AU2003248191A1 - Novel PRO717 polypeptides and nucleic acids encoding the same - Google Patents

Description

19/09 2003 18120T 61 3 92438333GRFIhHC i0 GRIFFITH HACK a0o6

AUSTRALIA

Patents Act 1990 COMIPLETE SPHCIflCLTION1 STANDARD PALTEN AppliCant:' invention GENENTECH, INC.

Title: NOVEL PR0717 POLYPEPTIDES AND FIUCLEIC THE SAME ACIDS ENC0O1ING The following statement is a full description of this invention, including the best method of performing it kno-r mefus; Ffl to COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:20 FAX 61t_19243833 GRIFFITH BACK .007 NOVEL rRO717 POLYPEPTIDES AND NUCLEIC ACIDS ENCODING T SAME FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of ndvl DNA and to recombinant production of novel polypeptides encoded by that DNA.

BACKGROUND OF T EE TNVENaTION Extracellular proteins play an important role in the formation, diffrentiation and maintenance of multicellular organisras, The fate of many individual cells, proliferation, migrati n, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the mmcdilto environment This infirmation is oftsen tranuitted by secrted polypeptides (for instance, mit genic factors, survi at factors, cytotoxic falors, differentiation factors, neuropoptides. and hormones) which are in .urn, received and inirpreted by diverso cell receptors or membrane-bound proteinL These secreted polj peptides or signaling molecules normally pass through the cellular asecretOry pathway to reach their site of aci in the exracell cnvironmcnt Secreted proteins have various industrial applcations, including pharmacenticals, diagnostics, biosens s and bioreactars. Most protein drugs available at present, such as thrombolytic agents, interferons, interleuld a.

erythropoiclins. colony stimulating factors, and various other cytokinos, are secretory ins. Their recep which are membrane proeins, also have potential as therapeutic or diagnostic agents. Effors are being undertal n by both industry and mcademisa to identify now. native secreted proteins. Many cforts ar flused on the scren g of mammalian recombinant DNA libraries to identify the coding sequences for novel secre d proteins. Examps of screening methods and techniqucs are described in the literature [see, for example, Kcin t al., E. NAL Sol., 23:7108-7113 996); US. Paent No. 5,536,637)].

Mmbrane-hound proteins and receptors can play an important role in the formallon, differemlation e d maintenance of multicellular organisms. The fate of many individual cells, p4lifration, migratian, differntiation, or intrraction with other cells, is typically governed by information received om other cells andor the imnmdiate environment This information is often tranmined by secreted polypcptdes for instance, mitog ic factors, survival factors, cycotoxic factors, differentiation factors. neuropoptides, and horm es) which are, in rn, received and interpreted by diverse cell receptors or membrane-bound proteins. Such mefbrane-bound proteins and call receptors include, but are not limited to, cytokine receptors, receptor kinases, jccptor phosphat es, receptors Involved in cell-cell interactions, and cellular adhesln molecules like selctins and integris. For insta ranaduction of signals that regulate cell growth and differentiation is regulated in part by phosphoazyltionl of vai us cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process. can als act as growth fa tor receptors. Examples include fibroblast growth factor receptor and nerve growth facto rtptor.

Mcmbrane-Ionnd protelins and receptor molecules have various indusbtrial apllcations, includin as pharmaceutical and diagnostic agents. Receptor immnunoadhesins, for instance, can be eployed as therapotic agents to block receptor-ligand interaction. The membrane-bound proteins can also be 4 loyed for screeig of COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:21 FAX 61 3 92438333 GRIFFITH HACK oo008 potential poptidc or small molecule inhibitors of the relevant receptor/ligand interactiol. Efforts are bei g undertaken by both industry and academia to identify new, native receptor proteins. Many ;fforts are focused m the screening of mammalian recombinant DNA librarics to identify the coding sequences for n vel receptor prote s.

We herein describe the identification and characterization of novel secreted and transmembra e polypeptides and novel nucleic acids encoding those polypeptides.

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

All references, including any patents or patent applications, cited in this speification are here y incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy a d perdinency of the cited documents. It will be clearly understood that, although a number of priar art publications a referred to herein, his reference does not constitute an admission that any of these docum ns forms part of f e common gcneral knowledge in the art, in Australia or in any other country.

In the claims of this application and in the description of the invention, except whert the context require s otherwise due to express language or necessary implication, the words "comprise" or variation such as "comprise" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated featus but not to precld the presence or addition of further features in various embodiments of the invention.

SUMMARY OF THE INVENTION 1PR 717 In a frst aspect, the present invention provides an isolated nucleic acid molecule oding a polypopli comprising an amino acid sequence having at least 80% sequence identity to the amino a sequence shown i Figure 2 (SEQ ID NO:420).

Applicants have identified a cDNA clone that encodes a novel 12 transmembrane polypeptide, wherein th polypoptide is designated in the present application as "PR0717".

In one embodiment, the invention provides an isolated nucleic acid molecule comprsing DNA encoding a PR0717 polypeptide. In one aspect the isolated nucleic acid comprises DNA encoding the PR0717 polypeptid having amino acid residues 1 through 560 of Figure 169 (SEQ ID NO:420). or is complemen ary to such encoding nucleic acid sequence, :nd remains stably bound to it under at least moderate, and optionally. tnder high stringenc conditions. The isolated nucleic acid sequence may comprise the cDNA insert of the vector posited on April 28 1998 with the ATCC as DNAS0988-1326 which includes the nucleotide sequence encoding R0717.

In another embodiment, the invention provides isolated PR0717 polypeptido. In pa ticular, the inventioi provides isolated native sequence PRO717 polypeptide, which in one embodiment, includes a 1 amino acid sequenc comprising residues 1 through 560 of Figure 169 (SEQ ID NO:420). An additional embo iment of the presen invention is directed to an Isolated extracellnlar domain of a PRO717 polypeptide. Optionally, the PRO71! 2 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:21 FAX 61 3 92438333 GRIFFITH HACK oo009 polypeptide is obtained or is obtainable by expressing the polypeptide encoded by the cDNA insert of the vect r deposiutd on April 28, 1998 with the ATCC as DNA50988-1326.

In other cmbcdiments of the present invention, the invention provides vectors comrsing DNA encoding any of the above or below described polypeptides. A host cell comprising any such vector is aSo provided. By way of example, the host cells may be CHO cells, E coli, or yeast A process for producing any of the above or bel w described polypeptides is futher provided and comprises cultuing host cells under conditions itable for expression of the desired polypepdde and recovering the desired polypeptide from the cell culture.

In other embdiments, the invention provides chimeric molecules comprising any 4f the above or below described polypeptides fused to a hotcrologous polypeptide or amino acid sequence. An examlie of such a chimeric molecule comprises alLy of the above or below described polypeptides fused to an epitope tag sequence or a Fe region of an immunoglobulin.

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

In yet other embodiments, the invention provides oligonucloolide probes useful for Isolating genomic a d eDNA nucleotide sequences, wherein those probes may be derived from any of the abov 4 or below described nucleotide sequences.

BRIEF DESCRITION QO THE DRAWINGS Figure 1 shows a nucleotide sequence (SEQ ID NO:419) of a native sequence PR 717 cDNA, whmin SEQ ID NO:419 is a clone designated herein as AUNQ385" andor ADNAS988-1326".

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

DiTAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS L Definitions The terms "PRO polypeptide" and "PRO" as used herein and when immediately followed by a numeric i designation refer to various polypeptides, wherein the complete designation PRO/number) rcfrs to slpcific

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polypeptide sequences as described herein. The terms 'PRO/number polypeptide" and "PIRO/numbcr" as use herein encompass native sequence polypeprides and polypeptide variants (which are further lefined herein). The PRO polypeptides described herein may be isolated from a variety of sources, such as from 4mn tissue types c from another source, or prepared by recombinant or synthetic methods.

A "native seqence PRO polypepdide" comprises a polypopide having the same an no acid sequence a the corresponding PRO polypcptide derived from nature. Such native sequence PRO polype fides can be isolate from nature or can be produced by recombinant or synthetic means. The term "native sequernc PRO polypeplide" specifically encompasses naturally-ooccurring truncated or secreted forms of the specific PRO polypeptide an extracellular domain sequence), naturally-occurring variant forms alternatively spliced orms) and naturally occurring allelic variants of the polypcptide. In various embodiments of the Invention, the nat ve sequence PRO7I7 polypeptide is a mature or full-length native sequence PR0717 polypeptide comprising amin acids I through 561] 3 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 200 FAX 61 3 92438333 GRIFFITH BACK IO010O of Figure 1 (SEQ ID NO:420).

