENM, polyethylene glycol (PEG), and PLURONICS The term "agonist" is used to refer to pcpdde and non-peptide analogs of the npti$e PRO polypeptids (where native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepr4PRO polypeptide, r mature PRO polypeptide) of the present invention and to antibodics specifically binding such native PRO polypoptides, provided that they retain at least ono biological activity of a native PRO polype Ptide. Preferably, t agonists of the preset invention retain the qualitative binding recognition properties receptor activation properties of the native PRO polypeptide.

Tbhe term "antagonist" is used to refer to a molecule inhibiting a biological acti ty of a native PRO polypeptide of the present invention wherein native PRO polypeptide refers to pro-PRO Rolypcptide, pre-P 0 polypeptide, propro-PRO polypeptide, or mature PRO polypeptide. Preferably, the antagonist& herein inhibit t e binding of a native PRO polypeptide of the present invention to a binding partner A PRO polypepide "antagoni t" is a molecule which prevents, or interfercs with, a PRO antagonist effector funaction a molecule which prevens or interferes with blndtng and/or activation of a PRO polypeptide receptor by PRO polypepide). Such molecule can be screened for their ability to competitively Inhibit PRO polypopide receptor activation by monitoring bindi g of native PRO polypeptide in the prescnce and absence of the test antagonist molecule, for csmple. An antagon at of the invention also encompasses an antisense polynuclentide against the PRO polypoplide Fene, which antisens polynucleotide blocks 'ranscription or translation of the PRO polypeptide gene, thereby inhibiting its expressionad biological activity.

"Stringency" of hybridization reactions is readily determinable by one of ordin4 skill In the art, an d generally is an empirical calculation dependent upon probe length, washing temperature, andsalt concentration. n general, longer probes require higher temperatures for proper annealing, while shortek probes need low temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complement y strands are present in an environment below their melting temperature. The higher the de of desired hounoloy between the probe and hybridizable sequence, the higher the relative temperature which call be used. As a res it follows that higher relative temperatures would tend to make the reaction conditions more bringen, while lowr temperatures less so. For additional details and explanation of stringency of hybridization ractions, see Ausubal at aL, Current Protocols in Moleclar Biology. Wiley Interscience Publishers, (1995).

"Stringent conditions" means employing low ionic strengh and high temnpeture for washing, f r example, 0.015 sodium chlorideJO.0015 M sodium citrate/O. 1% sodium dodecyl sulfata at 50 0 C, or employi n during hybridization a denauring agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1 bovine serum albumin/d0.1% Ficoll/0.1% polyvinylpyrrolidone/50nM sodiumphosphate buffer at pH 6.5 with mM sodium chloride. 75 mM sodium citrate at 42C. Another example is use of 50% forrnamidc, 5 x SSC (0.15 M NaCG, a0075 M sodium citrate), 50 mM sodium phosphae (pH 0.1% sodium pyrophcsphate, 5 x Denhard s solution, sonicated salmon sperm DNA (50 pghnl), 0.1% SDS, and 10% dextran sulfate atf42oC, with washes at 42"C in 0.2 x SSC anm 0.1% SDS. Yet another example is bybridization using a buffer of 10% dextran sulfate, 2 x SSC (sodium chloridehiodium citrte) and 50% formamide at 55 0 C, followed by a high-stency wash consisti g of 0.1 x SSC containing EDTA at 7 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 200AJAIt. FAX 61 3 92438333 GRIFFITH HACK 4014 "Moderately stringent conditions" arc described in Sambrook et al., supra, and includ the use of a wash ig solution and hybridization conditions tcmperature, ionic strength, and %SDS) less strngent than describ above. An example of moderately stringent conditions is a condition such as overnight in ubation at 37C in a solution comprising: 21% fomannide. 5 x SSC (150 mM NaCI. 15 mM trisodium citrate), 50 M sodium phosph te (pH 5 x Denhan t's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sper DNA, followed by washing the filters in 1 x SSC at about 37-50"C- The skilled artisan will recogiize how to adjust t e temperature, ionic strength, etc., as necessary to accommodate factors such as probe length nd the like.

"Southern analysis" or "Southern blotting" is a method by which the presece ol DNA sequences in a rcstriction endonucdene digest of DNA or a DNA-containing composition is confirmed by hy ridization to a kno n, labeled oligonucleotide or DNA fragment. Southorn analysis typically involves lectrophorehc separation of DNA digests on garosc gels, denatraion of the DNA after electrophoretic separation, and trnsfcr of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolaljecd, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook at aL, Molecular Cloning, A LlboratX Manual (New York: C(old Spring Harbor Laboratory Press, 1989).

"Northern analysis" or "Northern blotting" is a method used to identify RNA sequences that hybridize to a known probe such as an oligonucleotidc, DNA fragmen, cDNA or fragment thereof, or RN fiagwnL The pr be is labeled with a radioisotope such as or by biotinylatio, or with an enzyme. The RNA t be nalyzed is usualy electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitroueiulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques well known iI the art such as those described in sections 1.39-752 of Sambrook et al., supra.

As used herein, the term "immunoadhesin" designates antibody-like molecules whigh combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglolin constant doains.

Structirally, the immunoadhesins comprise a fuion of an amino acid sequence with the desired binding specifiity which is other than Ihe antigen recognition and binding sitc of an antibody is "lietrologos"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhcsin 4oleculo typically i; a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The immunoglob lin constant domain sequence in the immunoadhsin may be obtained from any lmmunoglobuli such as IgO-1. Ig 2, IgG-3. or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

"Chronic" administration refers to administration of the agent(s) in a continuous iode as opposed to an acute mode, so as to maintain the initial therapeutic cffect (activity) for an extended period of time. "Inbcimittet" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration "in combination with" one or more further therapeutic agents ]includes simultane as (concurrent) and con ecutive administration in any order.

The term "expression vector" is used to define a vector, in which a nucleic |acid encoding a P 0 polypeptide herein is operably linked to control sequences capable of affecting its expression is a suitable host ce Us.

Vectors ordinarily carry a replication site (although this is not necessary where chromosomal integration will occ r).

Expression vectors also include marker sequences which are capable of providing pienotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid erved from an E. 6li 8 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09_200Q 18:24 FAX 61 3 92438333 GRIFFITH HACK a015 species (Bolivar, et aL. Gene 2: 95 [1977)). pBR322 contains genes for ampicillin and tetratycline resistanced thus provides easy mans for identifying transformed cells, whether for purposes of ck4ning pr epressio Expression vectors also optimally will contain sequences which are useful for the control of transcription ad translation, promoters and Shine-Dalgarno sequences (for prokaryotes) or promotes and enhancers (for mammalian cells). The promoters may be. but need not be, inducible; even powerful constiutive promoters suh as the CMV promoter for mammalian hosts have been found to produce the LHR without host cell toxicity. Wh le it is conceivable that erpression vccutos need not contain any expression control. replicative iequences or selecti n genes, their absence m y hamper the identification of hybrid transformants and the achievemept of high level bid immunoglobulin exprtssion.

The term lipopolysaccharide" or "LPS" is used herein as a synonym of "endotoxin.' Lipopolysacchrides (LPS) are characteristic components of the outer membrane of Gram-negative bacteria, Eschrichia coL ey consist of a polysaccharide part and a far called lipid A. The polysaccharide, which varies fron one bacterial spec es to another, is made up of the 0-specific chain (built from repeating units of three to eight s ars) and the two-pt core. Lipid A virtually always includes two glucosarnine sugars modified by phosphate an a variable number of fatty acids. For fwther information see, for example, Rielsehel and Brade, Scientific Amerc August 1992,54- 1.

The termn "septic shock' is used herein in the broadest sense, including all deflnitina disclosed in Box Ann. Intern Mcd. 332-333 (1991), Specifically, septic shock starts with a systemic rsponse to infection a syndrome called sepsis. When this syndrome results in hypotension and organ dysfunction, il is called septic sb k.

Septic shock may be Initiated by gram-positive organisms and fungi, as well as endotoxin-cotaining Gram-negve organisms. Accordingly, the present definition Is not limited to "endotoxin shock." The phrases Agene amplification= and Ageno duplicatiors arc used intarchangeablf and refer to a proc s by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line! The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon". Usually, the amount OF the messenger RL (mRNA) produced, i.ue, the level of gene expression, also increases in the proportion of the lumber of copies ade of the particular gou expressed '"Tumor", as used herein, refers to all nooplastic cell growth and proliferation, whctlt malignant or beni and all pr-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe he physiological condition in mammals that is typically characterded by unrogulated cell grow4. Examples of caner include but are not liraited to, carcinoma, lymphoma, blastoma, saornoa, and leukemia- More particular exampls of such cancers include breast cancer. prostate cancer, colon cancer, squamous cell cancer, small-ccll long cancer, non-small cell lung cancer, gastrintestinal cancer, pancreatic cancer, glioblastoma, cervical 4ancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endomeirlal carcinoma, salivary gland carcinuma, kidy cancer, vulval nces, thyroid cancer, hepatic carcinoma and various types of head and nck cancer.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prcvqnts the finiction of c4s and/or causes destruction of cells. The term is intended to include radioactive isotopes cj 1131. 1125, Y90 and Rel86), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial fungal, plant or anial origin., or fragments thereof.

A "chcmotherapeuic agent" is a chemical compound useful in the treatment of cancer. Examples of 9 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:24 FAX 61 3 92438333 GRIFFITH HACK .016 chemothenpeutic agens include adriamycin, doxorubicin. pizubicin, 5-fluorouracil, cytosine rabinoside ("Ara-C), cyclophosphamide, thiolepa, busulfan, cytoxin, taxoids, e.g. paclitaxtl (Taxol, Bristol-Myr Squibb Oncoloy, Princeton, NJ), and doetaxel (Taxotrc*, Rhone-Poulene Rorer, Antony, France), loxotere, ethotrac, cisplatn.

melphalan, vinblastine, bleomycin, atoposide, ifosfamide, mitomycin C, mitoxantrone, vicriatine, vinrbie carboplalin, rtaeniposide. daunoiycin, carminomycin, aminoptorin, dactinomycin, mitomycins. speraicis (see U S.

Pat. No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this d4finition are hormo al agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapristone.

A "growth iuhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in itro or in vivo. Ths, the growth inhibitory LIgent is one which significantly reduces the percentage of cells overxpressing such ge as in S phase. Examples of growth inhibitory agents include agents that block cell cycle progr sion (at a placc Ohr than S phase), such as agents that induce G01 arrest and M-phase arrest. Classical M-phase blockers include b vincas (vincristine anwl vinblastine), taxol, and topo IT inhibitors such as doxorubicin, epiibicin, daunorubicin, etoposide, and bleomycin. Thuse agents that arrest Gl also spill over into S-phase arsn l for example, DNA alkylating agents sven as tamuxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, fluorourail, and n-C. Further infoirmadon can be found in The Molecular Basis of Cancer. Iendelsohn and Is el, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineaplastic drugs "by 4urakami ot al. (VB Saunders: Philadelphia, 1995), especially p.13- "Doxorubicin" is an athracycline antibiotic.

The term '"cynokine" is a gencric term for proteins released by one cell population w ich act on another cell as intercellular medialors. Examples of such cytokines are lymphokines, monokines, and tjaditional polypeptde hormones. Included among the cytokines are growth hormone such as human growth horm,t N-ntbionyl hu an growth hormone, and bovine growth hormone; parathyrold hormone; thyroxine; insuli; proinsulin; relaxin; prorelaxin; and the like. As used herein, the term cytokine includes proteins from natural sources or ftom recombinant cell culture and biologically active equivalents of the native sequence cytokin s.

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric lycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chins. Each light ch li is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linlages varies among he heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regu)arly spaced itrachdn disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of cons t domains. Each light chain has a variable domain at one end (VL) and a constant domain at its oter end; the constt domain of the light chain is aligned with the first constant domain of the heavy chain, and tic light chain variabc domain is aligned with the variable domain of the heavy chain. Particular amino acid rsiduts are believed to frm an interface between the light- and heavy-chain variable domains.

I

The term "vuriable" refers to the fact that certain portions of the variable do ir differ extensivel3 in sequence among antibodies and are used in the binding and specificity of each particular aw body for its partict lar antigen. However, the variability is not ovenly distributed throughout the variable domaits of antibodies. It is concentrated In three segments called comnplementarity-determining regions (CDRs) or hyp rvnriable regions b COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:24 FAX 61 3 91.431 33 GRIFFITH HACK .017 in the light-chain and the heavy-chain variable domains. The more highly consrvcd portion of variable domaas are called the framework The variable domains of native heavy and light chains ch comprise four FR regions, largely adopting a psheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the -shect structure. The CDRs in each chain are held together in close proximity y the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigon-blnding site f antibodies (see Kabat .t al,, NIH PubL No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are not involved directly in biding an antibody to an antigen, but exhibit various effector functions such as participation of the antibody in antibody-dependent cellular toxicity.

"Antibody frugments" comprise a portion of an intact antibody, preferably the antigp binding or varia le region of the intact anidbody. Examples of antibody fragments include Fab, Fab', F(ab'2, and Fv fragmens; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); singl-chan antibody molecule and multispecific antibodies formed from antibody fragments.

Papain diges ion of antibodies produces two identical antigen-binding fragments. cld 'Tab" ragment, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two anigen-combining sites lnd is still capable of cross-linking antigen.

"PV' is the minimum antibody fragment which contains a complete antigen-recognition and -binding sit.

This region consists of a dimer of one heavy- and one light-chain variable domain in tight, no -covalent associai n.

It is in this configuration that the three CDRs of each variable domain interact to define an anigen-binding site an the surface of the VB.-VL dimcr. Collectively, the six CDRs confr antigen-binding speci city to the antibody.

However, even a single variable domain (or half of an Fv comprising only thee CDRa specfi for an antigen) has the ability to recognizy and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragmn also contains the constant domain of the light chain and the first ontant domain 1) of the heavy chain. Fab fragments differ from Fab fragments by the addition of a few i sidues at the carbcxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'- H is the designation herein for Fab' in which the oysteine residue(s) of the constant domains t a free thiol gro p.

(ab)2 antibody fragments originally were produced as pairs of Fab' fagments which have hinge cysteines betwn them. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobultns) fomn any vertebrate species be assigned t one of two clearly distinct t)pes, called kappa and lambda. based on the amino acid squences of their constant domains.

Dcponding (n the amino acid sequence of the constant domain of their heavy chain immunoglobulins an be assigned to difforent clase. There re five major classes of immunoglobulins: IgA, IgD, IgG, and IgM, d several of these may be further divided into subclasses (isotypes), Ig1, IgG2, IgO3, lgO4, IgA, and lgA "Singlo-chan Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wher in these domains are present in a single polypcptide chain. Preferably, the Fv polypepti e further comprise a polypeptide linker between the VH and VL domains which enables the sFv to form the des' ed structure for and pa binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodi s, vol. 113, Rosenb rg and Moore eda., Springer-Verlag. New York, pp. 269-315 (1994).

11 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:25 FAX 61 3J 92438333 GRIFFITH HACK [018 The term "dibodies" refers to small antibody fragments with two antigen-binding qilcs, which fragmncts comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the s e polypeptide chain (VI[ VL). By using a linker that is too short to allow pairing between the two domains on tie same chain, the domanh s ame forced to pair with the complementary domains of another chain dnd create two antie nbinding sites. Diabodies are described more fully in, for example, EP 404,097: WO 93/1116); and Hollinger et a Proc. Nail. Acad. Sci. USA, 90:6444-6448 (1993).

An "isolated' antibody is one which has been identified and separated and/or recoved fom a compon t of its natural environment Contaminant components of its natural environment are materials which would interf er with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, apid other proteinacecus or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified to greater than 95% 3y weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, tc a degree sufficient to obtain at leas 15 residues ofN-terminal or internal amino acid sequence by use of a spinni g cup sequenator, or to homogeneity by SOS-PAGE under reducing or nonreducing conditions using Coomas ie blue or. preferably, silver stain. Isolated antibody includes the antibody in situ within recombiant cells since at last one component of the antibody's natural environment will rot be present Ordinarily, howdver, isolated antiboly will be prepared by at least one purification step. i The word "l;bel" when used herein refers to a detectable compound or compositidn which is conjugal d directly or indirectly to the antibody so as to generate a "labelled" antibody. The label mayi be detectable by itslf radioisotope labels or flunrescent labels) or, in the case of an enzymatic label, may catalze chemical alterati of a substrate compound or composition which is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody of the preset invention can adhe.

Examples of solid phases encompassed herein include those formed partially or entirely o' glass controll pore glass), polysaccharides agarose). polyacrylanides, polystyrene, polyviny] alcohol aId silicones. In cein embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column an affinity chromatography column). This term also Includes a diScontinuous solid ph sc of discrete particles, Nuch as those described in U.S. Patent No. 4,275,149.

A "liposomc" is a small vesicle composed of various types of lipids, phospholipids nd/or surfactant whi h is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herin and, optionally, a chcmotherapeutic agent) to a mammal. The components of the liposome are commonl arranged in a bila er formation, similar to the lipid arrangement of biological membranes.

IT. mrnpositions and Methods of the Invention Fu-enfth PRO717 Polvpeotides The present invention provides newly identified and isolated nucleotide sequences encoding polypepti cs referred to in the prrsent application as PRO717. In particular. Applicants have idcntifd and isolated cDl A encoding a PR0717 polypcptide, as disclosed in further detail in the Example below. rTo Applicants present knowledge, the UNQ385 (DNA50988-1326) nucleotide sequence encodes a novel factor; uing BLAST and Fai A sequence alignment computer programs, no significant sequence identities to any know{ human proteins wee 12 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

I

19/09 2003 18:25 FAX 61 3 92438333 GRIFFITH HACK 1oi9 revealed.

PRO Polypepide Variants In addition to the full-length native sequence PRO polypeptides described herein, iI is contemplated at PRO polypcptide variants can be prepared. PRO polypeptide variants can be prepared by introducing appropriatr nucleotide changes into the PRO polypeptide DNA, or by synthesis of the desired PRO polyi ptide. Those skilld in the art will appreciate that amino acid changes may alter post-translational processes of ]e PRO polypepddes, such as changing the number or position of glycosylation sites or altering the membrane anloring characteristics.

Variations in the native full-length sequence PRO polypeptides or in various domains of the PRD polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the PRO polypcptide that results in a change n the amino acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide Optionally the variation is by substitution of at least one amino acid with any other amino aci in one or more of the domains of the PRO polypeptide. Guidance in determining which amino acid residue may b 9 inserted, substitutd or deleted without adversely affecting the desired activity may be found by comparing the equence of the PR polypeptide with that of homologous known protein molecules and minimizing the number o amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result if replacing one amino a d with another amino acid having similar structural and/or chemical properties, such as the reacement of a leucr e with a sorine, conservative amino acid replacements. Insertions or dclctions may optionhly be in the range of I to 5 amino acids. The variation allowed may be determined by systematically making itsertions, deletions or subsuttutions of amino acids in the sequence and testing the resuling variants for activity in the in vitro assy described in the Examples below.

In particular :mbodiments, conservative substitutions of interest are shown in Tabi 1 under the hadig of preferred substitutions. If such substitutions result in a change in biological activity, ien more substantial changes, denominated exemplary substitutions In Table 1. or as further described below in re erence to amino ad classes, are introduced and the products screened.

13 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

I

19/09 249Q1 :26 FAX 61 3 92438333 GRIFFITH HACK Q1020 Table I Original Exenplary Prcferred Residue Substitutions Substit Ala val; leu; ile val Arg lys; gin; asn lys Asn gln; his; lys; ar gin Asp glu glu Cys ser ser Gln(Q) ain asn Glu asp asp Gly pro; ala ala His asn; gin; lys; arg Us ie leI; val; met; ala; ph; norloucine leu Leu norleucine ile; val; met; ala; phe il Lys arg; gn: asn arg Met len; phe; ile leu Phe leu; val; ile; ala; tyr leu Pro ala ala Ser Ihr thr Thr cr ser Trp tyr: phe tyr Tyr trp; phe; ihr; ser Phe Val ile; lou; met; phe; ala; n;ducine leu Substantial modifications in function or immunological identity of the PRO polypebtide are accmplish by selecting substitutions that differ significantly in their offect on maintaining the stnruare of the polypoptIde backbone in the aren of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain. Naturall occurring residues are divided into groups based on common side-chain properties: hydrophobic: norloucine, met, ala, val, le, ile; neutral hydrophilic cys, Por, thr: acidic: asp, glu; basic: asn, gin, his, lys, arg; residues that influence chain orientation; gly, pro; and aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of those casses for another cbms.

Such substituted residues also may be introduced into the conservative substitution sites morc preferably, into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such as oligoncotideodiatd (she-di -d) nunagenesis, alanine scanning, and PCR mutagenesis. Sit-directed mutagenesis [Carter t Nucl. Ads .11:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis (W611s at al., en1, 14 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:28 FAX 81 3 924U1 GIFTHHC J2 GRIFFITH HACK Z021 (1985)], -restriction selection mutagenoshs [Wells Ci aL, Philos. Tnt IR. Soc. 1,ndon L orJ745 18) other known techniques can be performed on the clon ed DNA to produce the desired PRO poll pptide variant DNA Scanning amiao aid analysis can also be employed to identify one or MOre amino acids along a configo sequence- Among the prefrrrcd scanning amino acids are relatively small, neutral amino acqs. Such amino aokhs include alanine, glci nt, saint, and cysteine. Alanine is typically a prefened Scanning amino ricid among ibis gwp because it eliminiates the side-hain beyond the beta-carbon and is less likely to alter the main-chain conformnatio of the varint. Alanine is also typically preferrd beCause it is the most common wmino acid. ~urffict, it is frequen ly found in both buried and exposed positions ECreigt, Thrtis W.Femn&C. Y) Chotbia, J- MAL.

Hint 150:1 (1976)). if alanine substitution does not yield adequate amounts of variant, an Aotric amino acidca be usedL Modiftenlions; of PRO P01VDC3114Ms Covalent modifications of PRO polypeptides are included within the scope ofthis i~wcntion. One type of Covalent mnodification includes reacting targeted amino a"i residues of the PRO polypelpdde with an orgai derivatizing agent thai is capable of reacting with selected side chains or the N- or C- termingl residues of the PRO polypeptide. Derivatizatiun with bifunctional agents is useful, for instance, for crosshlnn a PRO polypeptid to a water-insoluble support matrix or surfacee far use in the method for purifying anti-PRO polypepflde antibodies. ed vice-versa. Commlonl y uised crosslikang agents include, 1 1. l-bisdiar~oBCCiyI).2-PhcUylithane, glusaraldehy ac, N-hydrotysuociinimdL esters, for example, estr3 with 4-azidomalicylk acid, homobifuhnti irniduesters, iud ing disuccindidkyl esters such as 3.3'-dithiobis(suciniidylopionhat), bifunctionial maleilnides such as his naaelnidol ,-ocaneand agents such as methyl-3-(RpazidopheDnYl)diiolPrPioi11idtC- Oihe modilications include demmidation or glum~minyl and asparaginyl rcsiduc4 to the correspondng glutamnyl and aspartyl residues, respectively, hydroxylalti of probate and lysine, phosphuiylahofl of hydroxyl grup of scryl or threonyl rcsidues, methylahion of the a-amino groups of lysine, arginine, and hista sdeS chains [T.E.

Creighton, gfjc tutr and Molecular Proertles W.H Freeman Co., San Fmancl o, pp. 79-86 (198:1)], acetylailon of the N-tnnninal amine, and amidation of any C-tcmonlna] carboxyl group- Another type of covalent modification of the PRO polypeptidam included within thj scope of this invent ion comprises altering thu native glycosylatlon pattern of the polypeptide. "Altering the nativ, glycoSylation patt n is intended for purposes herein to mean deleting one or more carbohydrate, moietis foiund inlia native sequence PR0 polypeptide, andhlof adding one or more glycosyladon sites that arc not present in the native sequence PR0 polypeptide, and/or alteration of the ratio and/or composition of the sugar residues attach~d to the glycosylation site(s).

Addition of glycosylation sites to the PRO polypeptide may be accomplished by Irering the amino aid sequence. The altendtaon may be made, for example, by the addition of. or substitution by, one or more serini or threonine residues to the native sequence PRO polypeptide (for 0-linked glycosylation itcs)' ThePRO polypepide amnino acid sequence may optionally be altered through changes ate DNA level, paricu~ bymtaigth, encoding the PRO polypeptde at preselected bases such That codons are generaed that will 6mansate into the de m amino acidsi-s COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09. 29q,.18:26 FAX 61 3 92438333 GRIFFITH HACK *022 Another meas of increasing the number of carbohydrate Ionieties on the PRO polyepide polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are desbed in the art, in WO 87/05330 poblisbe 11 September 1987, and in Aplin and Wriston, CRC Cdt, Rev. o m, pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may be accoD lished chemically r enzymatically or by mutational substitution of codons encoding for amino acid residues th serve as targets or glycosylation. Chemical deglycosylation techniques are known in the art and desctied, for instance, y flakimuddin, ci al., Ach. Biochem. Bionhys, 2:52 (1981) and by Edge et al., Anal. Biochem.,18:131 (198 1).

Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use ofa variety of endo- ad exo-glycosidases as described by Thotakura ct al,, Meth. Enanol.. 138:350 (1987).

Another type of covalent modification of PRO polypptides of the invention co ses linking the ILO polypeptid to one of a variety of nonproiinaccous polymers, polyethylene glycol, po ypropylene glycol, or polyoxyalkylcnes, in tte manner set forth in U.S. Patent Nos. 4,640,835; 4.496,689; 4,301.144 4,670,417; 4,791,192 or 4,179,337.

The PRO polypeptides of the present invention may also be modified in a way to for n a chimeric m!e ue comprising a PRO polypepride fused to another, beterologous polypeptide o amino acid sequnce. In ne embodiment, such a chimeric molecule comprises a fusion of the PRO polypeptide with a ag polypeptide whh provides an epitupo to which an anti-tag antibody can selectively bind. The epitope tag is 4 encrally placed at he amino- or carboxyl- aminus of the PRO polypeptide. The presence of such epitope-ta:ed forms of the P O polypeptide can be dowctd using an antibody against the tag polypcptide. Also, provision oflhIt epitope tag ena los the PRO polypeptide to be readily purified by affinity purification using an anti-tag antibddy or another type of affinity matrix that binds to the epitope tag. In on alternative embodiment, the chimeric ecule may compre a fusion of the PRO polypeptide with an immunoglobulin or a particular region of an immuno 1oblin. For abivalent form of the chimeric molecule, such a fusion could be to the Pc region of an ISG molcule Various tag polypepfidcs and their respective antibodies are well known in the art. Examples include plyhistidine (poly-his) or poly-histidins-glycine (poly-his-gly) tags; the flu HA tag polypeptide d its antibody 12CAS (Field at al., Molll. Biol., 8:2159-2165 (1988)]; the e-my tag and the IF9 3C7. 6110, G4, B7 and 9E10 antibodies thereto [Ean et al., Molecular and Cellular Biolov, 3610-3616 (1985)]; and th Herpes Simplex vrus glycoprotein D (gD) tag and its antibody [Paborsky ct aL, Protein Enginera.3(6):547-553 (1990)). Other tag polypeprides include the Flag-peptide [Hopp ot al., BioTechnolony. :1204-1210 (1988)]; tho KT3 epito pepide (Martin ct at., SCle,, 2,:192-194 (1992)], an a-bulin epitope peptide [Skinner etal., J. jiL Che., :15163- 166 (1991)]; and the 77 gene 10 protein pepdde tag [Lutz-Freyermuth at al., Proc. Natl Sd. §ISA. :693- 6397 (1990)J.

PrePratin of PRO Polyptides Tho descripcion below relaes primarily to production of PRO polypepides by culuring cells ansfom or transfected with a vector containing the desired PRO polypeptide nucleic acid. It is, of cc urse, contemplated that alternative methods, which are well known in the art, may be employed to prepare the j RO polypeptide. or instanco, the PRO polypcptide sequence, or porrions thereof, may be produced by direct pepdde synthesis ing 16 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:27 FAX 61S 3 92438333 GRIFFITH HACK [023 solid-phase techniques [see, Stewart et aL, Solid-Phasc Fpide Synthesis. W.H. Freemen Co., San Francis o, CA (1969); Merrifild, J. Am. Chem Soc.. :2149-2154 (1963)]. In vitro protein synthesis may be perfo using manual techniqus or by automation. Automated synthesis may be accomplished,: or insune, using in Applied Biosystemsn Pzptide Synthesizer (Foster City, CA) using manufacturer's instructon. Various portions of the desired PRO polypoptide may be chemically synthesized separately and combined using ihemical or enzym ic methods to produce the full-length PRO polypeptide.

A. Talation of DNA Eacoding PRO Polvnetides DNA encodijng PRO polypeptides may be obtained from a cDNA library prepredom tissue beieved to possess the desired PRO polypcptide mRNA and to express it pt a detectable level. Accordingly, human PRO polypeptide DNA can be conveniently obtained from a cDNA library prepared from human ti su ssuch as describd in the Examples. The PRO polypeptide-encuding gene may also be obtained from a -enomic library or y oligonucleotide synth(sis.

Libraries can be screened with probes (such as antibodies to the desired PRO polypoptide or oligonuclcotides of at least about 20-80 bases) designed to identify the gene of Interest or tie protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted usin; standard procedurs, such as described in Sambrook et al., Moleculr Clonine: A Laboratory Manual (New Yor Cold Spring Har or Laboratory Press, 1989). An alternative means to isolate the gene encoding the desired PR) polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach ci al., PCR Primer:A Laboratory Manual (Cold Sprig Harbor Laboratory Pess. 1995)].

The Examples below describe techniques for screening a cDNA library. The oli mucleotide sequen es soloced as probes should be of sufficient length and sufficiently unambiguous tha false p sitives are minimizd.

The oligonucleotide is preferably labeled such that it can be detected upon hybridization to D NA in the library being screened. Methods of labeling are well known in the art, and Include the use of radiolabel like "P-labeled A P, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency nd high stringency, ar provided in Sambroo k et sua.

Sequences identified in such library screening methods can be compared and aiigned to other kno n sequences deposited and available in public databases such as GenBank or other privat sequence databases.

Sequence idendty (at either the amino acid or nucleotide level) within defined regions of the molecule or across he full-length sequence can be determined through sequence alignment using computer soft .are programs such as BLAST, ALIGN, DNAstar, and INHERIT which employ various algorithms to measure h mology.

Nucleic acid having protein coding sequence may be obtained by screening alc ted cDNA or geno lic libraries using the deduced amino acid sequence disclosed herein for the first time, a d, if necessary, us ng conventional primer extension procedures as described in Sambrook et al., su to dtect p sors and process ng intermediates of mRNA that may not have been reverse-transcribed into cDNA.

B. Selection and Transfomtion of Hot Cells Host cells are transfucted or transformed with expression or cloning vectors de.cribed herein for PRO 17 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18&LLEhAI 61 3 92438333 GRIFFITH HACK 1024 polypeptide production and cultured in conventional nutrient media modified as appropriate ft selocting transformantLs, or amplifying the genes encoding the desired sequences. The cultu media, temperaturc, pR and the like, can be selected by the skilled artisan without unduec gencral, principles, protocols, and practical techniques for maximizing the productivity of cell in Mammalian Cell Biotechnologyv a Practical Aplrach, M. Butler, ed. (IRL Press, 1991 Methods of transfetion amr known to the ordinarily skilled artisan, for example, CaP Depending on the host cell used, transformation is performed using standard techniques apI The calcium trealmtt employing calcium chloride, as described in Sambrook ct al., jp generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infectit tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Q[G WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, i precipitation method of Graham and van der Eb, Vjflogy, 2:456-457 (1978) can be empl of mammalian cell hot system transformations have been described in U.S. Patent No. 4.399 Into yeast arc typically carried out acconlrding to the method of Van Solingen er al., LB Hsiao t al, Proc. Nal. Acad. Scl. (USA), 76:3829(1979). However, other methods for intro such as by nuclear microiqjection, elcetroporation, bacterial protoplast fusion with intact czl polybrene. polyornithmne, may also be used. For various techniques for transforming mamr ct al., Methods in ez. yms2D. L];527-537 (1990) and Mansour et al., N r, 336:348-2 Suitable host cells for cloning or expressing the DNA in the vectors herein includ higher eukaryote cell. Suitable prokaryotes include but are not limited to eubacteria, sue Gram-positive organisms, for example, Entarobacturiaceae such as E. coli. Various E. c available, such as E r ol 112 strain MM294 (ATCC 31,446); K coli X1776 (ATCC 31,537 (ATCC 27.325) and KS 772 (ATCC 53,635). Other suitable prokaryotic host cells include I as Esherichia, L col. Enterombcter, Erwbnia, Iebstella, Proreus, Salmonella, So SerraTia Serra6t nearcescans, and Shigella, as well as Bacilli such as B. subilir and B lichenfonrmls 41P dh closed in DD 266,710 published 12 April 1989). Pseudomonas such Streptomyces. Various E. coli strains are publicly available, such as colt K12 strain A coli X1776 (ATCC 31537); E. col swain W3110 (ATCC 27,325); and KS 772 (ATCC 51 am illustrative rather than limiting. Strain W3110 is one particularly preferred host or par common host strain fir recombinant DNA product fermentations. Prcferably, the host cells of protolytic enzymes. For example, strain W3110 may be modified to effeat a genetic encoding proteins endogenous to the host, with examples of such hosts including E coll W has the complete genotype tonA; coli W3110 strain 9E4t, which has the complete gene W3110 strain 27C7 (ATCC 55,244), which has the complotu genotype tonApr phA El.

ompT khan'; coli W3110 strain 37D6, which has the complete genotype tunA ptr3 phoA El ompT rbs7 LthrvG kan'; E. coUl W3110 strain 40B4, which is strain 37D6 with a non-k deletion mutation; and an E coU strain having mutant periplasmic pmtcase disclosed in U..

18 inducing promote 1 conditions, such experimentation.

cultures can be fou and Sambrook et i and electroporatic ropriate to such ce or electroporation n with Agrobacteri ,22:35 (1983) a c calcium phosph yed. General aspe 216, Transourmatic 1:30946 (197) a ngDNA into c b, Or polycations, e alian cells, see Kco 2 (1988).

prokaryote, yeast, as Gram-negative a strains are publi E cli strain W31 nterobacteriaceae sr mnaUa typhimuriz lichaniformis as P. aerginosa, iE 4294 (ATCC 31,44 These exam; t host because it i nesinrimal amo mutation in the ge i10 strain 1A2, wb yp tounA ptr3; E a (argF-lac)169 de '5 (argF-lac)169 d rmycin resistant di i. Patent No. 4,946,' COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 10/09 2003 1 AJ9A PA31 92436333 ~RIFFITH ACK 4025 _IJ Ir in/fO ~Art 1-9RFAT i 2 213521GRIFFITH HACK 02 issued 7 August 1990. Alternatively, in vitro methods of cloning, PCR or other nuleic acid polymor reactions, are suitable.

In addition to prukaryotes, euktaryotic microbes such as filamentous fungi or yeast dre suitable cloning expression hosts for PRO polypeptid-encoding vectors. Saccharomyces cerevisiac is a commonly used lon eukaryotic host microrganism. Others include Schizosaccharomyces pombe (Bach and Ntirsc, Nature, 290: 1 [1981]; EP 139,383 published 2 May 1985); Kliyveromyces hosts Patent No. 443,$529; Fleer at a BiolTachnolof, 2: 968-975 (1991)) such as, K. lactis (MW9-SC, CBS683 CBS4574; Luvencourt et e I. Bacterial., 737 K. fragilis (ATCC 12,4241 K bulgaricus (ATCC 16,045), K wicke if (ATCC 24,17 K- walii (ATCC 56,500), K drosophilarun (ATCC 36,906; Van den Berg t al, BioTechnblo. 8: 135 (199( K. thermnoleramns, and K maalanus; yarrowia (EP 402,226); Pichia pastoris (EP 183,074; Srcmokrishna etat.