The PRO polypeptide "oxtraellular domain" or "ECD" refers to a form of the PR 3 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 ansmnambrane and/or cytoplasmic douains and preferably, will have ]s than 0.5% of su h domains- It will be understood that any transmembrane domais identified for the PRO pol eptides of the prsEmn invention arc identified pursuant to criteria routinely employed in the art for identifying th4 type of hydropho ic domain. The exact boundaries of a transmembrane 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 transm mbra 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 disclos d 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. Ordinai y, a PRO polypeptide variant will have at least about 80% amino acid sequence identity, more p clerably at least abcut amino acid sequrncc identity, and even more preferably at least about 90% amino acid iequence identity, cv m more preferably at le st about 91% amino acid sequence identity, even more preferably at east about 92% ami io acid sequence identity, even more preferably at least about 93% amino acid sequence idcntit, even mo rerfera ly at least about 94% awinn acid sequence identity, even more preferably at least about 959 amino acid sequenbc 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 seque identity and most preferably at least about 99% amino acid sequence identity with the amino acid sequence of the full-length native amino acid sequence as disclosed herein.

"Percent amino acid sequence identity" with respect to the PRO polypeptid, sequences identifi d herein is defined as the percentage of amino acid residues in a candidate sequence that arc iJentical with the amio acid residues in the :.peific PRO polypeptide sequence, after aligning the sequences add introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutio s as part of rhe sequence identity. Alignment for purposes of determining percent amino aci sequence Identity cm 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 prefe ed software alignu nt program is BLAST. Those skilled in the art can determine appropriate parameters fol measuring alignmet, including any algorithms necded to achieve maximal alignment over the full Jength of the seq ences being compar d.

The identity values used herein have been generated using the WU-BLAST-2 computer rogram (Altschul et I., Methods in EnzvmnolV 266:460-480 (1996); htrp://blast.wust]/edu/blast/READMEt.ht). 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 thrshold 11, and scoring manix BLOSUM62. T HSP S and HSP S2 parameters, which are dynamic values used by BLAST-2, are establis d by the program its lf depending upon the composition of the sequence of interest and composition of the dala ase 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 d w aligned region.

"Percent nucleic acid sequence identity" with rvspect to PRO-encoding nucleic acid sequcncss identified herein is defined as the percentage of nucleotides in a candidate sequence thai are identical with die nucleotdes in the PRO nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of dltermining percent 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 art 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 paramethrs, with overlap sp and overlap fraction set to 1 and 0.125, respectively.

The term "positives", in the context of sequence comparison performed as dcsctibed above, includes residues in the sequerwes compared that are not identical but have similar properties as result of consurvative substitutions). The value of positives is determined by the fraction of residues scoring 1 positive value in tie 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 polypptidB comprising a PO 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 polypeptide of interest. The ag polypeptide preferably is also fairly unique so that the antibody does not substantially cross-reict with otlhr epitops.

Suitable tag polypcplides genrally have at least six amino acid residues and usually bctween 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. Contamim m components of its natural environment are materials that would typically interfere with diagnstic or therapeutic t es for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinccous soluts.

In preferred embodiments, the polypeptide will be purified to a degree sufficient to oblin 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 Coomassie blue or, preferably, silver stain. Isolaed polypeptide includes polypeptide in situ within recombinant 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 lea.t one contaminant nucleic acid molecule with which it is ordinarily asociated in the natu al source of the PRO polypeptide nucleic acid. An isolated PRO polypeptide nucleic acid milcule is other than in the form or setting in which it is found in nature. Isolated PRO polypeptide nucleic add moleculcs therefore re 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 molec les contained in c s 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 ryotes, for examp include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryo il 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 oxample, DNA for a presequence or secretory leader is operably linked to D A for a polypeptide if it is expressed as a prprotcin 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 ribsome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linke means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomilished 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 antibodin (including agonist, antagonist, and neutralizing antibodies) and anti-PRO polypeptide antibody compositions with polyepltopic specificity. The term "monoclonal antibody" as ted herein refers to i antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations tat may be present n minor amounts. i "Active" or "activity" for the purposes herein refers to form(s) of PRO polype4de which retain the biologic and/or immunologic activities of the specific native or naturally-occurring PRO po ypepide.

"Trcalmrnt" or "treating" refers to both therapeutic treatment and prophylactic or p cventative measu s.

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 mamnl. including bumans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, hor.s, cais, cows, and the like.

Preferably, the mammal herein is a human.

"Carriers" as used herein include pharmaccutically acceptable carriers, excipianis, o stabilizers which a e nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations Lmployed. Often te physiologically acceptable carrier Is an aqueous pH buffered solution. Examples of phys ologically acceptab e carriers include bufebrs such as phosphate, citrate, and other organic acids; antoxidants incld ng ascorbic acid; ow 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, arginino or lysine; monosaccharides, disaccharides, and other cubohydrates inclu ng glucose, manno 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; sal-forming counterlIS such as sodium; and/oi nonionic swfacants such as TWEENN, polyerhylene glycol (PEG), land PLURONICS The term "agonist" is used to refer to peptide 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 antibodies specifically bindihg such native PRO polypoptides, provided that they retain at least one 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 polypeptide "antagoni t" is a molecule which prevents, or interferes with a PRO antagonist effector function 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 presence 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 determnninable by one of ordina skill In the art, and 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 lowr 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 det of desired hounoloy between the probe and hybridizable sequence, the higher the relative temperature which cal 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 Ausall at aL, Current Protocols in Molecular Biology. Wiley Intescience Publishers, (1995).

"Stringent conditions" means employing low ionic strngth and high temnpe 4 ture for washing, f r example, 0.015 sodiura chlorideJO.0015 M sodium citrate/O. 1% sodium dodecyl sulfute 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% Ficoll0.1% polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride. 75 mM sodium citrate at 42C. Another example is use of 50% forrnamidc, 5 x SSC (0.15 M NaC, a0075 M sodium citrate), 50 mM sodium phosphae (pH 618). 0.1% sodium pyrophcsphate, 5 x Denhard 's solution, sonicated salmon sperm DNA (50 pg/hnl), 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 0% dextran sulfate, 2 x SSC (sodium chloridehodium cilrme) and 50% formamide at 55 0 C, followed by a high-strency 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 include the use of a washi g solution and bybridization conditions tcmperature, ionic strength, and %SDS) less stingent than desrib above. An example of moderately stringent conditions is a condition such as overnight inubation at 37C i a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 4M sodium phosu te (pH 5 x Denhan t's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sper DN, followed by washing the filters in 1 x SSC at about 37-50"C- The skilled artisan will recogilze how to adjust t e tIperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like.

"Southern analysis" or "Southern blotting" is a method by which the presence ol DNA sequences in a rcstriction endonudense digest of DNA or a DNA-containing composition is confirmed by hy ridization to a kno n, labeled oligonucleotide or DNA fragment. Southrnm analysis typically involves lectrophorehc separation of DNA digests on agarose gels, denatnraion of the DNA after elcctrophoretic separation, and tr sfcr of the DNA to nitrocellulose, nylon, or another suitable membrane support for analysis with a radiolaljld, biotinylated, or enzyme-labeled probe as described in sections 9.37-9.52 of Sambrook at aL, Molecular Cloning, A LlbOrat Manual (New York: Cold 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 fragment, cDNA or fragment thereof, or RN fiagmwn The pr be is labeled with a radioisotope such as or by biotinylatio, or with an enzyme. The RNA t be analyzed is usully electrophoretically separated on an agarose or polyacrylamide g cl traisfered to nitroceuulose, nylon, or oter suitable membrane, and hybridized with the probe, using standard techniques well known iI the art such as those described in sections 1.39-7.52 of Sambrook et al., supra.

As used herein, the term "immrunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglotlin constant domains.

Siructurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specifi ty which is other than Ihe antigen recognition and binding site of an antibody is "i.terologos"), and an imtunoglobulin constant domain sequence. The adhesin part of an immunoadhesin 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 immunoadhcain 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, ID or IgM.