Basic Microbiol.. Uj: 265-278 [19881); Candda; Trichoderma resia (EP 244,234); Ncur4 panra crssa (Cav at, Proc. Natl, Acad. Sci. USA, 2: 5259-5263 [1979]); Schwwmionoces such as Schwannilyces occidenlaUr 394,538 published 31 October 1990); and filanmenrous fungi such as, Neurospra, Pent1iium, TolypocLadi (WO 91100357 published 10 January 1991), and Asparglus hosts such as A. nidulans (Balance ser at. Bioh Biophvs. Rea. Comm in., 112: 284-289 1 983]; Tilburn et atl, f,m 2: 205-221 (19831; Y ton ar at, Pro Acad. Sci. USA. 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO 4: 475479 [1985] Methylotrpic yeasts are suitable herein and include, but are not limited to, yeast capable df growth on motha selected from the genera consisting of Hansenoda, Candida, KIoseckera, Pichia. Saccharodiyces. Torulopsis. a Rhodorula. A list oF specific species that are exemplary of this clias of yeasts my be £oud in C. Anthony, 3 Biochemistry ofMetlvlotosphS. 269 (1982).

Suitable host cells for the expression of glycosylated PRO polypeptides are derived from multicolit organisms. Examples of invcrtebrate cells include insect cells such as Drosophila 52 and Spoptrn 5f9, as v as plant cells. Examples of usefl mammalian host cell lines include Chinese hamster ovary CHO0) and COS ce Mom specific examples include monkey kidney CVI line tranformed by SV40 (COS-7, ATC CRL 1651); hur embryonic kidney line (293 or 293 cells subaloned for growth in suspension culcte, Grahm at al., 3. CnY ii -59 (1977)); Chinc hamster ovary cells/-DHFR (CHO, Urlaub and Chasn, Prog. Nall. ad. Sci. USA, 22:4' (1980)); mouse ertoli cells (TM4, Mather, Biol. Repro, 21:243-251 (1980)); human lWig cells (W138, AT CCL 75); human livar colls (Hop G2, I 8065); and mouse mammarny tumnor (IuM T 060561, ATCC CCL51).

selection of the apprcpriate host cell is deemed to be within the skill in the at C. Selection and Use of a Renlicable Vector The nucleic acid cDNA or gonomic DNA) encoding a desired PRO polypepe may be inserted j areplicable vector for cloning (amplification ofthe DNA) or for expression. Various vector are publicly availa The vector may, fbr exampe, be in the form of a plasmid, cosmid, viral particle, or phage. h appropriate nac acid sequence may be inserted into the vector by a variety of procedures. In general, 4A is inserted ine appropriate restrictin endonuclease sial(s) using techniques known in the art. Vector c mpontts generally inch but are not limited to, one or mre of a signal sequence. an origin of replication, one or flO marker genes enhancer element, a promoter, and a tmnscription termination sequence. Construction of suliable vectors contai one or more of these components employs standad ligation techniques which are known tp the skilled artisan COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

Y

19/09 203 18:28 FAX 61 3 92438333 GRIFFITH BACK 1026 The PRO polypeptide of interest may be produced recombinantly not only directl but also a afin polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypcpride having a spec o cleavage site at the N.terminus of the mature protein or polypeptide. In general, the sig;d sequence may be a component of the vectur, or it may be apart of the PRO polypeptide DNA that is inserted into vector. The sig al sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphata, penicillinase, pp, or heat-stable enterotoxin I leaders. For yeast secretion the signal sequence may be, the yeast invertae loader, alpha factor leader (including Saccharomyces and Kluyveramyce a-factor leaders, the latr described in U.S. Patsnt No. 5,010,3 82), or acid phosphatase leader, the C. albicans glu osamylase leader 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion f the protein, such as signal sequences fmrom secreted polypeptides of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables th vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication fi-om the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 L plasmid orl in is suitable for yeast, and various viral origins (SV40, polyona, adenovirus, VSV or BPV) are useful for cloning vectors in mnammalian cells.

Expression and cloning vectors will typically contain a selection gene. also termed a sclotable rarkr Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, ampicillin, neomycin, methotrexute, or LOtracycline, complement auxotirophic deficiencies, or s y critical nut s not available from complex media, the gene encoding D-alanine racemase for Bacilli.

An example of suitable selectable markers for mammalian cells are those that enae the identification of cells competent to take up the PRO polypeptide nucleic acid, such as DHFR or thymidinc .inas An appropriate host cell when wild-cype DHFR is employed is the CHO cell line delicient in DHFR activity, prepared nd propagated as described by Urlaub et al. Proc. Nat. Acad. Sci. USA. f;4216 (1980). A sitable selection gne for use in yeast is the rrpl gene present in the yeast plasmid YRp7 [Stinchcomb ct al., atg, =:39 (1979); Kingsman et al, Lzenq. 2:141 (1979); Tachemper at al., Qn=. Jl: 157 (1980)1. The rupl pe provides a ciect on marker for a mutant strain of yeast lading the ability to grow in trypphan, for example, ATCC No. 44076 aor P 4- I poncs, *nfA:g, j: 12(1977)].

Expression and cloning vectors usually contain a promoter apcrably linked to the PtO polypeptide n eic acid sequence to direct mRNA synthesis. Promoters rcoognized by a variety of potential hot cells are well known.

Promoters suitable for use with prokaryotic hosts include the f-lactamas and lautose prorratcer systems (Chang et al., Nature, 27:6 15 (1978); Goeddel ct al., Nature 281:544 (1979)], alkaline phosphatLc, a tryptophan (Irp) promoter system [GOacddel, Nucleic Acids Res., f:4057 (1980); EP 36,776], and hybrid prjmoters such as the tac promotor [deBoer et al., Proc. Natl. Acad. Sci USA 3R:21-25 (1983)]. Promoters for use i bacterial systems aso will contain a Shine-D)algarno sequence operably linked to the DNA encoding the doired PRO polypep do.

Examples of ;uitable promoting sequences for use with yeast hosts include the promoters for 3phosphoglycerate kinase (Hitzoman et al., J. Biol. Chem.. 29;2073 (1980)] or other glyc lytic enzymes [ess et al., I. Adv, Enzyme R, 2:149 (1968); Holland, Bigochenistr .172:4900 (1978)], such as enlase, glyceraldehyd 3- COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:28 AL_!01 3 92438333 GRIFFITH HACK .027 phosphate dehydrogenese, hexokinase, pyruvate dcarboxylase, phosphofructokinase, glucose-6-phosphte isomermse, 3-phosphoglycerate muta, pyruvate kinase, triosephosphate isomerase, phospholucose isomerase, ad glucokinase, Other yeast Fromoten, which are inducible promoters having rho additional advttage of transcrpt n controlled by growth conditions, arc the promoter regions for alcohol dehydrogenase 2, itocytoechromne C, ac phosphatase, degradalive enzymes associated with nitrogen metabolism, metallothionein, gIucraldehyde-3-phosphaic dehydrogenrc, md enzymes responsible fr maltose and galacose utilization. Suitable vectors and promo s for use in yeast expression are further described in EP 73,657.

PRO polypoptide transcription from vectors in mammalian host cells is contro led, for example, y promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 publihe July 1989), adenovirus (such as Adonovirus bovine papilloma virus, avian sarcoma vis, cyromegalovirus a retrovirus, hepatitis-B virus and Simian Vimrus 40 (SV40). from hterologous mamrnmalian promolers, the an in promoter or an immunoglobulin promoter, and from heat-shock pronmours, provided such pmouters arc campatile with the host ccll systms.

Transcription of a DNA encoding the desired PRO polypptide by higher eukaryows may be increased y inserting an enhancer sequcnce Into the vector. Enhancers are cis-acting elements of DNA, jusually about from t0 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequcnce4 are now known fi m mnammalian genes (globin, clastase, albumin, a-fetoproteln and insulin). Typically, hoviever. one will use enhancer from a eukmayolic cell virus. Examples include the SV40 enhancer on the lain side 6f the replication aui n (bp 100-270), the cytcnegalovirus early promotur enhancer, the polyoma onhancer on the la' side of the replicat3 origin, and adenovirus enhancrs. The enhancer nmay be spliced into the vector at a posit"o 5' or 3' to the O polypeptide coding saiquence, but is preferably located at a site 5' from the promoto.- Expression vectors used in oaukaryoric host cells (yeast, fungi, insect, plant, anil, human, or nucleatd cells from other multicdllular organisms) will also contain sequences necessary for the lerm nation of transcripti n and for stabilizing the mRNA. Such sequences ae commonly available from the 5' and, oc ionally untrans d regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide cents transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO polypetdes.

Still other nethods, vectors, and host cells suitable for adaptation to the synthesis 4f PRO polypeptides In recombinant vcrictbnite cell culture are described in GOthing et al., Natue 29:620-625 (1981); Mantci ct E, Nafis. 281:40-46 (1979); EP 117,060; and EP 117,058.

D. Deteetl Gene malificLation/Exrenuion Gene ampli.lcation and/or expression may be mcasured in a sample directly, for ample, by conventio al Southern blotting, Northern blotting to quntitalte the transcription of mRNA [Thomas, Pro. Acad. Sci. US 217:5201-5205 (1980)1, dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled pro, based on the sequentcs provided hcrein. Alternatively, antibodies may be employed tha can rccognize apeci duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DN4-procin duplexe. he antibodies in turm my be labeled and the assay may be carried out where the duplex is bnmd to a surface, so tat 21 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:29 FAX 613 92438333 GRIFFITH HACK .028 upon the formation of duplex on the surface, the presence of antibody bound to the duplex n be detected.

Gene expression, alternatively, may be measured by immunological methods, such as wmunohistocbemnc a staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate dir tly the expression rf gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be propae against a native sequence PRO polypeptide or against a synthetic peptide based on the DNj sequences provid hrcin or against exogonous sequence fused to a PRO polypeptido DNA and encoding a spe ific antibody epitop.

E. Purification of Poleieptide Forms of PRO polypeptides may be recovered from culture medium or from host cell lysales. If membraebound, it can be released from the membrane using a suitable detergent solution Triton-i 100) or by enzyma cleavage. Cells cmployed in expression of PRO polypeptidcs can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents hmaybeed to purify PRO polypeptides from recombinant cell proteins or polypcptides, The folliwing procedures are exemplary of suitable purification procedures; by fractionation on an ion-exchange column; ethanol precipitaic n; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEA chromatofoctsig; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex C-7k: protein A Sephr se columns to remove contaminants such as IgG; and metal chelating columns o bind epitopet ged forms of the PI.0 polypeptide. Various methods of protein purification may be employed and such methods known in the art id described for example in Deutscher, Method in Envmoloev. 182 (1990); Scopes, Prin P cat:Prin s and Practice Springei-Verlag, New York (1982). The purification sep(s) selected will depend, for example on he nature of the production process used and the particular PRO polypeptide produced.

Uses for PRO Polventides Nucleodide sequences (or their complement) encoding the PRO polypeptides of the present invention lu v various applicatons ili the an of molecular biology, including uses as hybridization probes, in chromosome and ge n mapping and in the generation of anti-sense RNA and DNA- PRO polypeptlde-encoding n clcic acid will also be useful for the preparation of PRO polypeptides by the rcombinant techniques described herein.

The full-length native sequence PRO polypeptide-encoding nucleic acid or prdoios thereof, may be u cd as hybridization probes for a cDNA library to isolate the full-length PRO polypeptide gene or to isolate still oth c genes (for instance, those Encoding naturally-occurring variants of the PRO polypcptide or RO polypeptides fr m other species) which have a desired sequence identity to the PRO polypeptide nucleic acid equonces. Optiona ly, the length of the pmres will be about 20 to about 50 bases. The hybridization probes mi y be derived from he nucleotide sequence of any of the DNA molecules disclosed herein or from genomic sequencs including promou rs, enhancer elements anm introns of native sequence PRO polypeptide encoding DNA. By way bf examplc, a screun ng method will comprise: isolating the coding region of the PRO polypeptide gone using the kr own DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a vaiety of labels, iclud ng radionucleotides sucil as "P or or enzymatic labels such as alkaline phosphatase co pled to the probe via 22 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:29 FAX 61 3 92438333 GRIFFITH HACK .029 avidln/biotin coupling systems. Labeled probes having a sequence complementary to tha of the specific PRO polypeptide gene of the present invention can be used to screen libraries of human cDNA, geoomic DNA or m A to determine which mumbers of such libraries the probe hybridizes to. Hybridizalion techniques are described in fwther detail in the Examples below, The ESTs diclosed in the present application may sihilarly be employed as probbs, using the methols disclosed herein.

The probes may also be employed in PCR techniques to generate a pool of scquencs for identification of closely related PRO polypeptide sequences.

Nucleotide sequences encoding a PRO polypeptide can also be used to construct hfbridizaion probes or mapping the gene whcch encodes that PRO polypeptide and for the genetic analysis of individuals with gne ic disorders. The nucleotide sequences provided herein may be mapped to a chromosome an spucific regions o a chromosome using known techniques, such as in situ hybridization, linkage analysis agains known chromoso al markers, and hybridization screening with libraries.

When the coding sequence for the PRO polypcptide encodes a protein which binds to another protein, ti PRO polypeptide can be used in assays to identify its ligands. Similarly, inhibitors of the r+ccptorllgand bindig interaction can be identified. Proteins involved in such binding interactions can also be used to screen for p Cil or small molecule inh bitors or agonists of the binding interaction. Screening assays can bl designed to find lead compounds that mimic the biological activity of a native PRO polypepide or a ]igand for he PRO polypeptid Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making the M particularly suitable fur identifying small molecule drug candidates. Small molecules conteom latd include synthleic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-pro! in binding assays, biochmiical screening assays, immunoassays and cell based assays, which ac well characterized in the art.

Nucleic acids which encode a PRO polypeptide or its modified forms can also be psed to generate cit r transgcnic animals or "knock out" animals which, in turn, are useful in the developaept and screening of thcrapeutically useful reagents. A transgcnic animal a mouse or rat) is an animal havng cells that contain a transgene, which traigene was introduced into the animal or an ancestor of the animal at a prenatal n embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a trasgenic anital develops. In one embodiment, cDNA encoding a PRO polypeptide of interest can be used to clone genomic D A encoding the PRO polypeptlde In accordance with established techniques and the genomic sequences used to generate trasgenic animals that contain cells which express DNA encoding the PRO pol vepiide. Methods or generating transgenic animals, particularly animals such as mice or rats, have become con entonal in the art and ar described, for example, in U.S. Paten Nos. 4,736,866 and 4.870,009. Typically, p icular cells would be targeted for PRO polypeptide transgene incorporation with rissue-specific enhancers. 'jransgenic animals tl at include a copy of a transgene encoding a PRO polypeptide introduced into the germ line of the animal at n embryonic stage can be used to examine the effect of increased expression of DNA encodi4 the PRO polypept le.

Such animals can be used as tester animals for reagents thought to confer protection from, fc example, pathologi l conditions associated with its overexpression. In accordance with this facet of the invnti n, an animal is treaed 23 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 18/09 2003 18:3 fA 01l 3 92438333 GRIFFITH HACK l030 with the reagent and a reduced incidence of the pathological condition, compared to unireate animals bearing 1e trianagene, would indicate a potential therapeutic intervention for the pathological condition Alternatively. non-human humologues of PRO polypeptides can be used to constr t a PRO polypeptide "knock out" animal which has a defoctive or altered gene encoding the PRO polypeptide of interest as a result f homologous recombinantion between the endogenous gene encoding the PRO polypeptide and terod genomic D A encoding the PRO polypeptide introduced Into an embryonic cell of the animal. For exaie, cDNA oncodins a PRO polypeptide can lie used to clone genomic DNA encoding the PRO polypeptide in accordance with establish techniques. A portion of the genomic DNA encoding a PRO polypeptide can be deleted or replaced with anothe gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically. seveal kilobases of unaltered flanking DNA (both at thc 5' and 3' ends) are included in the voctor tsee Thomas ad Capecbi, Cell, 51:501 (1987) for a description of homologous recombination vectors]. Te vector is introdu into an embryonic stei cell line by electropration) and cells in which the introduced D4A has homologo ly recombined with the endogenous DNA are selected Lsee Li ct al, Cell. :915 (1992)]. The selected cells re then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras [see Bradey, in Tcratocarcinornmas and Embryonic Stem Cells: A Pracical Approach, B. J. Robrtson, ed (MIL, Oxford. 198 pp. 113-152]. A chimric embryo can then be implanted into a suitable pseudopregnant feiale foster animal a d the embryo brought to term to create a l"knock out" animal. Progeny harboring the homolog aly recombined D A in their serm cells can be identified by standard techniques and used to breed animals in whit all cells of the a al containl the homologously recombined DNA. Knockout animals can be characterzed for instance, for their abity to defend against certain pathological conditions and for their development of pahological cdiions due to abs ce of the PRO polypeptide.

When in vive administration of a PRO polypeptide is employed, normal dosage ajnounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably a] out pg/kg/day to mg/kg/day, depending upon the route of administration. Cidance as to particular dosages and methods of delivy is provided in the litejature; see, for example, US. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipd that different formulations will be effective far different treatment compounds and dirorent disorders, tat administration targeting one organ or tissue, for example, may necessitate delivery in a man ner different from tt to another organ or ti sue.

Wher sustained-release administration of a PRO polypeptide is desired in a roimulation with relase characteristics suitable for the treatmennt of any disease or diasorder requiring administration the PRO polypeptile microoncapsulation of the PRO polypeptide is contemplatd. Microcnapsulation of red mbinant proteins or sustjined release has been successfully performed with human growth hormone (rhOH) interfron- (rhIFN intarlcukin-2, and MN rgpl20. Johnson at aL, Na. Me. 2: 795-799 (1996); Yasuda Biome Ther., 27; 1221-123 (1993); Hors at al., ijEehnalogy. 755-758 (1990): Cleland, "Design and Production f Single Immni on Vaccines Using Polylactide Polyglycolide Microsphere Systems," in VcciE Design: E Subuit adAdiat A@gwag, Powell and Newman, eds, (Pleanum Press: New York 1995), pp. 439462; WO 9103692, WO 96/40072, WO 96/07399; and U.S Pat. No, 5,654,010.