"Chronic" administration refers to administration of the agent(s) in a continuoiu Iode as opposed to an acute mode, so as to maintain the initial therapeutic cffect (activity) for an extended period of time. "Intcrmitent" 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 simultaneous (concurrent) and con ecutive dministration 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 afrctling its expression is a suitable host c( Us.

Vectors ordinarily carry a replication site (although this is not necessary where chrmosomal 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 rived 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 rampicillin and tctraiycline resistanced thus provides easy mans for identifying transformed cells, whether far purposes of clcning or exprfssio Expression vectors also optimally will contain sequences which are useful for the control of transcription ad trnslatlon, promoters and Shine-Dalgarno sequences (for prokryotes) 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 epression vcutors need not contain any expression control. replicaive iequences or selcti n genes, their absence m y hamper the identification of hybrid transformants and the achievcmept of high level bid immunoglobulin cxprtssion.

The tcrm "lipopolysaccharide" or "LPS" is used herein as a synonym of "endotoxin.' Lipopolyscchrides (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 polysaccharidc, which varies foIn one bacterial spec es to another, is made up of the 0-specific chain (built from repeating units of three to eight s gan) and the two-pt core. Lipid A virtually always includes two glucosarnine sugars modified by phosphate an a variable numwnber 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 deflniins disclosed in Box Ann. Intern MarL 332-333 (1991), Specifically, septic shack starts with a systemic riaponse to infacion 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-poaitive organisms and fungi, as well as endotoxin-coitaining Gram-negve organisms. Accordingly, the present definition is not limited to "endotoxin shock." The phrases .gcne amplification= and Agen duplicatior are used intcrchangeablf and refer to a proc es by which multiple copies of a gene or gene fragment are farmed in a particular cell or cell line! The duplicated region (a stretch of amplified DNA) is often referred to as "amplicon". Usually, the amount f 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 guc expressed '"Tumor", as used herein, refers to all ncoplastic cell growth and proliferation, whctlt malignant or banil, and all pro-cancerous and cancerous cells and tissues. The tcrms "cancer" and "cancerous" refer to or describe he physiological condition in mammals that is typically charactcded by unregulated cell grow4. Examples of caner include but are not liraited to, carcinoma, lymphoma, blastoma, sarcomorna, and leukemia. More particular exampls of such cancers include breast cancer. prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical Cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endomerial carcinoma, salivary gland carcinoma, kidy cancer. vulval cances, thyroid cancer, hepatic carcinoma and various types of head and nck cancer.

The term "cytoroxic agent" as used herein refers to a substance that inhibits or prcvqnts the fuinction of ll1 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 fngal, plant or anial origin. or fragments thereof.

A "chemotherapeuic agent" is a chemical compound useful in tho 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 cyclophosphamide, thiotepa, busulfan, cytoxin, laxoids, e.g. paclitaxel (Taxol, Bristol-Myh Squibb Oncoloy, Princeton, NJ), and doetaxel (Taxotrc*, Rhone-Poulene Rorer, Antony, France), loxotre, jethotr cisplatn.

melphalan, vinblastine, bleomycin, atoposide, ifosfamide, mitomycin C, mitoxantrone, vicriatine, nrbie carboplalin, rtaeniposide. daunoniycin, carminomycin, aminopterin, dactinomycin, mitomycins. speaicis (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 iunhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer coll overexpressing any of the genes identified herein, either in itro or in vivo. Thus, the growth inhibitory LIgent is one which significantly reduces the percentage of cells ovoxxpressing 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-phaso 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 sen as tamuxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate. fluorourail, and n-C. Further informatdon 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- "Doxorbicbr" is an athracycline antibiotic.

The term '"cynokine" is a generic term for proteins released by one cell population w ich act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and jaditional polypeptde hormones. Included among the cytokines are growth hormone such as human growth hormo,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 cytokins.

"Native antibodies" and "native immunoglobulins" are usually heterotetrameric lycoprotins 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 itrachn disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of const 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 tc light chain variabc domain is aligned with the variable domain of the heavy chain. Particular amino acid residuts are believed to frm an interface between the light- and heavy-chain variable domains.

I

The rm "vuriable" refers to the fact that certain portions of the variable do ir differ extensivel3 in sequence among antibodies and ar 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 coxnplementarity-determining regions (CDRs) or hyp rvariable regions b h 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 each comprise four tR regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-shet structure. The CDRs in each chain are held together in close proximity Iy the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigcn-blnding site f antibodies (see Kabat et al., NIH PubL No.91-3242, Vol. I, pages 647-669 (1991)). The constant domains are ot involved directly in biiding 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 antibody. Examples of antibody fragments include Fab, Fab', F(ab'2, and Fv fragmes; diabodies: linear antibodies (Zapaa cet al., Protein Eng. 8(10): 1057-1062 [1995]); singl-chan antibody molecul and multispecific antibodies formed from antibody fragments. I Papain digeslion of antibodies produces two identical antigen-binding fragments. cld "Fab" fragment 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 antigen-combining sites knd is still capable of cross-linking antigen.

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

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

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

However, even a single variable domain (or half of an Fv comprising only three CDRa specic for an antigen) ,as the ability to recogni'y and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first ontant domain (C of the heavy chain. Feb 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 cysteina from the antibody hinge region. Fab'- H is the designation herein for Fab' in which the cysteine residue(s) of the constant domains I a free thiol gro p.

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

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

Dcpnding on the amino acid sequence of the constant domain of their heavy chain immunoglobulins an be assigned to difforent claes. Thee are ive major classes of immunoglobulins: IgA, IgD ,IgG, and IgM, several of theme may be further divided into subclasses (isotypes), IgOl, IgG2, IgO3, g4, IgA, and lgA "Singlo-chai Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wher in these domains are present in a single polypoptide 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 asructure for antid binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodi s, vol. 113, Rosenb rg and Moore eds., Sprnger-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 fragen ts comprise a heavy-chain variable domain (VH) connected ro a light-chain variable domain (VL) in the s e polypeptide chain (VI- VL). By using a linker that is too shor to allow pairing between the two domains on tie same chain, the domlh is ame forced to pair with the complementary domains of another chain dnd create two antig nbinding sitos. 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 recovrd from a compo t of its natural environment Contaminant components of its natural environment are materials which would intlrftre with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, apid other proteinaceus 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 least 15 residues ofN-terminal or internal amino acid scquenct 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 antibo y will be prepared by at least one purification step. i The word "l;bel" when used herein refers to a detectable compound or composiin 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 catalyze chemical alterati of a substrate compound or composition which is detectable.

By "solid phase" is meant a non-aqueos 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 ein 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 cr formation, similar to the lipid arrangement of biological membranes.

IT. omnpositions and Methods of the Invention Fu-enfth PRO717 Polyvuetides 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 cDi 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 Fas 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

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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 nuclcotide 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 anoing 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 vaiation 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 bt 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 rep acing one amino a d with another amino acid having similar structural and/or chemical properties, such as the re acemen of a leucr e with a serine, conservative amino acid replacements. Insertions or dcletions may optionhily 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 Imbodiments, conservative substitutions of interest are shown in Tabi 1 under the hading 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 reerence to amino a d classes, are introduced and the products screened.

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

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19/09 249Q1 :26 FAX 61 3 92438333 GRIFFITH HACK Q1020 Table I Original Exemplary Prcferred Residue Substitutions Substiti Ala val; leu; ile val Arg lys; gin; asn lys Asn gln; his; lys; arg gn Asp glu glu Cys ser ser Gln(Q) ian asn Glu asp asp Gly pro; ala ala His asn; gln; lys; arg Ug e lenI; val; met:; ala; ph; norloucine leu Leu norleucine ile; val; met; ala; phe ile Lys arg; gn: asn arg Met lan; 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 lOu Substantial modifications in functidon or immunological identity of the PRO polypebtide are accmpli sh by selecting substitutions that differ significantly in their offect on maintaining the struiure of the polypptide backbone in the aren of the substitution, for example, as a sheet or helical conformadon, 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: norleucine, met, ala, val, leu, ilc; neutral hydrophilic cys, Por, thr: acidic: asp, glu; basic: asn, gin, his, lys, arg; residues that inflience chain orientation; gly, pro; and aromatic: trp, tyr, phe.

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

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

The variations can be made using methods known in the art such as oligonlcotidcdiatpd (she-di -d) mnutagenesis, alanine scanning, and PCR mutagenesis. Sit-directed mutagenesis [Carter t al., Nucl. Ads .11:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis (W611s at al., enns, 14 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:26 FAX 81 3 924U1 GIFTHHC J2 GRIFFITH HACK Z021 (1985)], -restriction selection mutagenosis [Wells Ci aL, Philos. Tnt R. Soc. 1,ndon L orJ:45(18) other known t~echniques can be performned on the cloned DNA to produce the desired PRO pol. pptido variant DNA Scanning amino aid analysis can also be employed to identify one or more amino acds along a configo sequence- Among the prefrrrcd scanning amino acids are relatively small, neutral amino acqs. Such amino aokhs include alanine, glyc it, saint, and cysteine. Alanine is typically a preferred Scanning amino ricid among this gwp because it eliiniates the side-hain beyond the beta-carbon and is less likely to mter 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- MA1.

Hint 150?:1 (1976)). if alanine substitution does not yield adequate amounts of variant, an Aotric amino acidca be usedL Modiftenlions; of PRO0 Polvpetides Covalent modifications of PRO polyprptides are included within die scope ofthis iJivcntion. One type of Covalent mnodification includes reacting targeted amino a"i residues of the PRO polype~idde with an orgai derivatizing agent that 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 puifying anti-PRO polypepflde antibodies. ed vice-venta Comnuonl y uised crosslikang agents include, e.g..1 1. l.bis~diazaaceiyD..2..Phcnylithane, ghnaraldehy ac, N-hydroitysuccinimnidt: asters, for example, estrs with 4-azidomalicylic acid, homohifunctionnj irniduesters, id ing disuccindidflyl esters such as 3.3'-dithiobis(sucininidylropionhat), bifunctional nialilnides such as his naaelnldol ,-ocaneand agents such as methyl.3-(.RazidcnYldiiolPrPioi11idtC- Quie modilications include demmidation or gluminyl and asparaginyl rcsiduc4 to the correspondig glutamyl and aspartyl residues, respectively, hydroxylati of proline and lysine, phosphwiyhhoii of hydroxyl grup of scryl or threonyl rcsidues, methylahlion of the a-amino groups of lysine, arglnine, and hist dma side 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-icnninal amine, and amidation of any C-tAoina] carboxyl group- Mnother type of covalent modification of the PRO polypeptides included within chj scope of this invent ion comprises altering thu native glycosylatlon pattern of the polypeptide. "Altering the nativ, glycoSylation patt n is intended for puposes herein to mean deleting one or more carbohydrate. moieties foiund ut In native sequence PR0 polypeptide, andllof 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 shte(s).

Addition of glycosylation sites to the PRO polypeptide may be accmplished by .lering the amnino aid sequence. The altendtaan may be made, for example, by the addition of, or substitution by, one or more sorini or threonine residues to the native sequence PRO polypeptide (for 0-linked glycosyhitiot'sitcs) ThePRO polypepide amnino acid sequence may optionally be altered through changes ate DNA level, parriuO bymtaigth, encoding the PRO polypeptide at preselected bases such That codons are generated that will 16anslate into the derd i-s COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09. 9A,U 18:26 FAX 81 3 92438333 GRIFFITH HACK .022 Another nea is of increasing the number of carbohydrate rnoieties on die PRO Polt Pride polypeptide is by chemical or enzylmtic coupling of glycosides to the polypeptide. Such methods arc des~bed in the aft, in WO 87/05330 pobllsbeci 11 September 1987. end in Aplin and Wriston, -CRC- Rev- Ti2gbsuL. pp- 259-306 Removal of carbohydrate moieties present on the PRO polypeptidoc may he accoDlished chemically a enzymatically or by mutational substitution of codons encoding for amino acid residues th+ serve as targets o:r glycosylation. Chemical deglycosylation techniques art known in the art and desctd rd for inistance, b flak mudd n, c at ch, io Bi io h ~:52 (1981) and by Edge et aW., A na l. B o he .I M l 1 8 Enzymatic cleavage of carbohydrate moieties on polypeptides can he achieved by the use ofla variety of endo- and exo-glycosidasos; as described by Thotakura. ot al., Moth. Eazyol-.. 38:350 (1987).

Another type of covalent modification of PRO polypepddcs of the invention cl ses linking the 0IL polypeptidc to one of a variety of nonproteinaceous polymers, polyethylene glycol, Pojypi'opylene glycol, or polyozalkylcnes, in the manneir set forth in U.S. Pant Nos, 4,640,835; 4,496,689; 4,301.14 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 a chimeric molcule comrisin a PRO polypeptide fused to another, beterologous polypepfide 01 amnino aci udnc. Ini ne embodiment such a chimeric molecule comprises a fusion of die PRO polypeptide with a agpolypeptide which provides An epitopo to which an anti-tag antibody can selectively bind. The epitope tag Is ;ecriilly placed at h amino- or carboxyl- Ierminus of the PRO polypeptide. The presence of such epitope-tae forms of the P1 0o polypepide can be dowctrd using an Antibody againt the tag polypeptd&. Also, provision of~ft epitope tag ena Its the PRO polypeptide to bc readily purified by a~ffinity purification using an anti-ta entibdidy or another typ of affinity matrix that binds to the epitapo tag. In on alternative embodiment, the chimeric rneeuie may complie a fusion of the PRO polypeptide with an nmmunoglobulin or a particular region olint immuno 1obulin. For abiv at form ofte ehimeric molecule, such a fusion could be to the Pc region of an ISG molccule~ Vaious tag polypepfidct and their respective antibodies =r well known in the Art Examples include p dyhiStldilB (poly-hil) or poly-histidins-glycine (poly-his-gly) tags; the flu HA tag polypeptide nd its antibody 12CA (Field at: al., Moltill. Biol.,8-2159-2165 (1988)1; the e-myc tag and die If. C7610G4. 7 and 9E10 antibodies therto LE'an et al., Molecular and CddelllrBiolr. 1-3610-3616 (1995)]; and tl} Heorpes Simplex Viu glycoprotein D (gD) tag aid its antibody [Paborsky ct Proniukginiin-n 3(6),547-553 (1990)). Other tag polypeprides include thie Flag-peptide [Hopp et al., Bi~o elioo, 6:1204-l2lO (1988)]; t~o KT3 ertiope peptd (Martin CL al., Sclens, 3,:9-194 (1992)], an u-tibulin epitope peptide (Skinner et al.,.-0W C~MtLho ,6.1~i:5163- 166 (1991)]; ad the 27 gene 10 protein peptide iag (Lutz-Freyemmuth st at., Proc. adv. Sci.XISA §2-.6391- 6397 (1990)].

Prwatn*fPOQol~rdde Itoe desciption below relaxe primarily to production of PRO polypeptides by cull ring cell irmifo or transfoeted wit a vector containing the desired PRO polypeptide nucleic acid, It of cc urse, contenmplated ht alternative mthods, which are well known in the art, may be employed to prcpare the j'RO polypeptide. For instanco, the PRO po~lypcptide sequence, or portions thereof, my be produced by direct peptide synthesis waig 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 Fptide Synthesis W.H. Freemen Co., San Francis o, CA (1969); Merriild, 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 instuce, using i Applied Biosystenms Pzptide Synthesizer (Foster City, CA) using manufacturer's instruction. Various portions of the desired PRO polypoptide may be chemically synthesized scparately and combined using chemical or enzym ic methods to produce the full-length PRO polypeptide.

A. Talation of DNA Encoding PRO Polvneptides DNA encoding PRO polypeptides may be obtained from a cDNA library preparedom 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 suE ssuch as described in the Examples. The PRO polypeptide-encuding gene may also be obtained from a -enomc library or y oligonucleotide synthsis.

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 ite protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted usin; standard procedurpa, 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 it al., PR Primer:A Laboratory Manual (Cold Sprig Harbor Laboratory Piess, 1995)].

The Examples below describe techniques for screening a eDNA library. The oli nucleotide scquen es solcred as probes should be of sufficient length and sufficiently unambiguous that false p sitives are minimied.

The oligonucleotide is preferbly labeled such that t can be detected upon hybridiztion 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, provided in Sambroo k et supa.

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 databass.

Sequence identty (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 sot are programs such as BLAST, ALIGN, DNAstar, and INHERIT which employ various algorithms to measure h imoloy.