The sustained-release formulations of those proteins were developed using poly-lactic-coglycolic aid 24 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:30 FAX 61 3 92438333 GRIFFITH HACK (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation produ of PLGA, lactic and glycolic acids, can be cleared quickly within the human body. Moreovcr, the degradability this polymer can be adjusted from months to years depending on its molecular weight and composition. Lew "Conrolled release of bioactive agents from lactide/glycollde polymer," in: M. Chasin and R. Langer (Ed Biodegradable Polyms as Dr .Doelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

For example, for a formulaton that can provide a dosing of approximately 80 g/kg day in mannals w a maximum body weight of 85 kg, the largest dosing would be approximately 6.8 mg of the PRO polypepLide I day. In order to achieve this dosing level, a sustained- release formulation which contains a maximum possil protein loading (15-20% w/w PRO polypeptide) with the lowest possible initial burst is necessary.

continuous (zero-order) release of the PRO polypeptide from inicroparticles for 1-2 week Is also desirable.

addition, the eneapsul:ned protein to be released should maintain its integrity and stability o ler the desired relei period.

The compounds of the present invention can be formulated according to know methods to prep' pharmaceutically usel compositions, whereby the PRO polypeptide hereof is combinc in admixture wit pharmaceutically acceptable carrier vehicle. Suitable carrier vehicles and their formulation, inlusive of other bun proteins, human serum albumin, are described, for example, in Remington's Pharmaceur cal Sciences, 16th c 1980, Mack Publishing Co., edited by Oslo et al the disclosure of which is hereby incorpo ated by reference.

Dosages and desired drug concentrations of pharmaceutical compositions of the pres nt invention may vi depending on the panicular use envisioned. For example, in the treanent of deep vein tl ombosis or pcriphe vascular disease, "bolus" doses, will typically be preferred with subsequent administrations t eing given to main an approximately constant blood level, preferably on the order of about 3 pgnl.

However, for use in connection with emergency medical care facilities where infusion capability generally not available and due to the generally critical nature of the underlying disease (e embolism, infan it will generally be desirable to provide somewhat larger initial doses, such as an intraveno s bolus.

Andt-PRO Podcptide Antibodies The present invention further provides anti-PRO polypcptide antibodies. Exemplary antibodies inlu polyclonal, monoclonal, humanized, bispecific, and hereroconjugate antibodies.

A. Polydvonal Antilmdies The anti-PRO polypeptide antibodies may comprise polyclonal antibodies. Methods of prepari polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be rted in a mammal, example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizi agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrape toneal injections. 7 immunizing agent maty include the PRO polypeptide or a fusion protein thereof. It may beluseful to conjugate 1 immunizing agent to a protein known to be immunogenic in the mammal being Immuni4ed. Examples of st immunogenic proteins include but ar not limited to keyhole limpet hemocyanin, m albumin, bovi thyroglobulln, and soybean trypsin inhibitor. Examples of adjuvants which may be em oyed include Freun 1031 ts of is, Cr le

A

Tn se a an a., Iry adn in is

I),

de ng "or ng he he ch ne Ns COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 I I II 19/09 2003 18:31. EAKL3 92438333 GRIFFITH HACK complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose diorynomycolate). ch immunization protocol may be selected by one skilled in the art without undue cpcrximenta 'ion.

B. Monoclonal Antibodies The anti-PRO polypeptide antibodies may, alternmatively, be monoclonal antibodies. uoonal antibod s may be prepared using hybridom methods, such as those described by Kohler and Milstin,. ture, 256-495 (197).

In a hybridoma method, a mouso, hamster, or other appropriate host animal, is typicall immunized with immunizing agent to elicit lymphocytes that produce or are capable of producing estibodi that will specifi y bind to the immunizing agent Altematively, the lymphocytes may be imnmunized in vitro.

The immunizing agent winll typically include the PRO polypeptide of intarest or a ion protein theref.

Generally. either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or splern cells or lymph node clls are used if non-human mammalian sources are desired. The lymp ocytes are then fund with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to orm a hybridoma ca [Onding, Monoclonal Antibodies: PrincinlsndPractica. Academic Press. (1986) pp. 59-143]. Immortalized clI lines are usually transformed mammalian cvlls, particularly myeloma cells of rodent, bovie and humant orig, Usually, rat or mouse mycloma cell lines ne employed. The hybridoma calls may be cultd in a suitable culti medium that prefcrally contains one or more substances that inhibit the growth or suqvival of the unha4d immortalized cells. For example, if the parental cells lack the enzyme hypoxanlhne guann phosphoriboyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthie, aminopterin, and ihynidine ("HAT medium"), which substances prevent the growth of HGPRT-dficient cells Prmforred immortalized call lines are those that fuse efficiently, support stable hikh level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Mre preferred inummortalized cell lines are murine myclonma lines, which can be obtained, for I stanee, from the S k Institute Cell Distribution Center, San Diego, California and the American Type Culture Follection, Rockvi le, Maryland, Human myeloma and mous-human beraromyeloma cell lines also have bcon deslibed for the product on of human monoclonal antibodies [Kozbor, J. ImmunrnoL, 133:3001 (1984); Brodeur et at. iMonoclonal Antibdy Production Techniques and Applications, Marcel Dekker, Inc.. New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayd far the presence of monocloial antibodlhs directed against the PRO polypeptide of interest Preferably, the binding specificity of monoclonal antibodirs produced by the hybridoma cells is determined by immunoprecipitioun or by an in vit binding assay. such as radioimmunoassay (RIA) or easymc-linked immunoabsorbant Lsay (ELISA). S ch techniques and assays are known in the art. The binding affinity of the monoclonal antiboc y can, for example, be determnincd by the Scatchard analysis of Munson and Pollard. Anal. Biochenm, 107:220 (1 After the dsired hybridoma cells are identified, the clones may be subon by limiting dilution procedures and grown by standard methods [Goding, aulwj. Suitable culture media fur ils purpose include, or example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, tt1 hybridoma cells ay be grown in vive as ascites in a rnammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified im the culture medi 26 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:31 FAX 61 3 92438333 GRIFFITH HACK 033 or ascites fluid by conventional immunnglobulin purification procedures such as, for example, protein A-Sepharo e, hydroxylapalite chromatography, gel electrophoresis, dialysis, or affinity Chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can ba readily isolated a id sequenced using conventional procedures by using oligonucleotido probes that are capable of bindi g specifically to gens cancoding the heavy and light chains of murine antibodies). The ~bridorna cells of tc invention serve as a pn-ferred source of such DNA. Once isolated, the DNA may be placed ino expression vecto s, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myelona cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in e recombinant host cells. The DNA also may be modified, for example, by substituting the codix sequence for hmn m heavy and light chain constant domains in place of the homologous maine sequences 'atent No. 4,816457; Morrison et aL, suara] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-imnunoglobulin polypeptidc. Such a non-inmunoglobulin polypeptide be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable d mains of one antigncombining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovaleit antibodies are wll known in the art. For example, one method involves recombinant expression of immunoglhbulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region o as to prevent hea y chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another mnino acid residue or are deleted so as to pnsvent crosslinking.

In virro methlds are also suitable for preparing monovalent antibodies. Digestion of antibodies to produ fragments thereof, particularly, Fab fragments, can be accomplished using routine techniqu known in the art.

C. HMlllaniaed Antibodies The and-PRO polypeptide antibodies of the invention may further comprise huz human antibodies. Humanized forms of non-human murine) antibodies are chime immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab, F(ab)z or other antigenof antibodies) which contain minimal sequence derived from non-human immunoglobulin. include human immunoglobulins (recipient antibody) in which residues from a complemenLt (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor an rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv ran human immunoglobulin arc replaced by corresponding non-human residues- Humanizes comprise residues which are found neither in the recipient antibody nor in the importec sequences. In general, the humanized antibody will comprise substantially all of at least 0 variable domains, in which all or substantially all of the CDR regions correspond to 1t immnoglobulin and :ul or substantially all of the PR regions are those of a human immu sequence. The humanized antibody optimally also will comprise at least a portion of an imn region typically that of a human immunoglobulin [Jones or aL, Nature, 321: 522-525 (19 27 ianized antibodies .ic immunoglobulii binding subsequenc umanized antibodi ry determining regil ibody) such as moui ework residues of f antibodies may al CDR or framewo ic, and typically tw lose of a non-hum ioglobulin consns iunoglobulin coasts 36); Riechmann etr COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

I

19/09 2003 18:31 FAX 61 3 92438333 GRIFFITH HACK Z034 Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. BioL, 2:593-596 (1992)].

Methods for humanizing non-human tibodies are well known in the art. Generally, humanized antib y has one or more amino acid residues inroduced into it from a source which is non-human. Ti ese non-human am o acid residaes are often referred to as "import" residues, which arc typically taken from an "import" variable dom Humanization can be essentially performed following the method of Wjnter and co-workeri [Jones er at, Na e, 321: 522-525 (1986); Ricchmann et aL, Nature, 332:323-327 (1988); Verhocyen et aL, Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

Accordingly, such "humanized" antibodies are chimeric antibodies Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the coarsp nding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in whi h some CDR residics and possibly some FR residues are substituted by residues from analogous sites in rodent a itibodies.

Human antibdies can also be produced using various techniques known in the art. ipcluding phage disp ay libraries [Hoogenboom and Winter, J. Mo ot, :3 (1991); Mat al Mo Biol (1991; M s a. M81(1991)]. he techniques of Cole et aL and Boenmer r at are also available for the preparation of human monoclonal antibud es (Cole et alp Monoclotal Antibodie and Cancer Thempy, Alan R. Liss, p. 7 7 (1985) and Bosncr etal., I. m n L, 14711t86-95 (1991)].

D. BIpedfle Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have bind ng specificities for at least two diffcront antigens. In the present case, one of the binding specficities is for the P .0 polypeptido, the other one is for any other antigen, and preferably for a cell-surface protein ar receptor or rcce tor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the rombinant product ion of bispecfllc antibodies is based on the co-expression of two imununoglobulin heavy-cthain/-htchain pair, were the two heavy chains have different specificities [Miltein and Cucllo, Nature, 305:537-53 (1983)]. Becaust of the random assortment of immunoglobulln heavy and light chains, these hybridomas (quadr pas) produce a potendal mixture of ten different antibody molecules, of which only one has the correct bispecific sm ture. The puification of the correct molecule is usually accomplished by affinity chromatography steps. Similar p ocedurs are discloed in WO 93/08829, published 13 May 1993, and in Trannecker et aL, EMBO 1&03655-3 59 (1991).

Antibody variable domains with thf desired binding specificities (antibody-antig n combining sites)an be fused to immunoglobulin constant domain sequences. The fusion preferably is with an i nmunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is peferrcd to have the irst heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and ir desired, the imm noglobulin light chain, are inserted into sepacate expression vectors, and are co-transfected into a suitable host orgaz ism. For further details of generating bispecific antibodies see, for example, Suresh et aL. Methods in E ymato 1, :210 (1986).

E. Hete- o aite Aitibodies 28 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:32 FAX 61 3 92438333 GRIFFITH HACK a035 Heteroconjugate antibodies are also within the scope of the present invention. Hetew are composed of two iovalently joined antibodies. Such antibodies have, for example, be immune system cells to unwanted cells Patent No. 4,676,980], and for treatment o; 91/00360; WO 92/200 173; EP 03089]. It is comemplated that the antibodies may be prepare methods in synthetic protein chemistry, including those involving crosslinklag agents. For ex may be constructed using a disulfide exchange reaction or by forming a thioethcr bond.

reagents for this purpose include iminothiolate and r ethyl-4-mnraptobutyrimidat and those c in U.S. Patent No. 4,676,980.

Uses for Anti-PRO Polvnentide Antibodies econ n p

SHI

in amp Exa i.%cl The anti-PRO polypeptide antibodies of the invention have various utilities. Fcr ex polypeptide antibodies may be used in diagnostic assays for a PRO polypeptide, dete ting specific cells, tissues, or serum. Various diagnostic assay techniques known in the art ay competitive binding assays, direct or indirect sandwich assays and immunoprecipitation a.sa s cc heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual ofTechn ucs, (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labeled with a dt tect detectable moiety should be capable of producing, either directly or indirectly, a detectable sign the detectable moiety may be a radioisotope, such as 3 H, 32 p 5 S. or '1I. a fluorescent OT c compound, such as fluorescein isothiocyanate, rhodamine, or lucifrln, or an enzyme, such as ala beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugati g th detectable moiety may be omployed, including those methods described by Hunter ial., N ure, David ct at, Biochemistry, .1:1014 (1974); Pain et at, J. ImmunoL Meah., 4:219 (1 )81) Hisochem. and Cyrochem, 30:407 (1982).

Anti-PRO polypeptidc antibodies also are useful for the affinity purification of PO recombinant cell culture or natural sources. In this process, the antibodies against the IRO immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well nov immobilized antibody then is contacted with a sample containing the PRO polypeptide to be urifi the support is washed with a suitable solvent that will remove substantially all the material in the s PRO polypeptide, which is bound 10 the immobilized antibody. Finally, the support is washe wit solvent that will release the PRO polypeptide from the antibody.

ljugatc antibodj roposed to tar l V infection [V vitro using kno It, immunotoxi ples of suital sed, for examp ample, anti-PP its expression be usud, such nducted in eitt CRC Press, Ii able moiety. T al. For examp hemiluminesc line pho phatia e antibody to t 144:945 (196: and Nygren, polypoptide frc polypeptide a m in the art. T ed, and thcreaf ample except t h another sutat Lving the desir rs (Remingtoi tions or aquco Id concentralio eluding ascorb bumin, geladt Therapeutic Compositions Therapeutic compositions are prepared for storage by mixing the active ingredie degree of purity with optional physiologically acceptable carriers, excipients or stal Pharmaceutical Sciences 16th Edition, Osol, A. Ed. 1980) in the form of lyophilized for solutions. Acceptable carriers, cxcipients or stabilizers are nontoxic to recipients at the dosa employed, and include buffers such as phosphate, citrate and other organic acids; anioxida acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as ser 29 nt h filize nula es ar its in mal COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:32 FAX 61 3 92438333 GRIFFITH HACK 4036 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such is glycine, glutaine, asparagine, arginine or lysine; monosaccharidcs, disaccharides and other carbohydrates including glucose, manno e, or dextrins: cholating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterios such as sodium; and/or nonionic surfactants such as Tween. Pluronics or PEG.

The active ingredicntls may also be entrapped in microcapaules prepared, for example, by cowervati n techniques or by interficial polymerization, for example, hydroxymethylccllulose or golatin-rm crocapsules and po y- (methylmetbacylate) nicrocapsules, respectively), in colloidal drug delivery systems (for example, liposom s, albumin microsphers. microomulsions nano-particles and nanocapsules) or in nncronmuls ons. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

The forudations to be used for in vive administration mast be sterile. This is rdily accomplished y filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a container having a erile access port, for example, an inaveous solution bag or vial having a stopper pierccable by a hypodermic i iection needle.

The route of administration is in accord with known methods, e.g. injection or infusion by intravcnoms, intrapeitoncal, intracerebral intramuscular, intraocular, intraartrial aor intralesional routes, opical adminisati or by sustained release systems.

Suitable examples of sustained release preparations include semipermeable polyrnmr matrices In the for of shaped articles, e g. filns, or microcapsulc. Sustained release matrices include jjolyesters. hydrog s, polylactides Palent 3,773,919, HP 58,481), copolymers of L-gluamic acid and gamma ethyl-L-glutamatc J.

Sidman at ak Bloalymrs 2Z 547-556 [1983]), poly (2-hydroxyethyl-mthacrylate Langer, s Al-, Biomed. Mater. Res. 5: 167-277 [19811 and R. Langer, Chm. Tech, 98-105 [1982J), ethylene vinyl ac te Langerg Id-) or poly-D-(-)-3-hydroxybutyric acid (EP 133.988)- Sustained release compositions also include liposomes. Liposomes containing a molecule within theb scope of the present invention arc prepared y mlethods known per DE 3,218,121 Epstein g L, Proc. Natl. Acad. Sci. USA 82: 368E -3692 (1985): Hwa g g at, Proc. Nail. Acd. ci. USA 22: 4030-4034 (1980); EP 52322; EP 36676A; EP 8046i BP 143949; P 142641; Japanese patent application 83-118008; U.S. patents 4,485,045 and 4,544,545; and rP 102,324. Ordinarly the liposomes are of the small (about 200-800 Angstrom) unilamelar type In which the lipid content is greater than about 30 mol. 9% cholesterol, the selected proportion being adjusted for the optimal NT-4 ll erapy.

An cffective amount of the active ingredient will depend, fir oxample upon the thazaeutic objectives, t route of administration, and the condition of the patient. Accordingly, it will be necessary 4r the therapist to til er the dosage and modify the route of administration as required to obtain the optimal therapcuic effect A typical dly dosage might range hofrom about 1 pg/kg to up to 100 mg/kg or more, depending on the fact ors mentioned abov.

Typically, the clinician will administer a molecule of the present invention until a dosage is reached that provid.s the required biological effect. The progress of this therapy is easily monitored by convna mal assays.

The following examples are offered for illustrative purposes only, and are not iniu id to limit the scoe of the present invcntiim in any way.

All patent and literature references cited in the present specification a=e hereby incborated by referen in their entirety.

COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:33FEAX 61 3 92438333 GRIFFITH HACK i 037 r's nd

EXAMPLES

Commercially available reagents referred to in the examples were used accord instructions unless olerwise indicated. The source of those cells identified in the foil throughout the specification, by ATCC accession numbers is the American Type Culture Maryland.

i.g to manufactun wing examples, a :ollection, Rockvillc, EXAMPE1: Exracellular Domain Honolov Screcnint to Identiy Novel Polypeptidre and cDNA Encodi Ig Therefor The extracelllar domain (ECD) squences (including the scretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Proc public database were used to search EST databases. The EST databases included public databases Dayhoff. GenBank), and proprietary databases (e g. LIFESEQT, inc: Pharmaceuticals, Pale Alto, CA). The search was performed using the computer progran BLAST or BLAST2 (Allschul and Gish, Mcthods in Enzvmoloy 26fi: 460-480 (1996)) as a comparison of the CD protein sequen as to a 6 frame translation of the EST sequences. Those comparisons with a Blast score of 70 (or in some cases or greater that did not encode known proteins wee clustered and assembled Into consensus DNA sequences ith the program "phrap" (Phil Green, University of Washington. Seattle, WA; (http://bozcman.mbtrwashington.edulphrap.docs/phraphtml).