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

B. Selection and Transformtion of Hot C Host cells are transfucted or transformed with expression or cloning vectors de.cribed herein for P O 17 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 1&12LEhAI 61 3 92438333 GRIFFITH HACK 1024 polypeptide production and cultured in conventional nutrient media modified as appropriate ft selocting transformnLts, or amplifying the genes encoding the desired seqpences. The cultu media, temperature, 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. (IR,.Press, 1991 Methods of transfection amr known to the ordinarily skilled artisan, fbr example, CaP Depending on the host cell used, transformation is performed using standardt techniques apI The calcium trealmett employing calcium chloride, as described in Sambrook ct al.. pr generally used for prokaryotes or other cells that contain substantial cell-wall harriers. 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, Vjjgloy, 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 ar typically carried out acconlrding to the method of Van Solingen or al., L3n Hsiao et al, Proc. Nal Acad. Scl. (USA), 76:3829 (1979). However, other methods for intro such as by nuclear microiqjection, elcetroporaion, bacterial protoplast fusion with intact czl polybrene. polyornithmne, may also be used. For various techniques for transforming mamr ct al., Methods in ez. yms2D. Lj;S27-537 (1990) and Mansour et al., Na-ra_ 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. cofl. Various E. c available, such as E r ol 112 strain MM294 (ATCC 31,446); K coli X3776 (ATCC 31,537 (ATCC 27.325) and KS 772 (ATCC 53,635). Other suitable prokaryotic host cells include I as Esherichia, L col. Enterobacter Erwnia, Iebstella, Proreus, Salmonalla, e.g. So Serrtia, Serra nearcescans, and Shigella, as well as Bacilli such as B. subfilir and B licheniformis 41P dh closed in DD 266,710 published 12 April 1989), Pseudomonas such Streptomyces. Various E. coli strains are publicly available, such as col KZ12 strain A ccli X1776 (ATCC 31537); E. col swain W3110 (ATCC 27,325); and KS 772 (ATCC 5 are illustrative rather than limiting. Strain W3110 is one particularly preferred host or par common host strain fir recombinant DNA product fermenlIations,. Preferably, the host cell of protolytic enzymes. For example, strain W3110 may be modified to effelt a genetic encoding pmteins endogenous to the host, with examples of such hosts including E coll W has the complete genotype tonA; col W3110 strain 9E4t, which has the complete gene W3110 strain 27C7 (ATCC 55,244), which has the comploltu genotype tanAp 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-ka deletion mutation; and an E coU strain having mutant periplasmic promtase disclosed in U..

18 inducing promote e conditions, such experimentation.

culntures can be fou and Sambrook et i and electroporatic ropriate to such cel or electroporation n with Agrobacteri ,22:35 (1983) a c calcium phosph yed. General aspe 216 Transformatic 1:30946 (197) a ngDNA into c or polycations, e.

alian cells, see Kco 2 (1988).

prokaryote, yeast as Gram.negative a strains are publi E cli strain W31 nterobacteriaceaa si lmneUa typhimuriz lichaniformis as P. aerginosa, i 4294 (ATCC 31,44 These exam; t host because it i nes minnimnal 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 polypeptide-encoding vectors. Saccharomyccs 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 BiolTachnolory, 2: 968-975 (1991)) such as, K. lactis (MW9-SC, CBS683 CBS4574; Lonuvencourt et e I. Bacterial., 737 K. fragilis (ATCC 12,4241 K bulgaricus (ATCC 16,045), K wicke7" (ATCC 24,17 K- walii (ATCC 56,500), K drosophilarun (ATCC 36,906; Van den Berg t at, Bioech nblo. 8: 135 (199( K. thermatolerans, and K maalanus; yarrowia (EP 402,226); Pichia pastoris (EP 183,0714; Srccmkrishna etat.

Basic Microbiol.. Uj: 265-278 [19881); Candda; Trichoderma resia (EP 244,234); Neur para crssa (Caue at, Proc. Natl, Acad. Sci. USA, 2: 5259-5263 [1979]); Schwwmnioyces such as Schwanni yces occidenaltLr 394,538 published 31 October 1990); and filamnrous fungi such as, Neurospra, Pent1iium, Tolypocladi (WO 91100357 published 10 January 1991), and Asperglus hosts such as A. nidulans (Balance ser at. Bich Biophys. Res. Commin., 112: 284-289 1 983]; Tilburn et atl, fp, 2: 205-221 (19831; Y ton er 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, Klockera, Pichia. Sacchardiyces. Torulopsis. a Rhodorukla. A list oF specific species that are exemplary of this close of yeasts may be £oud in C. Anthony, 3 Biochemistry ofMetlvlotosphS. 269 (1982).

Suitable host cells for the expression of glycosylared PRO polypptides are derived from multicolit organisms. Examples of invertebrate cells include insect cells such as Drosophila 52 and Spdoptern 5f9, as v as plant cells. Examples of usefll mammalian host cell lines include Chinese hamster ovary CHO0) and COS ce Mom specific examples include monkey kidney CV! line transformed by SV40 (COS-7, ATC CRL 1651); hur embryonic kidney line (293 or 293 cells subaloned for growth in suspension cultnre, Grah M at 3. CnY ii -59 (1977)); Chinc hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Prog. Nadll. Aad. Sci. USA, 22:4 (1980)); mouse ertoli cells (TM4, Mather, Biol. Reproc, 21:243-251 (1980)); human lWg cells (W13, AT CCL 75); human livar colls (Hop G2, RD 8065); and mouse mammarny tnumr (IvMvT 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. appropriate nac acid sequence may be inserted into the vector by a variety of procedures. In general, DA is inserted ine appropriate restriction endonuclease sial(s) using techniques known in the art. Vector c mpontts generally inch but are not limited L, one or mre of a signal sequence. an origin of replication, one or ore 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 20Q3 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 polypEpride having a spec ic 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 Klayvermnryce a-factor leaders, the latir described in U.S. Patsnt No. 5,010,3 82), or acid phosphatase leader, the C. albicans glu oamylase leader 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In manmmalian cell expression, mammalian signal sequences may be used to direct secretion f the protein, such as signald 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 markr Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, ampicillin, neomycin, methotrexute, or LOtrucyclino, complement auxotirophic deficiencies, or s y critical nutri 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 DPHFR or thymidinc inas An appropriate host cell when wild-cype DHFR is employed is the CHO cell line delicient in DHFR Letivity, prepared nd propagated as described by Urlaub et al. Proc. Nat. Acad. Sci. USA. 24216 (1980). A sitable selection gne for use in yeast is the rrpl gene present in the yeast plasmid YRp7 [Stinchcomb ct al., Natg, m :39 (1979); Kingsman et al, Lzenq. 2:141 (1979); Tachemper at al., .,gjl: 157 (1980)1. The rupl ep jt provides a acect on marker for a mutant strain of yeast lading the ability to grow in trypphan, for example, ATCC No. 44076 or P 4- I poncs, *nfA:g, j: 12(1977)].

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

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

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3phosphoglycerate kinase [Hitzoman et al., Biol. Chem.. 29;2073 (1980)] or other glyc lytic enzymes [ess et al., Adv, Enzyme R, 2:149 (1968); Holland, Bigochemnistr .12:4900 (1978)], such as enahlac, glyceraldehyd 3- COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:28 M 0 3 92438333 GRIFFITH HACK .027 phosphate dehydroge ase, hexolcinase, pyruvare dowaboxylase. phosphofructokinase, g jlucose-6-phosphato isomerumc, 3-phospho0glycerate mats,, pyruvatz ldnase, tniosepliosphate isomerase, phospboolucosC isomerase,mi ghacokinase.

Other yeast Fnimoen, which are inducible promoters having Eho additional advttage of tlrp controlled by growth conditions, arc the promoter regions for alcohol dehydrosenase 2. itocytoebromne C, aci phoaptatase. degradalivo enzymes associated with nitrogen metabolism, metallothionein, gI~ucraldehyde-3-phosphaic dhydrogenc,. md enzymes responsibleflbr maltose and galacoutLization. Suitable Ivectors and prOOI II for use in yeast expression are further described in EP' 73,657.

PRO polypeptide transcription from vectors in mammalian host cells is contrf loed, for example, )y promoters obtained from the genonies of virues; such as polyoma virus, fowipox virus (UK 2,211,504 pulibe July 1989), adenovirus (such as Adenovinus bovine papillosna virus, aylan sarcoma viucyromegsovirtii a refrovirus, hepatitis-B virus and Simian Vims 40 (SV40). from beterologous marmmalian promoters, the ao n promoter or an immunoglobulin promoter. and from beat-shock prorneors3, provided such picmuters arc compatible with the host ccli systms.