Using this extracellular domain homology screen, consensus DNA sequences wer assembled relative to the other identified EST sequences using phrap. In addition, the consensus DNA sequences o tained were often mt not always) extended using repeated cycles of BLAST and phrap to extend the consensus seq a a as f a possi le using the sources of 1ST sequences discussed above.

Based upon the consensus sequences obtained as described above, oligonuclootide, were then synthesied and used to identify by PCR a cDNA library that contained the sequence of interest and for t e as probes to isol te a clone of the full-length coding sequence for a PRO polypeptide. Forward and reicrac PCR prim tr generally range from 20 to 30 nucleotides and are often designed to give a PCR product 01 about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides ire synthcsized when the consensus sequence is greater than about 1-1 -kbp. In order to scree several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per usubcl et aL, Cun Protocols in Molecular Biology, with the PCR primnr pair. A positive library was then used to isolate cloes encoding the gene of interest using the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed by tdard methods using commercially available reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed with ohigo dT containing a NotT site, linked with blunt to Sall hemikinased adaptors, cleaved with Nod sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (so h as pRKB or pRD; is a precursor of pRKSD that does not contain the Sill site: see, Holmes et al., i A 2531278-1280 (1991)) in the unique XhoI and NotI sites.

31 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:33 FAX 61 3 92438333 GRIFFITH HACK [a038 EXAMPEL 2: Isolation of cDNAcloncs by Amvlaas Screening 1. Preoaration of oliz dT primed cDNA library mRNA was isolated from a human tissue of interest using reagents and protocols from Invitrogen, Sm Diego, CA (Past Track This RNA was used to gcnerate an oligo dT primed cDNA libra3 in the vector pRI D using reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script Plarmid System). In I is procedure, the double stranded cDNA was sized to greater than 1000 bp and the SalI/Not linkored cDNA *as cloned into XhoI/Notl cleaved vector. pRKSD is a cloning vector that has an sp 6 trans ription initiation site followed by an Sfi restriction enzyme site preceding the Xhol/NotI cDNA cloning sites.

2. Priaration of random primed cDNA library A secondary eDNA library was generated in order to preferentially represent the ends of the primary eDNA clones. Sp 6 RNA was generated from the primary library (described above), and his RNA was used to generate a random primed cDNA library in the vector pSST-AMY.0 using reagents anl protocols from Lfe Technologies (Super Script Plasmid System, referenced above). In this procadure the doub o stranded cDNA was sized to 500-1000 bp. linkered with blunt to Not adaptors, cleaved with Sfi, and cloned tor Sfil/NI cleav d vector. pSST-AMY.I is a cloning vector that has a yeast alcohol dehydrogenase promoter precoding the cD A cloning sites and the imose amylase sequence (the mature sequence without the secretion i gnal) followed by E e yeast alcohol dehydrogenaso terminator, after the cloning sites. Thus, cDNAs cloned into th s vector that are fs d in frame with amylase sequence will lead to the secretion of amylse from appropriately tra sfected yeast colon 3. Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice to which was add x electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was th n electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Te hnologies, 1 ml) was added and the mixturs was incubated at 37C for 30 minutes. The transformants were then I lated onto 20 stand rd 150 rnm LB places cortaining ampicillin and incubated for 16 hours (37C). Positive coloni s were scraped offt plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCI gradient. The purified DNA was thei carricd on to the yeast protocols below.

The yeast methods were divided into three categories: Transformation of yeast ith the plasmld/cD A combined vector: Detection and isolation of yeast clones secreting amylase; and PR amplification of te insert directly from die yeast colony and purification of the DNA for sequencing and furthr analysis.

The yeast stain used was HD56-SA (ATCC-90785). This strain has the following genotype: MAT alp a, ura3-52, ]eu2-3, leu2-112. his3-11, his3-15. MAL, SUC*, GAL. Preferably, yeast mutant can be employed that have deficient post-translational pathways. Such mutants may have translocation deficient llelcs in sec7l, e 72, sec62, with truncated sec71 being most prferred. Alternatively antagonists (including antisnse nuclotides andr ligands) which interfire with the normal operation of these genes, other proteins implicated in this post translation pathway SEC61p, SEC72p. SEC62p, SEC63p, TDJlp or SSAlp-4p) or the compex formation of tm proteins may also be prefcrably employed in combination with the amylase-expressing yet, 32 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:33 FAX 61 3 92438333 GRIFFITH HACK 1039 Transformation was performed based on the protocol outlined by Gitz ct al., Nuc Acid. Res. 20:14: (1992). Transformed cells were then inoculated from agar into YEPD complex media broth (100 ml) and gro overnight at 300C. The YEPD broth was prepared as described in Kaiser et al., Methods in Yeast Genetics, Co Spring Harbor Pres, Cold Spring Harbor, NY, p. 207 (1994)- The overnight culure was th diluted to about 1 106 ells/ml (approx. OD 0 oo-0.1) into fresh YEPD broth (500 ml) and regrown to 1 x i0 ccmls/ml (appro ODsaxO.4-05).

The cells wern then harvested and prepared for transformation by ransfer into S53 n tor bottl in a Son GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and then resuspended i to sterile water, ai centrifuged again in 50 ml falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The supcrnatant w discarded and the cells were subsequenily washed with LIAcl'E (10 ml, 10 mM Tris-HCI, 1 m V EDTA pH 75, 11 mM LigOOCCH 3 and resuspended into LiAc/TB (2.5 ml).

Transformation took place by mixing the prepared cells (100 l) with freshly den tuhrd single strand4 salmon testes DNA (Lofstrand Labs, Galthersburg, MD) and transforming DNA (1 pg, vol. e 10 pl) in microful tubes. The mixture was mixed briefly by vortexing, then 40% PE/TE (600 pl, 40% polyelh ylne glycol-4000, mM Tris-HCI, 1 mM EDTA, 100 mM LiOOCCH3, pH 7.5) was added. This mixture vas gently mixed a incubated at 30°C while agitating for 30 minutes. The cells were then beat shocked at 42"C fir 15 minutes, and d reaction vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted and resu pended into TE pl, 10 mM Tris-HC, 1 mM EDTA pH 75) followed by recentrifugation. The cells were then liluted into TE (I I and aliquots (200 pl) were spread onto the selective media previously prepared in 150 mm rowth plates (VWR Alternatively. instead of multiple small reactions, the transformation was perform I using a single, larj scale reaction, wherein reagent amounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lacking uracil (E CD-Ura) prepared described in Kaiser et Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Sprin Harbor, NY, p. 201 210 (1994). Transfonnants were grown at 30°C for 2-3 days.

The detection .of colonies secreting amylasc was performed by including red starch i the selective growl media. Starch was coupled to the red dye (Reactive Rod-120, Sigma) as per the procedure de cribed by Biely et al Anal-ichem 172:176-179 (1988). The coupled starch was incorporated into the SCD-Ur agar plates at a fini concentration of 0.15% and was buffered with potassium phosphate to a pH of 7 0 (50-100 mM fin concentation).

The positive colunics were picked and streaked across fresh selective media (onto 1 0 mun plates) in ordt to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase sccretio were detected by direct incorporation f red starch into buffered SCD-Ura agar. Positive col nies were determine by their ability to break down starch resulting in a clear halo around the positive colony vis alized directly.

4. Isoation of DNA by PC Anmlification When a positive colony was isolated, a portion of it was picked by a toothpick and di uted into sterile wate p) in a 96 well plhae. At this time, the positive colonies were either frozen and stored fTr subsequent analysi or immediately amplified. An aliquot of cells (5 pl) was used as a template for the PCR reac ion in a 25 pl volumr 33 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:34 FAX 81 3 92438333 GRIFFITH HACK 1040 containing: 0.5 pl Klcntaq (Clontcch, Palo Alto, CA); 4.0 1 pl 10 mM dNTP-s (Perkin Elmerbuffer (Clontech); 0.21 pl forward oligo 1; 0.25 p1 reverse oligo 2; 12.5 p1 distilled water.

forward ollgonucleoticte 1 was: 5'-TGTAAAACGACGGCCArTTAAATAACCTCAAAIAATC-3 (Si The sequence of reverse oligonucleotide 2 was: 5'-CAGGAACAGCrATGACCACCTGACAC4CCAMTCCAIL3- (SEQ PCR was then performed as follows: a. Denature 92 0 C, 5 minutes Celus); 2.5 p Kent The sequence oft Q ID NO;324) bD NO:325) b. 3 cycles of: Denature Anneal Extend c. 3 cycles of: Denature Anneal Extend d. 25 cycles of: Denature Anneal Extend 92 0

C,

590C, 72C.

92C, 57C,.

72C, 92C,.

55 0

C,

72C,.

30 acseconds 30 seconds 60 seconds 30 seconds 30 seconds 60 seconds 30 seconds 30 seconds 60 seconds Hold The underlinud regions of the oligonucleorides annealed to the ADH promoter re region, respectively, and amplified a 307 bp region from vector pSST-AMY.0 when nc Typically, the first 18 nuclotidcs of th 5' end of these ollgonucleotides contained annealing a primers. Thus, the total product of the PCR reaction from an empty vector was 343 bp. How fused cDNA resulted in considerably longer nucleotide sequences.

Following the PCR, an aliquat of the reaction (5 pi) was examined by agrose get el agnmse gel using a Tris-Borate-EDTA (TEE) buffering system as described by Sambrook resulting in a single strong PCR product larger than 400 bp were further analyzed by D purification with a 96 Qiaquick PCR clean-up colunm (Qiagen Inc., Cbhatsworth, CA).

EXAMPLE 3: Iawjtion of cDNA Clonesp Encoding Human PR0717 A consensus sequence was obtained relative to a variety of EST sequences as doi above, wherein the consnsus sequence obtained Is herein designated DNA42829. Basei consensus sequence, oligonucleotddes were synthesized: 1) to identify by PCR a cDNA libr ecqurnce of intcrest, and 2) for use as probes to isolate a clone of the full-length coding s A pair of PCR primers (forward and reverse) were synthesized: forward PCR imn 5'-AGCITCICAGCCCTCCTGGAG CCAG-3' (SEQ ID NO:42 1); reverse CR Drimer 5'-CGCGTCAATAAACCTGGACGCTrGO-3' (SEQ ID NO:422).

Additionally, a synthetic oligonuclotide hybridization probe was constucted from the sequence which had the following nucleotide icquence; 34 ion and the amyla insert was presni tes for the sequenci ver, signal acquen ectrophoresis In a 1 at al.. ruig. Clon A sequencing aft Icribed in Example Son the DNA4282 ny that contained C Ience for PRO 17.

NA42829 consensus COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:34 FAX 61 3 92438333 GRIFFITH HACK l041 hybridination pnhj 5'-TATGTGGACCGGACCAAGCACITCACTGAGGCCACCAAGATTG3' (SEQ ID NC:423).

In order to screen several librmies for a source of a full-length clone, DNA from the ibraries was screen by PCR amplification with the PCR primer pair identified above. A positive library was then sed to isolat clon encoding the PRO717 gene using the probe oligonucleoide and one of the PCR primers. Rb A for construction the cDNA libraries was isolated from humrnan fetal liver tissue (LIB229).

DNA sequencing of the clones isolatwd as described above gave the full-length DNA equence for PR07] [herein designated as UNQ3S5 (DNA50988-1326)] (SEQ ID NO:419) and the derived rotein sequence fi PRO717.

The entire nuclcotide sequence of UNQ385 (DNA50988-1326) is shown In Figure 1 8 (SEQ 1D NQ:419 Clone UNQ385 (DNA50988-1326) contains a single open reading frame with an apparent t anslational initiatic site at nucleotide positions 17-19 and ending at the stop codon at nucleotide positions 1697-161 (Figure 168). TI predicted polypepddo precursor is 560 amino acids long (Figure 169). The full-length PRO 17 protein shown i Pigure 169 has an estimated molecular weight of about 58,427 dahltons and a pl of about 6.86. Clone UNQ38 (DNA50988-1326) has been deposited with the ATCC on April 28, 1998. Regarding the seqience it is understo that the deposited clone contains the correct sequence, and the sequences provided herein are based on know sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO717 polypeptide suggests that PR0717 may b a novel 12 tremmasembcane receptor. The reverse complement strand of DNA50988 has stretch that matche identically with human regulatory myosin light strand.

Still analyzing the amino acid sequence of SEQ ID NO:420, transmembrane domaivs are at about amin acids 30-50, 61-79, 98-112, 126-146, 169-182, 201-215, 248-268, 280-300, 318-337, 341-3 7, 375-387, and 42( 441 of SEQ ID NO:420. N-glycosylation sites are at about amino acids 40-43 and 43-46 of SEQ LD NO;420. i glycosaminoglycan attachment site is at about amino acids 468471 of SEQ ID NO:420, The corresponda, aucleotides can be routinely detanermined given the sequences provided herein.

EXAMPLE 4: Use of RO Polwpnlid-Wnnodins Nuclei Acid as Hbridatwion Probes The following method describes use of a nucleodtide sequence encoding a PM hybridization probe.

DNA comprising the coding scquence of of a PRO polypeptide of interest as disc employed as a probe or used as a basis fum which to prepare probes to screen for homologous encoding naturally-occun-ing variants of the PRO polypopide) in human tissue cDNA librn genomic libraries.

Hybridization and washing of fiters containing either library DNAs is performed u stringency conditions. Hybridization of radiolabeled PRO polypeptido-encodlang nucleic ack filters is performed in a solution of 50% formamnide, 5x SSC, 0.1% SDS, 0.1% sodium py sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42C for 20 h flitors is performed in an aqueous solution of 0.1x SSC and 0.1% SDS at 42C.

0 polypeptide as losed herein may b DNAs (such as thos ries or human tissu or the following hig -derived probe to the ophosphate, 50 mM uMrs. Washing of the COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 S19/09 2003 18:35 FAX 61 3 92438333 GRIFFITH HACK [042 DNAs having a desired sequence identity with the DNA encoding full-length r polypeptide can then be identified using standard techniques known in the art.

EXAMPLE Expression of PRO Polvoentides in E coli This example illustrates preparation of an unglycosylated form of a desird recombinant expression in E. coli.

The DNA sequence encoding the desired PRO polypeptide is initially amplifie( primers. The primers should contain restriction enzyme sites which correspond to the restri the selected expression vector. A variety of expression vectors may be employed. An examn is pBR322 (derived from E. coli; see Bolivar at al., Gene. 2:95 (1977)) which contains gmc tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylatec sequences are then ligated into the vector. The vector will preferably include sequences antibiotic resistance gmne, a trp promoter, a polyhis leader (including the first six STII cod and entcrokinase cleavage site), the specific PRO polypeptide coding region, lambda trnscri an argU gene.

The ligation mixture is then used to transform a selected E coli stain using the Sambrook et al., siuna. Transformiants are identified by their ability to grow on LB plates colonies are then selecied. Plasmid DNA can be isolated and confirmed by restriction analysis Selected clones can be grown overnight in liquid culture medium such as LB bro antibiotics. The overnight culture may subsequently be used to inoculate a larger scale cultl grown to a desired optical density, during which the expression promoter is turned on.

After culturing the cells for several more hours, the cells can be harvested by centrf obtained by the centrifugation can be solubllized using various agents known in the art, an polypeptide can then lie purified using a metal chelating column under condidons that allo protein.

In EP Application No. 99912321.9, PRO polypeptides were expressed in E. col in a using the following procedure. The DNA encoding the PRO polypeptide was initially ampli primers. The primers contained restriction enzyme sites which correspond to the restrictie selected expression vector, and other useful sequences providing for efficient and reliable trant purification on a meoal chelation column, and protoolytic removal with enterokinase. The PC tagged sequences wernt then lgated into an expression vector, which was used to tranform a strain 52 (W3110 fuhA(tonA) Ion galE rpoHls(htpRts) clpP(lcTq). Transformants were first mg/ml carbeniclllii at 30"C with shaking until an O.D.600 of 3-5 was reached. Cultures wt fold into CRAP media (prepared by mixing 3.57 g (N-)SO4, 0.71 g sodium ciTrae2H20. I.

yeast extract, 5.36 g Sliefield hycas SF in 500 mL water, as well as 110 mM MPOS, pH 7. and 7 mM MgSO4) anr grown for approximately 20-30 hours at 30 0 C with shaking. Samples expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the ccls. C until purification and refolding.

36 ative sequence PIo PRO polypeptide by using selected PCR :tion enzyme sites in le of a suitable vect r ies for ampicillin aid .The PCR amplifi d which encode for as, polyhis sequenc;, tional teminator, a d methods described n id antibiotic resista i and DNA sequencin h supplemented wi h re. The cells are then gation. The cellpell t the solubilized PRO v tight binding of de poly-His tagged fou i, d using selected PC Z a enzyme sites on the adon initiation, rapi t-amplified, poly-H s E. coi host based a ownin LB conain e then diluted 50-10 7g KC, 5.36 gDif 0.55% glucos wre removed to verit 11 pellets were loze COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:35 FAX 61 3 92438333 GRIFFITH HACK @043 E. coli paste trom 0.5 to 1 L fermentations (6-10 g pellets) was resuspended in 10 guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionale is concentrations of 0.M and 0.02 M, respectively, and the solution was stirred overnight at 4 in a denatured protein with all cystelne residues blocked by sulfitolization. The solution was rpm in a Beckman Ulticentifug for 30 min. The supernatant was diluted with 3-5 volumes of buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to cl clarified extract was lkaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrate column buffer. The cclumn was washed with additional buffer containing 50 mM imidazol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole. Fractions protein were pooled and stored at 4C. Protein concentration was estimated by its absorbanc calculated extinction cocflicient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshly prepared refnldin mM Tris, pH 8-6, 0.3 M NaCI, 2.5 M urea, 5 mM cystelno. 20 mM glycine and 1 m volumes were chosen so that the final protein concentration was between 50 to 100 microgra solution was stirrd gently at 4 0 C for 12-36 hours. The refolding reaction was quenched by a final concntration of 0.4% (pH of approximately Before further purification of the pro filtred through a 0.22 micron filter and acetonitrile was added to 2-10% final concentrationwas chromatographed on a Poros RI/H reversed phase column using a mobile buffer of 0.1 a gradient of acetonirile from 10 to 80%. Aliquots of fractions with A280 absorbance polyacrylamide gels and fiacliuns containing homogeneous refolded protein were pooled- C refolded species of most proteins are eluted at the lowest concentrations of accionitrile sinc most compact with their hydrophobic inteiors shielded from Interaction with the reversed phi species are usually eluied at higher acetonitrile concentrations. In addition to resolving misfo from the desired form the reversed phase step also removes cndoroxin from the samples.