Transcriptioi of a DNA encoding the desired PRO polypcptkle by higher euiaxyows may be increased y inserting an enhancer bequcuce Into the vector. Enhancers are cit-acting elements of DNAJusually about from 0 to 300 bp, that act oFL a promoter to increase its transcription. Many enhancer sequcnce4 are now known fi ms nwmrnalian genes (globin, clastase, albumin, a-fttoprotoln and insulin). Typically, hovirever. one will use a enhancer from a eukmyotic cell virus. Examples include the 8Y40 enhancer on the laic side of the replication ai< (bp 100-270), the cytconegalovimus carly promotur enhancer, the polyonia enhancer on the 'a4side of the replicadi3 origin, and adenovirus enhanocra. The enhancer mnay bo spliced into the vector at a posit"o 5' or 3' to the 0 polypeptido coding sitquenc, but is preferably locatod at a siteS Nfrom the promoo- Expression vectors used in oukaryoric host cells (yeast, fungi, insect, plant, anil, human, or nucleated cells from other multiccllular organisms) will also contain sequences necessary for the termlination of transcription and for stabilizing the mRNA. Such sequences we commonly available from the 5' andi, occaionally untase regions of eukazyotiu or viral DNAs or e]DNAs. These regions contain nucleotide tcutes transcribcd as polyadenylated fragments in the untransated portion of the mRNA encoding PRO polypcqtides.

Still other neathods. vectors, and host cells suitable for adaptation to the synthesis 6ff PRO polypeptides In recombinant vcritmirte cell culture are described in Curbing et al-, Nature 293:620-675I1(1991); Mantelci tE.

No ir. 281:40-46 (1979); EP 117,060; and EP 117,058.

D. Dntecftn &WMlijLtion/BxtnudofI Gene ampliflcation and/or expression may be measured in a sample directly, for 4mph, by conventox al Southern blotting, Niathen blottng to qunui tate the transcription of mENA [Thomas, Pro24NaAad. Si.US& 77:5201-5205 (1980)1, dot blotting (DNA analysis), or in situ hybridization, usn an aprIatl laeepo based on the sequentcs provided hcrein. Alternatively, antibodies may be employed thai can recognize s dc; duplexes, including DNA duplexes, RNA duplexes, and DNA-RNlA hybrid duplexes or 0N4-protcin duplexesa Te antibodies in turn am y be labeled and the assay may be curried out where the duplex is bnlmd to a surface, so ta 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 nmunohistocbical 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 horcin or against exogonous sequence fused to a PRO polypeptido DNA and encoding a sp ific antibody epitop.

E. Purification of Poleieptide Forms of PRO polypeptides may be recovered from culture medium or from host cell lysaees. If membraebound, it can be released from the membrane using a suitable detergent solution Triton-i 100) or by enzyma ic cleavage. Cells cployed in expression of PRO polypeptidcs can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or call lysing agents h maybe ed to purify PRO polypaptides from recombinant cell proteins or polypoptides. The folliwing procedures are exemplary of suitable purification procedures; by fractionation on an ion-exchange colump; ethanol precipitati n; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAB; 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 To bind epitopta ged forms of the PI.0 polypeptide. Various methods of protein purification may be employed and such methods a known in the art id described for example in Deutscher, Method in Envmoloev. 182 (1990); Scopes, P in riicato Prin and Practice Springei-Verlag, New York (1982). The purification step(s) selected will deped, for example, on the nature of the production process used and the particular PRO polypeptide produced.

Uste for PRO Polventides Nucleotide 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 gineration of anti-sense RNA and DNA- PRO polypeptide-encoding n clcic acid will also be useful for the preparation of PRO polypeptides by the recombiant techniques described herein.

The full-length native sequence PRO polypeptide-encoding nucleic acid or porrioA thereof, may be u ed as hybridization probes for a cDNA library to isolate the full-length PRO polypeptide gene or to isolate still othcr 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 nucloic acid equences. Option ly, the length of the pmres will be about 20 to about 50 bases. The hybridization probes Ly be derived from he nucleotide sequence of any of the DNA molecules disclosed herein or from genomic sequeno including promou rs, nhancer elements and introns of native sequence PRO polypeplide encoding DNA. By way bf examplc, a scTuen ng method will comprise: isolating the coding region of he PRO polypeptide gone using the r own DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a vr iety of labels, includi n radionucleotidsc suclh as "P or or enzymatic labels such as alkaline phosphatase copled 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 1 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 mR A to determine which mnmbcrs of such libraries the probe hybridizes to. Hybridizalion techniques ae described in further detail in the Ex amplos below, The ESTs diclosed in the present application may similarly 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 rcuptor/lgand bind g interacion can be identified. Proteins involved in such binding interactions can also be used to screen for p Cil or small molecule inhibitors or agonists of the binding interaction. Screening assays can bl designed to find lead compounds that mimic the biological activity of a native PRO polypeptide 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 conteo latd include synthc ie organic or inorganic compounds. The assays can be performed in a variety of formats, inc luding protein-prot in binding assays, biochemical 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 gcncrate citi r transgcnic animals or "knock out" animals which, in turn, are useful in the develop*apt and screening of thcrapeutically useful reagents. A transgnic 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 gonome of a cell from which a transgenic anal 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 transgenic animals that contain cells which express DNA encoding the PRO poll 'epide. Methods or generating transgenic animals, particularly animals such as mice or rats, have become con entonal in the art and ae described, for example, in U.S. Palen Nos. 4,736,866 and 4.870,009. Typically, p ticular cells woud be targeted for PRO polypeptide transgene incorporation with tissue-specific enhancers. 'ransgcnic animals tl at include a copy of a transgne encoding a PRO polypeptide Inroduced 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, fcr example, pathologiCl conditions associated with its overexpression. In accordance with this facet of the invont 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 unireatedanimals bearing 1e trianagene, would indicate a potential therapeutic intorvention 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 recombintion between the endogenous gene encoding the PRO polypeptide and ltred genomic D A encoding the PRO polypeptide introduced Into an embryonic cell of the animal. For exam&e 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]. Tle vector is introdu into an embryonic stem cell line by electroparadon) and cells in which the introduced DA has homologo ly recombined with the endogenous DNA are selected Lsee Li ct al. Cell. L:915 (1992)]. The selected cells re then injected into a blastocyst of an animal a mouse or rat) to form aggregation chidras [see Bradey, in Tratocatcinornmas and Embryonic Stem Cells: A Pracical Approach, Robrtson, ed (ILL, Oxford, 198 pp. 113-152]. A chimric embryo can then be implanted into a suitable pseudoprognant feiale foster animal a d the embryo brought to term to create a o"kock 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 whi4 all cells of the a al containl the homologously recombined DNA. Knockout animals can be characterized 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 umployed, 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 deliviy is provided in the liteature; 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.

Where sustained-release administration of a PRO polypeptide is desired in a folmulation with relase characteristics suitable for the treatmennt of any disease or diasorder requiring administration the PRO polypeptie microoncapsulation of the PRO polypeptido is contemplated. Microcneapsulation of red mbinant proteins or sustaind release has been successftlly performed with human growth hormone (rhOH) interferon- (rhIFN intarlcukin-2, and MN rgpl20. Johnson at aL, N, Me&, 2: 795-799 (1996); Yasuda Biome L 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 Vaccine Design: Subuit audAdiat A@gwag, Powell anti 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. Moreover, 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/glycolide polymer," in: M. Chasin and R. Langer (Ed; Biodegradable Polyms as DrDelivery 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 manmals 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 imicroparticles for 1-2 week Is also desirable.

addition, the encapsul:ied protein to be released should maintain its integrity and stability o ler the desired rele period.

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

Dosages and desired drug concentrations of pharmaceuical compositions of the pres nt invention may vi depending on the panicular use envisioned. For example, in the treatment of deep vein tl ombosis or pcriphe vascular disease, "bolus" doses, will typically be preferred with subsequent administrations t eing given to maint 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 natnre 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 incl polyclonal, monoclonal, humanized, bispecific, and hereroconjugate antibodies.

A. Povlydonal Antibldiea The anti-PRO polypeptide antibodies may comprise polyclonal antibodies. Methods of prepari polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be traed 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 Itoneal injections. 'I immunizing agent maiy 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 Lmmuni4ed. Examples of st immunogenic proteins include but are not limited to keyhole limpet hemocyanin, m albumin, bovi thyroglobulln, and soybean trypsin inhibitor. Examples of adjuvants which may be em oyed include Freun ~031 ts of is, th Cr ile

A

Tn se re a an d., Iry aj 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 I 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 cpcrimenta ton.

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 Milstei 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 entibodi that will specifi y bind to the immunizing agent Altematively, the lymphocytes may be immunized 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 rmamalian sources are desired. The lymp ocytes are than fund with an immortalized cell line using a suitable fusing agent, such as polyethyleno glycol. to orm a hybridoma ca [Onding, Monoclonal Antibodics: Princinls ndPractica. Academic Press. (1986) pp 59-13]. Immortalized cl lines are usually transformed mammalian cvlls, particularly myeloma cells of rodent, bovie and human orig, Usually, rat or mouse mycloma cell lines ne employed. The hybridoma cells may be cultd in a suitable culti medium that prefcralPly contains one or more substances that inhibit the growth or suqvival of the unlsA4 immortalized cells. For example, if the parental cells lack the enzyme hypoxanlhne guan 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 high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Mire 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. Humnan myeloma and mous-human beraromyeloma cell lines also have bcon dslibed for the product on of human monoclonal antibodies [Kozbor, J. ImmunrnoL, 133:3001 (1984); Brodeur et at. iMonoclonal Antibdy Production Techniques and Applications, Marcel Dekkr, 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 antibodihs 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 enzymc-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, suawl. 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 mammal.

The monoclonal antibodies secreted by the sublones may be isolated or purified fm 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 b L readily isolated a i 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 lbridomna cells of tc invention serve as a p-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 (CkO) cells, or myelor a 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 hun m heavy and light chain constant domains in place of the homologous murine 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-immnunoglobulin polypeptidc. Such a non-irnmunoglobulin polypeptide :n 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 monovalea antibodies are well 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 Eo as to prevent hca y chain crosslinking. Alternatively, the relevant cyseine residues are substituted with another lmino acid residue r arc deleted so as to pnsvent crosslinking.

In virro methods are also suitable for preparing monovalent antibodies. Digestion ou antibodies to produ fragments thereof, paricularly, Fab fragments, can be accomplished using routine techniqu<s known in the art.

C. Hfmaniaed Antibodies The and-PRO polypeptide antibodies of the invention may further comprise hur 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 complements (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 fran human immunoglobulin arc replaced by corresponding non-human residues- Humanizec 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 a variable domains, in which all or substantially all of the CDR regions correspond to 1t immunoglobulin and :dl 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 -ie immunoglobulii binding subsequenc umanized antibodi cy determining regil ibody) such as mou.

ework residues of antibodies may al CDR or framewo ie, and typically tw lose of a non-hum toglobulin consens iunoglobulin coasta 36); Richmann eta 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 antibodies are wellknown 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 amio acid resides are often referred to as "import" residues, which arc typically taken from an "import" variable dom n.

Humanization can be essentially performed following the method of Wintr and co-workeri [Jones er at, Na re, 321: 522-525 (1986); Ricohmann et at, 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 corrsp 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 antiboies can also be produced using various techniques known in the art. ipcluding phage disp ay libraries [Hoogenboom and Winter, J. Mot Bfot, 227:381 (1991); Marks at. J. MoL io, 222:581 (1991)]. The techniques of Cole et aL and Boerner er at are also available for the preparation of human monoclonal antibud es (Cole et alp Monoclonal Antibodie and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boeacr etal. J. l n)L, 147(1186-95 (1991)].

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

Methods for making bispecific antibodies are known in the at. Traditionally, the rombinant product on of biapecilc antibodies is based on the co-expression of two iunmunoglobulin heavy-chain/l ht-chain pair, were the two heavy chains have different specifcities [Miltein and Cuello, Nature, 305:537-53 (1983)]. Becaust of the random assortment of immunoglobuln heavy and light chains, these hybridomas (quadr las) produce a poten al mixture of ten different antibody molecules, of which only one has the correct bispecific smcture. 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&3655-3 59 (1991).

Antibody variable dumains with the desired binding specificities (antibody-antig n combining sites) can be fued 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 (CH1) containing the site necessary for light-chain binding present in at least one of the fusions, DNAs encoding the immunoglobulin heavy-chain fusions nd, idesired, the imm noglobulin light chain, are inserted into sepacate expression vectors, and are co-transfected into a suitable host orga l ism. For further details of generating bispecific antibodies see, for example, Suresh et aL. Methods in Enymato 1:210 (1986).

E. H -rEco imte Antibodies 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 Hcteroconjugate antibodies are also within the scope of the present invention. Heter 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 73; EP 03089]. It is comemplated that the antibodies may be prcpare methods in synthetic pnotein chemistry, including those involving crosslinklg agents. For ex may be constructed using a disulfide exchange reaction or by forming a thioethcr bond.

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

Uses for Anti-PRO Polvnentide Antibodies acon 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 ing specific cells, tissues, or serum. Various diagnostic assay techniques known in the art ay competitive binding assays, direct or indirect sandwich assays and immunoprecipitation aIsa Vs c heterogcncous 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 toct 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 "1C, 32 p, sS, or a fluorescent OT c compound, such as fluorescein isothiocyanate, rhodamine, or ludcirin, 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 ture, David at at, Biochemistry. .1L:1014 (1974); Pain et at, J. ImmunoL Merh., 40:219 (11 81); Hislochem. and Cyrochem., 30:407 (1982).

Anti-PRO polypeptidc antibodies also are useful for the affinity purification of P O I recombinant cell culture or natural sources. In this process, the antibodies against the ?RO 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.

jugatc antibodj roposed to tarl V infection [V vitro using kno It, immunotoxi ples of suital sed, for examp ample, anti-PP its expression be usud, such nducted in eith 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 n in the art. T ed, and ihcreaf ample except t h another sulta Lving the desir rs (Remingtoi tions or aquco Id concntralio eluding ascorb bumin, geladn Therapeutic Compositions Therapeutic compositions are preparod 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, glutmine, asparagine, arginine or lysine; monosacharidcs, disaccharides and other carbohydrates includn gluose, manMo e, or dextrins: chelating agents such as EDTA; sugar alcohols such as manaltol or sorbitol; salt-forming counterios such as sodium; and/or nonionic surfactants such as Tween. Pluronics or PEG.

The active ingredicnls may also be entrapped in mlicrocapsules prepared, for example, by cowervati n techniques or by interficial polymerization, for example, hydroxymethylcellulose or gelatin-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 mncroamuli ons. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

The fornudations to be used for in vive administration mnst 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 inavnous solution bag or vial having a stopper pierccabic by a hypodermic i iection needle.

The route of administration is in accord with known methods, e.g. injection or infiusion by intravcnoms, intraperitoncal, 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 fo of shaped articles, c g. filns, or microcapsulc. Sustained release matrices include jolyesters. hydrog s, polylactides Palent 3,773,919, HP 58,481), copolymers of L-gluamic acid and gamma ethyl-L-gltamat J.

Sidman at ak Bloalymrs 2Z 547-556 [1983]), poly (2-hydroxyethyl-mthacrylate Langer, s Al., aL.

Biomed. Mater. Res. 5: 167-277 [19811 and R. Langer, Chm. Tech, j12: 98-105 [1982J), ethylene vinyl acU te Langerg il.. 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 invtion 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 P 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 tlerapy.

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 convn mal assays.

The following examples are offered for illustrative purposes only, and are not ini bled to limit the scoe of the present inventiim in any way.