Fractions containing the desired folded PRO proteins were pooled and the aceton gentle stream of nitrogen directed at the solution. Proteins were formulated into 20 M He; M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfit equilibrated in the formulation buffer and sterile filtered.

EXAMPLE 6: Exfnre'in of PRO Polvpeptides in Mammalian Colls olumes in 7: added to make fin hC. This stcpresuJ entrifuged at 40,0( metal chelate colun aify. Depending tl Sin the metal chela i (Calbiochem, Utr ,ontaining the desin a at 280 nm using tl Sbuffer consisting c A EDTA. Refoldir ns/ml, The refoldii te addition ofTFA 1 tein, the solution wi The refolded prote rFA with elution wil no analyzed on SD nerally, the properl those species are tsc resin. Aggregate ded forms of protei rile removed using es, pH 6.8 with 0.1 S(Pharmacia) resir This example illustrates preparation of a glycosylated form of a desired PRO polyp expression in mannnalian clls.

The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as t Optionally, the PRO polypeptide-encoding DNA is ligated into pRK5 with selected restric insertion of the PRO polypeptide DNA using ligation methods such as described in Sambr resulting vector is called pRK5-PRO polypcptide.

In one embodiment, the selected host cells may be 293 cells. Human 293 colls grown to confluence in tissue culture plates in medium such as DMBM supplemented wit 37 ptido by recombin t co expression vector ion enzymes to allow pok eL al., si=. Thw LTCC CCL 1573) ar Sfetal calf scrum a COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:35 FAX 61 3 92438333 GRIFFITH HACK [044 optionally, nutrient components and/or antibiotics. About 10 p.g pRK5-PRO polypeptide DN 1 gg DNA encoding the VA RNA gone [Thimmappaya et al., CllL :543 (1982)] and dissol Tris-HCI. 0.1 mM EDTA. 0.227 M CaC 1 To this mixture is added, dropwise. 500 Pl of 50 n 280 mM NaCI, 1.5 mM NaPO 4 and a precipitate is allowed to form for 10 minutes at 25' suspended and added co the 293 cells and allowed to settle for about four hours at 37C, aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are th free medium, fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium is removed and medium (alone) or culture medium containing 200 gCiml SS-cystcine and 200 pCi/ml 25

S-

hour Incubation, the cc nditioned medium is collected, concentrated on a spin filter, and loade The processed gel nmy be dried and exposed to film for a selected period of time to revea polypeptide The cultures containing transfcted cells may undergo further incubation (in se the medium is tested in selected bioassays.

In an alternative technique, PRO polypeptide may be introduced Into 293 cells ransi sulfate method described by Somparyrac et al, Proc. Natl. Acad. Sci., 2:7575 (1981). 2 maximal density in a spinner flask and 700 jg pRKS-PRO polypeptide DNA is addc concentrated from the spinner flask by centrifugation and washed with PBS. The DNA incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 tissue culture mediuni, and re-introduced into the spinner flask containing tissue culture insulin and 0.1 Ig/ml bovine tansferrin. After about four days, the conditioned media is c to remove colls and debris. The sample containing expressed PRO polypeptide can then be co by any selected ilethod, such as dialysis and/or column chromalography.

In another embodiment, PRO polypeptides can be expressed in CHO cells- The can be transfected into CHO cells using known reagents such as CaPO 4 or DEAE-dextran.

cell cultures can be incubated, and the medium replaced with culture medium (alono) o radiolabel such as SS -methionine. After determining the presence of PRO polypeptide, the a replaced with serum free medium. Preferably. the cultures ar incubated for about 6 days, a medium is harvsted. The medium containing the expressed PRO polypeptide can then be cc by any selectcd method.

Epitope-tagged PRO polypeptide may also be exprssed in host CHO cells. The P subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fue in frame ta such as a poly-his tag into a Baculovirus expression vetor. The poly-his tagged PRO pol be subcloncd into a SV40 driven vector containing a selection marker such as DHFR for se Finally, the CHO cells can be transfected (as described above) with the SV40 driven ve performed, as described above, to verify expression. The culture medium containing the cx PRO polypeptide can then be concentrated and purified by any selected method, such as chromatography.

Stable expression in CHO cells was performed using the following procedure. The 38 is mixed with abeat ed in 500 pl of 1 mv M HEPES (pH 73), SThe precipitate is he culture mediumis n washed with serm replaced with cultue ethionine. After a 2 Sonto a 15% SDS g l.

the presence of PRO u em e medium) ad atly using the dextr 93 cells are grown to The clls are fist detran precipitate is seconds, washed with iumn, 5 jg/nm bovi ntrifuged and filter.

nccntraed and purifi UKS-PRO polypepti e s described above, t i medium containing a ulture medium may x id then the condition d acentrad and purifi :c LO polypeptide may e with a selected opito rpcptide insert can th n action of stable clu s.

tor. Labeling may ressed poly-His tag ad y Ni+-chclate affinity roteins were expres COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 II 19/09 2003 18:36 FAX 61 3 92438333 GRIFFITH HACK i1045 as an IgG construct (immunoadhesin), in which the coding sequence for the soluble for s extracellul domains) of the respective proteins were fused to an IgG1 constant region sequence containirg the hinge. CH2 ax CH2 domains and/or i. a poly-His tagged form.

Following PCR amplification, the respective DNAs were subcloned in a CHO ex ression vector usil standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, n it 3.16, John Wili and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites and 3= of the DN of interest to allow the convenient shuttling of cDNA=s. The vector used expression in CHO cells is as describ in Lucas et aL, Nucl. Acids Res. 2A: 9 (1774-1779 (1996), and uses the SV40 early prom terlenhancer to dril expression of the cDNA of interest and dihydrofolate rtductase (DHFR). DHPR eopressior permits selection f stable maintenance of the plasmid following transfection.

Twelve micrograms of the desired plasmid DNA were introduced into approximately 10 million CHO eel using commercially ,vailable tranfection reagents Superfect 7 (Quiagen), Dosper 7 or Fugenc 7 (Boehring Mannheim). The cells were grown and described in Lucas ea al., supra. Approximately 3 x .0 cells arc rlozen an ampule for further growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement into war r bath and mixed 1 vortexing. The contents were pipetted into a centrifuge tnbe containing 10 mLs of media at d centrifuged at 1O rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL selective media (0 un filtered PS20 with 5% 0.2 pm dlafiltered fetal bovine serum). The cells were then ali uoted into a 100 a spinner containing 90 mL of selective media. After 1-2 days, the cells were transferred into e 250 mL spinner fil with 150 mL selective growth medium and incubated at 37°C. After another 2-3 days, a 250 L, 500 mL and 201 mL spinners were seeled with 3 x 10 cells/mL. The cell media was exchanged with fresh n edia by centrirugatl and Tesuspnsion in production medium. Although any suirable CHO media may be employe, a production medju described In US Patent No. 5,122,469, issued June 16, 1992 was actually used. 3L producti n spinner is seeded 1.2 x 10 cels/mL. On day 0, the cell number pHwere determined. On day 1, the spinner wa sampled and spargi with fillcted air was commenced. On day 2, the spinner was sampled, the temperature shinft to 33"C, and 30 n of 500 g/L glucose and 0.6 mL of 10% antifoam 35% polydimethylsiloxane emulsi m, Dow Coming 3( Medical Grade Emulsion). Throughout the production, pH was adjusted as necessary to ke p at around 7.2. Afi days, or until viability dropped below 70%, the cell culture was harvested by centrifugtion and filtering throul a 0.22 um filter. The filtrate was either stored at 4'C or immediately loaded onto columns for purification.

For the poly -His tagged constructs, the proteins were purified using a Ni-NTA col nn (Qiagen), Befo purification, imidazolo was added to the conditioned media to a concentration of 5 mM. The conditioned media w pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7 buffer containing 0.3 M NaCI and 5 m imidazole at a flow rateof 4-5 ml/mmn. at 40C. After loading, the column was washed with dditonal equilibratiL buffer and the protein cluted with equilibration buffer containing 0.25 M imidazole. The higdy purified protein w subsequently desalted into a storage buffer containing 10 mM Hepos, 0.14 M NaCI and 4% mannitol, pH 6.8, wi a 25 ml G25 Superfine (Pharmacia) column and stored at Immunoadbesin (Fc containing) constructs of were purified from the conditioned media as follows. T conditiond medium was pumped onto a 5 ml Protein A column (Pharmacia) which had beer equilibrated in 20 m 39 COMS ID No: SM81-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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I 9/09 2003 18:36 FAX 61 3 92438333 GRIFFITH HACK Ie 04 6 Na phosphate buffer, pH 6.8. Aftr loading, the column was washed extensively with equi elution with 100 mM citric acid, pH 3.5. The elated protein was immediately neutralized by c into tubes containing 275 pL of 1 M Tris buffer, pH 9. The highly purified protein was sub storage buffer as described above for the poly-His tagged proteins. The homogeneity polyacrylamide gels anid by N-terminal amino acid sequencing by Edman degradation.

EXAMPLE 7; ExB sign of PRO Polneutides in Yeas The following mrnethod describes recombiant expression of a desired PRO polypel First yeast expression vectors are constructed for intracellular productieon or secretio from the ADH2/OAPDH promoter. DNA encoding a desired PRO polypeptido, a selected promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct ii of the PRO polypeptide. Forat secretion, DNA encoding the PRO polypeptide can be cloned ini together with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secretory and linker sequences (if needed) for expression of the PRO polypeptide.

Yeast cells, rech as yeast strain AB 10, can then be transformed with the expressi above and cultured in selected farmentation media. The transformed yeast supernatant precipitation with 10% trichloroacetic acid and separation by SDS-PAGE. followed by S Coormassic Blue stain. I Recombinant PRO polypeptide can subsequently be isolated and purified by remov the fermentation medium by centrifugation and then concentrating the medium using selectc concentrate containing the PRO polypeptide may further be purifed using solccted column EXAMPLE X- Fxnrelnn of PRO Polvuentides In Baculovirus-Infected Inst Calls bration buffer bef lecting 1 ml fractii equently desalled I pgcsdby

S.

Vas assessed by SI ide in yeast.

Sof PRO polypcpti ignal peptide and tracellular express o the selected plasn zgnal/1eader tequeis on plasmids descril s can be analyzed dining of the ge1s V ng the yeast cells fr l cartridge fillers. I hromatography res] The following method describes recombinant expression of PRO polypeplides i Baculoviros-lafec insect cells.

The desired PRO polypeptide is fused upstream of an epitope tag contained with a vector. Such epitope tags include poly.his tags and inmmunoglobulin tags (like Fe regiom plasmnids may be employed, ncluding plasmids derived from conmnrcIally available pla 1 (Novagen). Briefly, the PRO polypeptide or the desired portion of ti PRO polypeptide encoding the extracellular domain of a transmembrane protein) is amplified by PCR withl to the 5' and 3' regions. The 5' primer may incorporate flanting (selected) restriction e than digested with those selected restriction enzymes and sublonoud into the expression v Recombinart baculovirus is gcnerated by co-transfcting the above plasmid and B (Pharmingen) into Spadopterafnrrgiperda cells (ATCC CRL 1711)using lipofectin from GlCO-BRL). Aftelr 4-5 days of incubation at 28*C, the released viruses ar harves amplifications. Viral infection and protein expression is performed us described by O'R expression vectors: A labhoratory Manual, Oxford; Oxford University Press (1994).

Expressed poly-his tagged PRO polypoptide can then be purified, for example, I ulovirus express o0 of IgG). A variety of ids such as pVL193 (such as the sequen rimetrs complemuenry e sites. The produc Is ctor.

culoGold" vims IA commercially availa te Bd and used for fter Hley ct al., Baculovi us )y Ni?-chelate affin ty COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:37 F.61 3 92438333 GRIFFITH HACK [047 chromatography as follows. Extracts are prepared from rocombinant virus-infected 519 cells et aL, Naure. 2:175-179 (1993). Briefly, Sf9 calls are washed, resuspended in sonication pH 7.9; 12.5 mM MgCI 2 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCI), and s seconds an ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50 mM phosphate, 300 mM NaCI, 10% Glycerol, pH 7.8) and filtered through a 0.45 p agarose column (comnaercially available from Qiagen) is prepared with a bad volume of5 mi of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded mt per minute. The 'olumn is washed to baseline A.n with loading bufTer, at which poin started. Next the columnis washed with a secondary wash buffer (50 mrM phosphate: 300 I pH which clutes nonspecifically bound protein. After reaching Amo baseline again, th with a 0 to 500 mM fIaidazole gradient in the secondary wash buffer. One mL fractions are by SDS-PAGE and silver staining or western blot with Ni?-NTA-conjugaed to alkaline Fractions containing the elated Hiso-magged PRO polypeptido are pooled and dialyzed aga Alternatively, purification of the IgG tagged (or Pc tagged) PRO polypeptide c known chromatography techniques. including for instance, Protein A or protein G column In EP Applic ation No. 99912321.9 PRO polypeptides were successfully expressed 519 insect cells. While the expression was actually performed in a 0.5-2 L scale, it can be re 8 L) preparations. The proteins were expressed as an IgG construct (imnmunoadhs extracellular region was fused to anIgG1 constant region sequence containing the hinge, and/or in poly-His tagged forms.

For expression in baculovirus infected Sf9 cells, following PCR amplification, sequences were subeloned into a baculovirus expression vector (pb.PI.Ig for IgO fusio poly-His tagged proteins), and the vector and Baculogold7 baculovirus DNA (Pharminge into 105 Spodoptera frugiperda cells (ATCC CRL 1711), using Lipofectin (Gibco pb.PH.His are modifications of the commercially available baculovirus expression vector p with modified polylinac regions to include the His or Pc tag sequences. The cells were r medium supplemented with 10% FBS (Hyclone). Cells were incubated for 5 days at 21l.

harvested and subsequently used for the first viral amplification by infecting Sf9 cells in H supplemented with 10% FBS at an approximate multiplicity of infoction (MOI) of 10. Cal days at 280"C. The supernatant was harvested and the expression of the constructs in the vector was detenmined by batch binding of 1 ml of supernarant to 25 mL of Ni-NTA beads tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IG tagged proteins analysis comparing to a known concentration of protein standard by Coomnassie blue stain The first viral amplification supernatant was used to infect a spinner culture (5 in ESF-921 medium (Expression Systems LLC) at an approximate M01 of0.1. Cells weri 28C. The supernatant was harvested and filtered. Batch binding and SDS-PAGi an necessary, until expression of the spinner culture was confirmned.

The conditioned medium from the transfcted cells (0.5 to 3 L) was harvested by e 41 Sdescribed by Rup buffer (25 mL He s, onicated twice for 0 fold in loading filter. A Ni-N A washed with 25 L nto the column at fraction collection is I NaCI. 10% Glycerl, column is developed olleefrd and analyzed hosphatase (Qiage).

ast loading buffer.

n be performed using .hromatography.

in baculovirus infctd ily scaled up for la or in which the prot in H2 and CH3 dome as the respective coding s and pb.PH.His.c or n) were co-tranfed BRL). pbPH.gG #1393 (Pharminge wn in Hink's TNM-F The supernatant as nk's TNM-FH mdLm s were incubated fo 3 aculovirus expression )IAGEN) for histidine lowed by SDS-PA ig.

md) of 519 cells grown ncuhaedl for 3 days at ysis was repeated, as utrifuigation to rorc COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:37 FAX 51 3 9243&3L GRIFFITH HIACK 048 I the cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the potein construct we purified using a Ni-NTA colurmn (Qiagen). Before purification, imidazole was added to the conditioned media a concentration of 5 mM. The conditioned media were pumped onto a 6 mil Ni-NTA column quilibrated in 20 m Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 r/min. at 40C. A loading, the column wus washed with additional equilibration buffer and the protein luted wi equilibration but containing 0.25 M imidazole. The highly purified protein was subsequently deselled into as buffer containi mM Hopes, 0.14 M NaCl and 4% mannitol, pl 6.8, with a 25 ml G25 Superfine (Pharmr ia) column and sto at Immunoadhesin (Fe containing) constructs of proteins were purified from the condi- ned media as folloi The conditioned media were pumped onto a 5 ml Protein A column (Pharmnacia) which had can equilibrated in mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equ ibration buffer bef< elation with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by c olecting I ml fractic into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purifled protein was sub equntly dosalted it storage buffer as described above for the poly-His tagged proteins. The homogeneity of thd proteins was verif by SDS polyacrylamde gel (PEO) eloctrophoresis and N-rerminal amino acid sequencing Edman degradatio In BP Application No. 99912321.9 PRO polypeptides were succesfally expressed n baculovirus infect HiS insect cells. While the expression was actually perfdbred in a 0.5-2 L scale, it can be rea ily scaled up for la 8 preparation.

For expression in baculovirus-infeccted Hi5 insct cells, the PRO polypeptide-c.coding DNA may amplified with suitable systems, such as Pfu (Straiagene), or fused upstream of an eiope tag contained w a baculovirus expressLon vector. Such epitope lags include poly-his tags and immunoglobul Eags (like Pc regk of gO). A variety of plasmids may be employed, including plasmids derived from comm ally available plasm such as pVLl393 (Novagen). Briefly, the PRO polypcptide or the desired partion of the RO polypoptide (st as the sequence encoding the exurncellulur domain of a transmembrane protein) is aiplifi by PCR with prim complementary to thu 5= and 3= regions. The 5= primer may incorporate flanking (sele cd) restriction enzy.

sites. The product is then digested with those selected restriction enzymes and subcloned int the expression vec; For example, derivatives of pVL1393 can include the Pc region of human IgG (pb.PH EgO) or an 8 histidi (pb.PH.His) tag dovustream (3-ol) the NAME sequenc. Preferably, the vctor con ruc is sequenced confirmation.

IS cells arv grown to a confluency of 50% under the conditions of, 270C, no C02. NO pac/strep. For ei 150 mm plate, 30 S g of pIE based vector containing PRO polypeptide is mixed with 1 ml E-Coll medium (Med Ex-Cell 401 1/100 LGla JRH Biosciences #14401-78P (note: this media is light sensh and in a separ tube, 100 ul of CellFctin (CellFECTIN (GibcoBRL #10362-010) (vortexod to mix)) is mix d with 1 ml of Ex-C medium The two solutions are combined and allowed to incubate at room temperature for 5 minutes. 8 ml of I Cell media is added to the 2mnd of DNAICcIRECTIN mix and this is layered on Hi5 cells that have booeen wast once with Ex-Coll media. The plate is then incubated in darkness for 1 hour at r mtemperature. I DNA/CIlFECTIN mix is then aspirated, and the cells are washed once with Ex-Cell to remve excess ClUlFECI ml of fresh Es-Coll media is added and the cells are incubated for 3 days at 280C. The upernaalnt is harves 42 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19Z09 2003 18:37 FAX 61 3 92438333 GRIFFITH HACK [049 J.I209 203 8:37FAX 1 39243333GRIFFITH HACK04 and the expression of the PRO polypeptide in the baculovirus expression vector can be determined by batch hindi of I ml of supornatcnt to 25 mL of N-NTA beads (QIAGEN) for histidine uagged prottins or Protein-A Sepharo CL-4B beads (Pharmcia) for IgG tagged proteins followed by SDS-PAGE analysi copang to a kno concentration of protein standard by Coomassle blue staining.

The conditioned media from the transfocted cells (0.5 to 3 L) is harvested by contri igation to remove ti cells and filtered through 0.22 micron filters. For the poly-His tagged constructs, the prote comprising the PR polypeptide is purified using a Ni-NTA column (Qlagen) Before purification, imidazole is ad led to the condition media to a copcentratitn of 5 mM. The conditioned media is pumped onto a 6 mi Ni-NTA clumn equilibrated mM Hopes. pH 7, buffer containng 0.3 M NC and 5 mM imidazole at a flow rate of 4- ml/in. at 4tC AD loading, tbo column is washed with additional equilibration buffer and the protein cluted wijb equilibration buff containing 0.25 M imidazole. The highly purified protein is subsequently deslated into a stuage buffer conini mM Hepes, 0.14 M NaC and 4% mannitol, pH 6.8, with a 25 ml 025 Superfine (Phamena) column and stan at Inunuoadhein (Fr containing) constructs of proteins are purified from the conditicned media as follo' The conditioned media i pumped onto a 5 ml Protein A column (Pharmacia) which had been .quilibratcd in 20 mf Na phosphate buffer. pH 6.8. ALtor loading, the column is washed extensively with equilibrntion buffer befo clution with 100 mM citric acid, pH 3.5. The eluted protein is irmediately neutralized by c lcting 1 ml fractia: into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein is subs quntly desalted in storage buffer as described above for the poly-His tagged proteins. The homogeneity of PRO polypeptide can I assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman iegradation and oth analytical procedures as desired or necessary.

EXAMPI 9: mpanrAtin of Antibodies that Bind to PRO Polvuotides This example illustrates preparation of monoclonal antibodies which can speci polypeptide.

Techniques ar producing the monoclonal antibodies are known in the art and are c in Goding. su ln Immunogens that may be employed include purified PRO polypeptide, fus the PRO polypaptide. and cells expressing recombinant PRO polypeptide on the cell sur immunogen can be mode by the skilled artisan without undue experimcnutation.

Mice, such as Balb/c, are immunized with the PRO polypeptide immunognca ae Pround's adjuvant and injected subculaneously or intraperitoneally in an amount from Alternatively, the immanogen is emulsified in MPL-TDM adjuvant (Rihi Imunocheminal Re and iiected into the animal's hind foot pads. The immunized mice are then boosted 10 additional immunogen emulsifled in the selected adjuvanLt. Thereafter, for several weeks, boosted with additional immunization injections. Serum samples may be periodically obta retro-orbital bleeding for testing in BLISA assays to detect anti-PRO polypeptide antibodic After a suitable antibody titer has been detected, the animals "positive" for antibod a final Intravenous injection of PRO polypeplide. Three to four days later, the mice are s 43 ally bind to a PR ascribed, for insm, on proteins containig hce. Selection of t nulsified in complele 1-100 micrograms March. Hamilton, M1 to 12 days later with the mice may also ncd from the mice by 9.

es can be injectedwili rificed and the spla COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

I

19/09 2003. J& FAX 61 3 92438333 GRIFFITH HACK Z050 cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a seletod murine myclor cell line such as P3X63AgU.1, available from ATCC, No. CRL. 1597. The fusions generate b bridoma cells whi can then be plated in 96 well tissue cultore plate; containing HAT (bypoxanthine, aminopirin, and thymidin medium to inhibit proliferation of non-Cased cells, myoloma hybrid, and spleen coll hybrid- The hybridona cells will be screened in an ELISA for reactivity against the PRO polypeptide Determination of "pusitive" hybridoma cells secreting the desired monoclonal antibodes against the PR polypoptide is within the skill in the art.

I

The positive hybridoma cells can be injected iniraperltoneally into syngenceic Balic cc sio produce ascii containing the anti-PRO polypeptide monoclonal antibodies. Alternatively, the hybridomrna lls can be grown tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can 1 accomplished using atomonium ulfate precipitation, followed by gel exclusion chromatog aphy. Alternative: affinity chromatography based upon binding of antibody to protein A or protein G can be eployed.

EXAMpIE 2: hinwric PRO PolvuMtides PRO polypptides may be expressed as chimeric proteins with one or nmore additions polypeptide domai added to facilitate protein purification. Such purification facilitating domains include, but aw not limited to, mci chelating peptides uch as histidine-tryptophan modules that allow purification on immobilned metals, protein domains that allow purification on immobilized immunoglobulin, and the domain utill ad in the FLAGS extension/affinity purification system (Immunex Corp., Seattle Wash.). The inclusion of a cle vable linker sequen such as Factor XA or enteroldinase (Invitrogen, San Diego Calif.) between the purification domain and the PR polypeptide scqunc may be useful to facilitate expression of DNA encoding the PRO pol ptide.

EXAMPE 11J: Purification of PRO Polvneutides Usins Succific Antibodies Native or recombinant PRO polypeptides may be purified by a variety of standard iohniques in the art protein purification. For example, pro-PRO polypoptide, mature PRO polypeptide. or pre-PRO polypeptide purified by immunoafl inity chromatography using antibodies specific for the PRO polypoptidl of interest. In gencr an iummunoafinity coluam is constructed by covalently coupling the anti-PRO polypeptide a tibody to an activaL chromatographic rosin.

Polyclonal inmunoglobulins am prepared from imneB sara either by precipitadtion ivith ammonium sulfi or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, Likewi4 monoclonal antibodies amr prepared from mouse ascites fluid by ammonium sulfate precipita on or chromatograp on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chro atographic resin so as CnBr-activated SEPHAROSE"' (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the res is blocked, and the derivative resin is washed according to the manufacturer's instructions.

Such an imn unoafinity column is utilized in the purification of PRO polypptide py preparing a frnciii from cells containing PRO polypeptide in a soluble form. This preparation Is derived by so biization of he who cell or of a subcellular fraction obtained via differential ccntrifgation by the addition o0 detergent or by oth methods well known in the art. Alternatively, soluble PRO polypeptide containing a signal seuence may be secret 44 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:38FAX 61 3 92438333 GRIFFITH HACK 0051 in useful quantity into the medium in which the cells are grown.

A soluble PRO polypeptide-comaining proparation is passed over the immunoaffnity column, and e column is washed under conditions that allow the preferential absorbance of PRO polype tide high io ic strength buffers in the iesence of detergent). Then, Oth cohmn is eluted under conditions that disrupt Antibody/P O polypeplide binding a low pH buffer such as approximately pH 2-3. or a high concentrati of a chaotrope suh as urea or thiocyanate ion), and PRO polypeptide is collected.

EXAMPLE 12: r Dtg Screcning This invention is particularly useful for screening compounds by using PRO pu ypeptides or binding fragment thereof in any of a variety of drug screening techniques. The PRO polypeptide or fragment employed in such a test may either lie fee in solution, affixed to a solid support, borne on a cell surface, or located intacellular y One method of drug screening utilizes eukaryotic or prokaryolic host cells which are st bly transformed w th recombinant nucleic atids expressing the PRO polypoptide or fragment. Drugs are screened a ainst such transfor d cells in competitive binding assays. Such cells, either in viable or fixed form, can be us4 for standard bindi g assays. One may measure, for example, the formation of complexes between PRO polypep de or a fingment aid the agent being tested. Alternatively, one can examine the diminution in complex founalion between the PI0 polypeptid and its target cell or target receptors caused by the agent being tested.

Thus, the prtsent invention provides methods of screening for drugs or any other gents which can aff ct a PRO polypeptidc-associated disease or disorder. These methods comprise contacting suc an agent with an P 0 polypeptide or fragment thereof and assaying for the presence of a complex between 1 e agent and the P.O polypeptide or fragment, or (ii) for the presence of a complex between the PRO polypepide c r fragment and the c 11, by methods well known in the art. In such competitive binding assays, the PRO polypeplide Dr fragment is typica ly labeled. After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound fr and the amoun of frue or uncomplexed label is a measure of the ability of the particular agent to bind to PO polypeptde or to interfere with the PRO polypeptide/coll complex.

Another tcclmique for drug screening provides high throughput scroeing for com ounds having suita e binding affinity to a polypeptide and is described in detail in WO 84/03564, published od September 13, 198.

Briefly stated, large iLumbers of different small peptide test compounds are synthesized on a solid substrate, v :h as plastic pins or some other surface. As applied to a PRO polypeptide, the peptide test comTn ounds are reacted w th PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known i the art Purified PR polypeptide can also be coated directly onto plates for use in the aforementioned drug sreenlng techniques. In addition, non-neutralizing antibodies can be used to capture the puptide and immobilize it n the solid support.

This invention also contemplates the use of competitive drug screening assay in which nutralizi g antibodies capable of binding PRO polypeptide specifically compete with a test compouid for binding to PRO pulypeptide or fragments thereof. In this manner, the antibodies can be used to detect the ce of any peptide which shares one or more antigenic determinants with PRO polypeptide.

EXAMPLE 13: Ratimal Drug Depsign COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 li IllI III il III n n, H:\cintae\Keep\speci\2003248191 amendments.doc 07/04/05 The goal of rational drug design is to produce structural analogs of biologically active polypeptide of 0 interest a PRO polypeptide) or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO S polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo Hodgson, Bio/Technologv, 9: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the PRO polypeptide must be ascertained Sto elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins.

00 In both cases, relevant structural information is used to design analogous PRO polypeptide-like molecules or to identify efficient inhibitors. Useful examples of rational drug design may include molecules which have improved O activity or stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or which act as CN inhibitors, agonists, or antagonists of native peptides as shown by Athauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides would then act as the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

EXAMPLE 14: Gene Amplification This example shows that genes encoding various PRO polypeptides are amplified in the genome of certain human cancers. Amplification is associated with overexpression of the gene product, indicating that the PRO polypeptide is a useful target for therapeutic intervention in certain cancers such as colon, lung and other cancers. The therapeutic agent may take the form of antagonists of PRO polypeptide-encoding genes, for example, murine-human chimeric, humanized or human antibodies against the PRO polypeptide.

The starting material for the screen was genomic DNA isolated from a variety cancers. The DNA is quantitated precisely, fluorometrically. As a negative control, DNA was isolated from the cells of ten normal healthy individuals which was pooled and used as assay controls for the gene copy in healthy individuals (NorHu).

The 5' nuclease assay (for example, TaqMan

TM

and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection SystemTM (Perkin Elmer, Applied Biosystems Division, Foster City, were used to find genes potentially amplified in certain cancers. The results were used to determine whether the DNA encoding 19/09 2003 18:39FAX 61 3 92438333 GRIFFITH HACK R053 the PRO polypeptlde is over-represented in any of the lung and colon cancers that were scr ened. The result v reported in Delta CT units. One unit corresponds I PCR cycle or approximately a 2-fold at plification relative normal, two units cornrsponds to 4-fold, 3 units to 8-fold and so on. Quantitation was obtai ed using primers a a Taqman T M fluorescent derived from the PRO polypeptide-encoding gene. Regions of the P O1 polypeptide wh are most likely to conTain unique nucleic acid sequences and which are least likely to have pliced out introns preferred for the primer derivation, 3=-untranslatcd region.

The 5' nuclease assay reaction is a fluorecent PCR-based technique which makes usf of the 5' exonucle activity of Taq DNA polymerase enzyme to monitor amplification in real dme. Two oligo ucleotide primers: used to generate an amplicon typical of a PCR reaction. A third oligontcleotide, or probe is designed to det nucleotide sequence located between the two PCR primers. The probe is non-extendible by aq DNA polyme enzyme, and is laboled with a reporter fluorescent dye and a quencher fluorescent dye. Any ascr-induced enmei from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are the probe. During the amplification reaction, the probe is cleaved by the Taq DNA po ymorase enzyme it template-dependent manner. The resultant probe fragments disassociate in solution, and si nal from the releat reporter dye is free from the quenching offect of the second fluorophore. One molecule of r porter dye is liberal for each new molccul: synthesized, and detection of the unquenched reporter dye provides td basis for quantiac interpretation of the data.

The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 77001 Sequence Detection. The system consists of a thcrmocyler, laser, charge-coupled devi c (CCD) camera a computer. The systcm amplifies samples in a 96-well format on a thormocycler. During amp iiation, laser-indui luorescent signal is collected in real-time through fiber optics cables for all 96 wells, and de ected at the CCD. 1 system includes software for running the intument and for analyzing the data NucIese ,ssay data are initially expressed as C, or the threshold cycle. This is dofined as the cyck which the reporter signal accumulates above the background level of fluorescence. The Ct values arc used quantitative measurement of the relative number of starting copies of a particular targot se] uence in a nucleic a sample.

EXAMPLE 15: I.rMu Hybridlzation In situ hybridization is a powerful and versatile technique for tIh detection and loclization of nucleic a sequences within cell or tissue preparations. It may be usoedl. for example, to identify si es of gene expressil analyze the tissue disl ribution of transcription, identify and localize viral infection, follow ch nges in specific mRT synthesis and aid in chrumosome mapping.

In situ hybridization was performed following at optimized version of the protoco by Lu and Gillett, C Mioan 1:169-176 (1994), using PCR-gencrated "P-labelod riboprobes. Briefly, fnrmalin-fi ad, parain-embedd human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 5 minutes at 37"C, a further processed for in situ hybridization as described by Lu and Gillett, supra. A UTP-labeled antisei riboprobe was generated from aPCR product and hybridized at 55C overnight. The slida were dipped in Koc NTB2 nuclear track amulsion and exposed for 4 weeks.

47 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:40 FAX 61 3 .24383 3 GRIFFITH HACK Ia054 -Ribaoprobe sn ss p (125 mCi) of 'P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vi containing dried 3P-UTT, the following ingredients wre added: gl Sx transcription buffer pIDT' (100 mM) l NTP mix (2.5 mM: 10 each of lo mM GTP CTP ATP 10pl H] 1 pL UTP (50 pM) pl Rnsin p DNA template (I g) p1 liO i. p RNA polymerase (for PCR products T3 AS. T7 S, usually) The tubes were incubated at 37C for one hour. 1-0 pl RQ1 DNase were added, f at 37C for 15 minutes. 90 p1 TB (10 mM Tris pH 7.6/1mM EDTA pH 8.0) were added pipetted onto DB81 paper. The zemaining solution was loaded in a Microcon-50 ultrafiltati program 10 (6 inute). The filtration unit was inverted over a second tube and spun using After the final recovery spin, 100 p1 TB were added. 1 pl of the final product was pipet counted in 6 ml of Biofluor 11.

The probe was ran on a TBEfura gel. 1-3 pl ofthe probe or5 pl ofRNA Mirk IL loading buffer. After heating on a 95C heat block for three minutes, the gel was immediat walls of gel were flushed, the sample loaded, and run at 1 80-250 volts for 45 minutes. The g wrap and exposed to XAR film with an intensifying screen in -700C freezer one hour to ov 33 P-Hybridizatio A. ftratmonnt of frown sections The slides ware removed from the freeer, placed on aluminium trays and thawed minutes. The trays were placed in 55 0 C incubator for five minutes to reduce condsation for 10 minutes in 4% paraformaidehyde on Ice in the fume hood, and washed in 0.5 x SSC temperature (25 ml 2(1 a SSC 975 mi SQ IO). After deproteination in 0.5 pCt/l protein 37C (12.5 ipl of 10 ng/ml1 stock in 250 ml prewarmed RNas-free RNAse buffer), the secti x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%. 95%, each.

B. Pretreatmem of araffin-Smbcdded sctio The slides were deparaffinized, placed in SQ H2O, and rinsed twice in 2 x SSC a minutes each time. The sections were deproteinared in 20 g/ml proteinass K (500 pa RNas-free RNase buffer, 370C, 15 minutes) human embryo, or 8 x proicinase K (100 pn1 37C, 30 minutes) forrmalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were p above.

C. Prihybridizasio i The slides were laid our in a plastic box lined with Box buffer (4 x SSC, 50% fl a 48 c dried. To each tube )llowcd by incubati and the mixture a n unit, and spun usi rogram 2 (3 minute d on DE81 paper a were added to 3 pl ly placed on ice. I I was wrapped in sal rnight.

room tmperawre The slides wereo hx or 5 minutes, at ro se K for 10 minutes ms were washed in ethanol, 2 minu room temperature. 1 f 10 mg/mi in 250 250 ml Anase buff erformed as describ mide) saturated fler COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:4-QFAX 61 3 92438333 GRIFFITH HACK .1055 paper. The tissue was covered with 50 p1 of hybridization buffer (3.

75 g Dextran Sulfate 6 SQ H0), vorto d and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18.15 ml formamide, 3.75 mrl 20 x SSC and 9 mi SQ HO were added, the tissue was vortexed well, and incubated at 42cC for 1-4 hours.

D. WIidixatian x 10 cpn probe and 1.0 pl tRNA (50 mg/ml stock) per slide were heated at 95C for 3 minutes. he slides were cooled on ice, and 48 p hybridization buffer were added per slide. After vortexig, 50 pl p nmix We added to 50 pl prehybridization on slide. The slides were incubated overnight at E. Waihe Washing was done 2 x 10 minutes with 2xSSC. EDTA at room temperature (400 ml 20 x SSC 16 ml 0.25M EDTA, V=4L followed by RNascA treatment at 37*C for 30 minutes (500 pl of 10 ng/m] in 250 ml Rni buffer 20 pg/m), The slides were washed 2 x 10 minute with 2 x SSC, EDTA at rqom tempcrarC. The stringency wash conditions were as follows: 2 hours at 55 0 C, 0.1 x SSC, EDTA (20 nil 20 x SSC 16 mi ED A.

V-4L).

F. Olizonucleoides In situ analysis was performed on a variety of DNA sequences disclosed herein.! The oligonucleoti es employed for these analyses were derived from the nucleotide sequences disclosed herein ad generally range about 40 to 55 nucleAdOdes In length.

49 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 -i 19/09 2003 18:40 FAX. 81 3 92438333 GIF~ AKll5 GRIFFITH HACK R056 EXAMLE 16: Tablet Piimnrv Lung za Cgon Tuio-r Profles Priman Tumor Stg &agW Human lung tumor Ade dcCa (3RCC724) [LTI1] 11A Human lung tumor SqCCa (SRCC72S) [LTl 11B3 Human lung tumor AdenoCA (SRCC726) [LT2] lB Human lung tumor Ad enoat (SkCC727) (LT3] UJA Human lung tumor AdericCa (3RCC728) [LT4J 113 Human lung tumor SqCCa (SRCC7Z9) P-TO] ID Human lung tumor Aden/SqCCa (SRCC73O) (LT7) 1A liuman lung mirnor AdonaCa (5RC073 1) (LT9] lB Human lung tumor Sc 1 CCa (SRCC732) [LTJOJ 1113 Human lung tumor SqCCa (SRCCI33) [LT1I] IIA Human lunig tumor AdcrioCa (SRCC734) [LT 12] IV Human lung tumor AdcaaqcCCa (SRCC73S)(LT13]IB Human lung tumor SqCC. (5RCC736) [LT1S] lB Human lung tumnor SqCCa (5RCC737) [LTI6] TB Human lung tumor SqCCa (8 RCC73 B) [LTI?] 111B Human lung tumor SqCCa (SR(CC739) [LTlS] lB Human lung tumor- SqCCa (SRCC74O) [LTl9j 1131 Human lung tumor LC(:a (SRCC741) [LTZ1) ITB luman lung AdenoCa CSRCCS1 1) tMflZ tA Human colon Adanoai (SRCC74Z) (CM] Ml Human colon Adena!a (61WC43) [CT3J Human colon AdenoCa CSRCC744) (CT8] Human colon AdonoCa (SRCC74S) [C710] Human colon AdenoCa (SRCC74S) [Cfl2] MO, R Humanl colon AdenoCa (SRC0747) (Cfl4] pMO, Humnan colon AdcnoCa (SRCC74S) [CTIS] Ml, R Human colon AdaneCa CSRCC749) [CT 16] pMQ Human colon AdenoCa (8RC0750) [Ori?] Human colon AdenoCa (SRCC75 1) (all3 I Ilman colon AdenoCa (SRCC7S2) [(a43 Human colon AdenoCa (SRCO7S3) (Cr5] Human colon AdenoCa (SRCC7S4) (CT6J Human colon AdenoCa (SRCC75S) (CI Human colon AdcnoCa (SRCC7SE) Human colon AdenoCa (SRCC7S7) [Te III Hluman colon AdenoCa (SRCC7SS) [Cal8 B] MO, Other Stu Dukes tage IT Sfl

TI

T3 T2

TI

T2 T2

TI

722 T2

TI

T21 T2 T21 T21 T2 T3

TI

D pT4 NO 13 p12 B T3 A p77 NO B3 n NO OB pT3 pNO 1) T4 N2 B p173 pNO Cl p173 pNl MO.,RI B pT -1 B on2 02 C1 l pMO, RO B3PT 0I A p22 03 D p1T4 B n2 NO B3 pn7 pNOj N Stol

NI

NO

NO

N2

NO

NO

NO

NO

NI

NI

NO

NO

NO

NO

NI

NO

NO

Ni1

NO

NO

NO

NO

MO

pl(O pNO pNO

PNZ

I1

R

'0 DNA PepWaration: MNA was prepared fromi cultured cell lines, primary tumtors, nomal human blood. 'Pe isolation was perfbrmed using purification kit, buffer set and proteae and all from Qulagen, according to thiJ manutlcture' instructions and the description below.

Cell culture ly. is: Ceills were wa* hod and trypainized at a concentrati 'on of 735 x 10 por tip and pelleted b$1 ocatrifuging a: 1000 rpm for 5 minutias at 40(J, followed by washing again with 1/2 volumc of PBS recenfligatiou. The pellets wore washed -A third time, the suspended cells collected and wushead 2x with PBS. The cells were then suspended into 10 ml PBS. Buffer Cl was equrilibratcd at VC. Qiagen proteane #1!15 walluted into 6.25 ml cold ddH 2 O to a finalI concentration of 20 mg/mI and equilibrated at 4 0 C. 10mnl of 02 Buffr was prepared by diluting Qiaen, RNAse A stock (100 mg/mi) to a final concentratdon of 200 0"/m.

COMS ID No: SMSI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:40 FAX 61 3 9243833 GRIFFITH HACK a057 Buff- C1 (10 iml, 4QC) and ddH20 (40 ml, 40C) were then added to the 10 ml of cell suspnsion, mixed by inverting and ncubated on ice for 10 minutes. The cell nuclei were pelleted by ceirifuging in a Beckman swinging bucket rotor at 2500 rpm at 4°C for 15 minutes. The supernatant was discarded and the nuclei were suspended with a vortex into 2 ml Buffer CI (at 4C) and 6 ml ddHiO, followed b a second 4°C centrifugation at 2500 rpm for 15 minutes. The nuclei were then resuspendcd into the residual buffor using 200 O1 per tip. 32 buffer (10 ml) was added to the suspended nuclei while gentle vortexing was applied. Upon completion of buffer addition, vigorous vortexing was applied for 30 seconds. Quiagen pr tease (200 01, prepared as indicated above) was added and incubated at 50'C for 60 minutes. The ncubation and centrifugation was repeated until the lysates were clear incubating additional 30-60 minu es, pelloting at 3000 x g for 10 min., 4"C).

Solid human A mnr sample preparation and lysir: Tumor samples were weighed and placed into 50 ml conical tubes and held on ice. Processing was limited to no more than 250 mg tissue per preparation (1 tip/preparation), The prpteasc solut on was freshly prepared by diluting into 6.25 ml cold ddH 3 O to a final concentration of 20 mg/ml and stored at 4°C. G2 buffer ml) was prepared by diluting DNAse A to a final conentration of 200 mg/ml (from 100 mgml stock). The tumor tissue was homogenated in 19 ml 02 buffer for 60 seconds using the large ip of th polytron in a laminar-flow TC hood in order to avoid inhalation of aerosols, and held at'room temperature. B twean samples, the polytron was cleaned by spinning at 2 x 30 seconds each in 2L ddH20, foUowcd by G2 buyer (50 ml). If tissue was still present on the generator tip, the apparatus was disassembled and cleaned.

Quiagen prot ase (prepared as indicated above, 1.0 ml) was added, followed by vortexing and incubation at 50C for hours. The incubation and centrifugation was repeated until the lysates vre clear inoubating additional 30-60 minutes, pelleting at 3000 x g for 10 min., Hlwnan blood preparation and lysis: Blood was drawn from healthy volunteers using standard infectious agent protocols an citrated into ml samples per tip. Quiagen protease was freshly prepared by dilution into 625 ml cold d H a O to a final concentration of 20 rrg/ml and stored at 4°C. 02 buffer was prepared by diluting RNAec A to a final concentration of 200 Oeml from 100 mg/ml stock. The blood (10 ml) was placed into a 50 ml sonical tube and ml Cl buffer and 30 ml ddH20 (both previously equilibrated to 4°C) were added, and the corponents mixed by inverting and held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging bucket rotor at 2500 rpm, 4*C for 15 minutes and the supernatant discarded. With a vortex, the nuclei were s upended into 2 ml Cl buffer (4C) and 6 ml ddHzO Vortexing was repeated until the pellet was white. he nuclei were then suspended into ths residual buffer using a 200 01 tip. G2 buffer (10 ml) were added the suspended nuclei while gently vornexing, followed by vigorous vortcxing for 30 seconds. Quiagen protase was added (200 c1) and incubated at 50°C for 60 minutes. The incubation and centrifgation was repeated until the lysates were clear incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min, 4"C.

Purfication ocleared lysates: slarion of nmic DNA=: Gcnomic DNA was equilibrated (1 sample per maxi tip preparation) with 10 ml Q T buffer, QP elution buffer wAs equilibrated at 50"C. The samples were vortexed for 30 seconds, th m loaded onto 51 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 H:\cinta\Keep\speci\200324891 amendmentrs.doc 07/04/05 equilibrated tips and drained by gravity. The tips were washed with 2 x 15 ml QC buffer. The DNA was eluted 0 into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 ml QF buffer Isopropanol (10.5 ml) was added to each sample, the tubes covered with paraffin and mixed by repeated inversion until the DNA precipitated. Samples were pelleted by centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at 4 0

C.

The pellet location was marked, the supernatant discarded, and 10 ml 70% ethanol was added. Samples were pelleted again by centrifugation on the SS-34 rotor at 10,000 rpm for 10 minutes at 4°C. The pellet location was marked and the supernatant discarded. The tubes were then placed on their side in a drying rack and dried minutes at 37 0 C, taking care not to overdry the samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) and placed at 50 0 C for 1-2 hours.

Samples were held overnight at 4°C as dissolution continued. The DNA solution was then transferred to 1.5 ml 00 tubes with a 26 gauge needle on a tuberculin syringe. The transfer was repeated 5x in order to shear the DNA.

Samples were then placed at 50°C for 1-2 hours.

Quantitation of genomic DNA and preparation for gene amplification assay: The DNA levels in each tube were quantified by standard A26, A 280 spectrophotometry on a 1:20 dilution 01 DNA 95 01 ddH20) using the 0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer.

A

2 60/A 280 ratios were in the range of 1.8-1.9. Each DNA sample was then diluted further to approximately 200 ng/ml in TE (pH If the original material was highly concentrated (about 700 ng/ 01), the material was placed at 50 0 C for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material (20-600 ng/ml) using the manufacturer's guidelines as modified below. This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst dye working solution (#H33258, 10 01, prepared within 12 hours of use) was diluted into 100 ml 1 x TNE buffer. At 2 ml cuvette was filled with the fluorometer solution, placed into the machine, and the machine was zeroed. pGEM 3Zf(+) (2 01, lot #360851026) was added to 2 ml of fluorometer solution and calibrated at 200 units. An additional 2 01 of pGEM 3Zf(+) DNA was then tested and the reading confirmed at 400 10 units. Each sample was then read at least in triplicate.

When 3 samples were found to be within 10% of each other, their average was taken and this value was used as the quantification value.

The fluorometricly determined concentration was then used to dilute each sample to 10 ng/ 01 in ddH 2

O.

This was done simultaneously on all template samples for a single TaqMan plate assay, and with enough material to run 500-1000 assays. The samples were tested in triplicate with Taqman T primers and probe both B-actin and GAPDH on a single plate with normal human DNA and no-template controls. The diluted samples were used provided that the CT value of normal human DNA subtracted from test DNA was 1 Ct. The diluted, lotqualified genomic DNA was stored in 1.0 ml aliquots at -80 0 C. Aliquots which were subsequently to be used in the gene amplification assay were stored at 4°C. Each 1 ml aliquot is enough for 8-9 plates or 64 tests.

Gene amplification assay: The PRO polypeptide compounds of the invention were screened in the following primary tumors and the resulting Ct values greater than or equal to 1.0 are reported in Table 3 below.

TSlc ACtv___ in hfg lfd colon pimkytimO and cel lb n=dels Tumwor r PRO PRO PRO PRO FOPRO PRO PRO PO PRO PRO PRO PRO PRO PRO PRO PR101330 and CIl Lim 213-L 237 324 351 352 615 L 853 101? 618 712 70 72 474 274 311 111 PRO6449 LT-1 1.0 ic .T I LT-It 134 i4 L- I- 1.70 1.7385 '1.33 1.22 1.16 194 1.24 Ibm MSZ 151 1.74 L 15 233 103 L.1I1 1.77 L.0 2.55 Li 1.55 1214 1.52 L4 7-U 1- IM 1.46 I j, 124 2.26 L6 1.56 116 IDD 2.07 2.80 1 IA 5 1.91 2.10 1 r~ Ln I 2 1 t.4 IA9 U 1.2 1- 1.44 1- 1.0(6 Z AS 1 0 34 LT4 1.21 14 I 1.10274 14 2 .24 1.14 2.1 -t 2 1 1150 1.77 -5 1.4 -m 16 1. 1.1 .1 3 1 1.44 M- 1.06 211 1.16 2 L.29 I 1 15 2.26 1 1.46 1.79 154 11.4 I1A4 120 I tnri f 1 I t1 rI i In- L f1n+-...Ir I I LTIZ- 11.92 m- 1.86 LIt 1 1S I- 1138 I- 1 1- 1.86 -19/09 2003 I A-LAX1 3 92438333 GRIFFITH HACK 060 I 1 Ic I jI I o I I' I- II -i I l()I I i -s I I C- i COMS ID No: SMBi-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 W -1/09 203 18:42 FAX 61 3 92438333 GRIFFITH HACK 1Q0O61 I wqqr

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r H:\cintae\Keep\speci2003248191 amendmcnts.doc 07/04/05 Deposit of Material The following materials have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): Material ATCC Deposit No. Deposit Date DNA50988-1326 ATCC 209814 28 April 1998 These deposits were made under the provisions of the Budapest Treaty on the International Recognition O of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The 00 deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement Cc between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the Sculture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of K1 any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC 122 and the Commissioner's rules pursuant thereto (including 37 CFR 1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit or material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

The entire disclosure in the complete specification of our Australian Patent Application No. 30721/99 (762055) is by this cross-reference incorporated into the present specification.

Claims (27)

1. An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420).

2. The isolated nucleic acid molecule of claim 1, encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420).

3. The isolated nucleic acid molecule of claim 2, encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420). 00

4. The isolated nucleic acid molecule of claim 3, encoding a polypeptide comprising the amino Sacid sequence shown in Figure 2 (SEQ ID NO:420). The isolated nucleic acid molecule of claim 1, comprising the full-length coding sequence from within the nucleotide sequence shown in Figure 1 (SEQ ID NO:419).

6. The isolated nucleic acid molecule of claim 1, which comprises the full-length coding sequence of the DNA deposited under accession number ATCC 209814.

7. A vector comprising the nucleic acid of any one of claims 1 to 6.

8. The vector of claim 7, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.

9. A host cell comprising the vector of claim 7 or claim 8. The host cell of claim 9 which is a CHO cell, an E. coli cell or a yeast cell.

11. A process for producing a polypeptide, comprising culturing the host cell of claim 9 or claim under conditions suitable for expression of said polypeptide, and recovering said polypeptide.

12. An isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420).

13. The isolated polypeptide of claim 12, comprising an amino acid sequence having at least sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420).

14. The isolated polypeptide of claim 13, comprising an amino acid sequence having at least sequence identity to the amino acid sequence shown in Figure 2 (SEQ ID NO:420). 1 H:\cintae\Keep\speci\2003248191 amendments.doc 07/04/05 The isolated polypeptide of claim 12, comprising the amino acid sequence shown in Figure 2 O (SEQ ID NO:420).

16. The isolated polypeptide of claim 12, comprising the amino acid sequence encoded by the DNA deposited under accession number ATCC 209814.

17. A chimeric molecule comprising a polypeptide according to any one of claims 12 to 16, fused to a heterologous amino acid sequence.

18. The chimeric molecule of claim 17, wherein said heterologous amino acid sequence is an 00 epitope sequence. (N

19. The chimeric molecule of claim 17, wherein said heterologous amino acid sequence is an Fc C( region of an immunoglobulin. An antibody which specifically binds to a polypeptide according to any one of claims 12 to 16.

21. The antibody of claim 20, which is a monoclonal antibody.

22. The antibody of claim 21, which is a humanized antibody.

23. The antibody of claim 21, which is a chimeric antibody.

24. The antibody of claim 21, which is a human antibody. A composition comprising an antibody according to any one of claims 21 to 24, together with a pharmaceutically acceptable carrier.

26. A nucleic acid which is antisense to a nucleic acid molecule according to any one of claims 1 to 6.

27. A composition comprising an antisense molecule according to claim 26, together with a pharmaceutically acceptable carrier.

28. A composition comprising a chimeric molecule according to any one of claims 17 to 19, together with a pharmaceutically acceptable carrier.

29. A method of treatment of cancer, comprising the step of administering an effective amount of an antagonist of PR0717 to a subject in need of such treatment. A method according to claim 29, in which the cancer is a colon or lung cancer. H:\cintac\Keep\speci\2003248191 amendments.doc 07/04/05 O 31. A method according to claim 29 or claim 30, in which the antagonist is an antibody according to any one of claims 20 to 24, or an antisense molecule according to claim 26.

32. Use of an antagonist of PR0717 in the manufacture of a medicament for the treatment of cancer. O

33. Use according to claim 32, in which the cancer is a cancer of the lung or colon.

34. Use according to claim 32 or claim 33, in which the antagonist is an antibody according to any 00 one of claims 20 to 24 or an antisense molecule according to claim 26. 32. An isolated nucleic acid molecule according to claim 1, substantially as herein described with C1 reference to any one of the examples and drawings. 33. A method according to claim 29, substantially as herein described with reference to any one of the examples and drawings. 34. Use according to claim 32, substantially as herein described with reference to any one of the examples and drawings. Dated this 7th day of April 2005 GENENTECH, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia EDITORIAL NOTh: The Gene 5ecjuence Listings has been filed on dliskette as physiCaI medila.