All patent and literature references cited in the present specification are 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 otherwise 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: Exracllular Doamain Honmolo Screcnint to Identiy Novel Polypeptidre and cDNA Encodi Ig Therefor The extracellar domain (ECD) csquences (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 (eg. LIFESEQT, in:e 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 consnsus DNA sequences with 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 werf assembled relative to the other identifled EST sequences using phrap. In addition, the consensus DNA sequences or ained were often mt not always) extended using repeated cycles of BLAST and phrap to extend the consensus seq ce as asara possi le using the sources of IST 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 at a clone of the full-length coding sequence for a PRO polypeptide. Forward and rei cra PCR prim er generally range from 20 to 30 nucleotides and are often designed to give a PCR product 0: about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length. In some cases, additional oligonucleotides ire synthesized when the consensus sequence is greater than about 1-1 5kbp. In order to scree several libraries for a full-longth clone, DNA from the libraries was screened by PCR amplification, as per usubel et at, Cun Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate cloies 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 dard 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 g el ectrophoresis, ind 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 Sll site: see, Holmes et al., i Ae, 253:1278-1280 (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 EXAMPLE 2: Isolation of cDNAcloncs by Amvlase Screening 1. Preoaration of olio 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 is procedure, the double stranded cDNA was sized to greater than 1000 bp and the SalI/Not linkered cDNA *as cloned into XhoI/Notl cleaved vector. pRKSD is a cloning vector that has an sp 6 transription initiation site followed by an Sfil restriction enzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Priaration of random primed cDNA library A secondary eDNA library was generated in order to preferentially represent the i 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 vctor pSST-AMY.0 using reagents anl protocols from Lfe Technologies (Super Script Plasmid System, referenced above). In this procedure the doub e stranded cDNA was sized to 500-1000 bp. linkered with blunt to Nod adaptors, cleaved with Sfil, and cloned ito Sfil/NouI leav d vector. pSST-AMY.0 is a cloning vector that has a yeast alcohol dehydrogenase promoter preceding the cD A cloning sites and the mouse amylase sequence (the mature sequence without the secretion i gnal) followed by E e yeast alcohol dehydrogenase terminator, after the cloning sites. Thus, cDNAs cloned into th s vector that are fusid in frame with amylase sequence will lead to the secretion of amylase from appropriately tra sfcted yeast coloni.

3. Transformation and Detection DNA from the library described in paragraph 2 above was chilled on ice to which was add d electrocompetent DH10B bacteria (Life Technologies, 20 ml). The bacteria and vector mixture was thin electroporated as recommended by the manufacturer. Subsequently, SOC media (Life Te hnologies, 1 ml) ws added and the mixturf was incubated at 37C for 30 minutes. The transformants were then I lated onto 20 stand rd 150 inm LB places cortaining ampicillin and incubated for 16 hours (37C). Positive coloni s were scraped offt t plates and the DNA was isolated from the bacterial pellet using standard protocols, e.g. CsCI gradient The purified DNA was then carried on to the yeast protocols below.

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

The yeast strain 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, sc72, sec62, with truncated sec71 being most preferred. Alternatively. antagonists (including antismse nucloodtdes and ligands) which interfier 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 tha proteins may also be preferably employed in combination with the amylase-expressing yert, 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 et al., Nuc Acid. Res. 20:14: (1992). Transformed cells were then inoculated from agar into YEPD complex media brotl (100 ml) and gro overnight at 30°C. The YEPD broth was prepared a described in Kaiser et al., Methods in Yeast Genetics, Co Spring Harbor Pres, Cold Spring Harbor, NY, p. 207 (1994). The overnight culre was th diluted to about 1 106 colls/ml (approx. OD 0 =oo0.1) into fresh YEPD broth (500 ml) and regrown to 1 x i07 cclls/hl (appro ODsaxO.4-05).

The cells wenr then harvested and prepared for transformation by cransfer into S53 i tor bottles 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 supernatant w discarded and the cells were subsequcntly washed with LIAcfl (10 ml, 10 mM Tris-HCI, 1 mr EDTA pH 75, 11 M LigOOCCH 3 and resuspended into LiAc/TB (2.5 ml).

Transformation took place by mixing the prepared cells (100 l) with freshly den tured 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 was gently mixed as 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 (1 i and aliquots (200 pl) were spread onto the selective media previously prepared in 150 mm rowth plates (VWR Alternatively. instead of muliple 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, concentration).

The positive colunic were picked and streaked across fresh selective media (onto 10 mmn plates) in ordt to obtain well isolated and identifiable single colonies. Well isolated single colonies positive for amylase secretio were detected by direct incorporation of 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 visi alized directly.

4. Isolation f 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 imrmediately amplified. An aliquot of cells (5 pl) was used as a template for the PCR reac ion in a 25 gl volumr 33 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:34 FAX 81 3 92438333GRFIHAC I00 GRIFFITH HACK 14040 containing: 0-5 p1 Klcntaq (Clontcch, Palo Alto, CA); 4.0 pd 10 mnM dNTP=s (Perkin Elmerbuffer (Clontech); 0.25 pl forward ohigo 1; 0.25 p1 reverse oligo 2; 12.5 gil distilled water.

forward ollgonucleotcte 1 was: 5'-TGTAAAACGACGCCACTrrAAATAACTCArrrAAC-3* (Si The sequence of reverte ollgonucleotide 2 was: 5'-CAGGAACACrATGACCACGC4&~QA&M&CCAIL-3- (SEQ PCR was them perormed as follows: a. Denature 92 0 C, 5 minutes C~etus); 2.5 p1 Kent The sequence oftI iQ ID NO;324) bn NO:325) b. 3 cycles of: Denature Anneal Extend c. 3 cycles of; Denature Anneal Extend d. '25 cycles of:- Denature Anneal Extend 92-C, 590c, 72'C.

92 0

C,

57 0

C.

72-C, 92 0

C.

55 0

C'

7200.

30 seconds 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 ac Typically, the fIrst 18 nuclcolidcs of the 5' end of these ollgonucleotides contained annealing primers. Thus, the total product of the PCR reaction from an empty vector was 343 bp. H-ow fused cDNA resulted ina considerably longer nucleotidc soquences.

Following the PCR, an aliquot of thc reaction (5 W1) was examined by agnrose get el aprose gel using a Tris-Borat&-EDTA (TEE) buffering system as described by Sambrook resulting in a single qtrong PCR product lae than 400 bp were futher analyzed by D purification with a 96 Qiaquick PCR clean-up coluum (Qiagen Inc., Cbutlswortb, CA).

EXAMPLPJ 3: Iwblion of cDNA Cicinas Encoding Human PR0 1 7 A consensus sequence was obtained relative to a variety of EST soquences as dei above, wherein the conseznsus sequence obtained Is herein designated DNA42829. Basd consensus sequence, olgonucleoddes were synthesized: 1) to identify by FOR a eDNA libr .equence of intcrest, and 2) for usc as probes to Isolate a clone of the full-length codingsa A pair of PCR pinters (forward and reverse) were synthcsized: f TwardKPM dmnr 5'-AGCrT7TCAGCCCTCCTGG0AGCAC-3' (SEQ ID NO:42 1); evers EMR primer S'-CGCGTCAATAAACCTCIACGCrrGO-3' (SEQ JID NO:422).

Additionally, a synthetic ollgonucleotide hybridization probe Was constructed from the sequence which had the following nucleotido scquence;1 ~Ion and the amyin Insert was presets w4te for the sequnnc sverb signal acquene etrophoresis In a 1' at aL. rnu Clon NA sequencing aft cribe in Example Son the DNA4282 ny that contiained C iecTIO for FROM 1.

NA42829 consensuis COMS ID No: SMBI1-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

I

19/09 2003 18:34 FAX 61 3 92438333 GRIFFITH HACK l041 hybridination pnhj 5'-TATGTGGACCGACCAAGCACIrCACTGAGGCCACCAAGATTG-3' (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 cnliro nuclcotide sequence of UNQ385 (DNA50988-1326) is shown In Figure I18 (SEQ D 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 1697-16 (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 4.86. Clone UNQ38 (DNA50988-1326) has been deposited with the ATCC on April 28, 1998. Regarding the seqtnce it is understo that the deposited clone contains the correct sequence, and the sequences provided herein are basd on know sequencing techniques.

Analysis of the amino acid sequence of the full-length PRO717 polypeptide suggests that PR0717 may b a novel 12 trasmembcane 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, transmnembrane 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, auclcotides can be routinely detanermined given the sequences provided herein.

EXAMPLE 4: Use of RO Polplhl-idcEnnodins NuclAel Acid as Hbridlwion Probes The following method describes use of a nucleodtide sequence encoding a PM hybridization probe.

DNA compriing 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 gonomic libraries.

Hybridization and washing of filters containing either library DNAs is performed uni stringency conditions. Hybridization of radiolabeled PRO polypoptido-eancodinag nucleic ack filters is performed in a solution of 50% formamrnide, 5x SSC, 0.1% SDS, 0.1% sodium py sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran lfate 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 humran tissu or the following hig -derived probe to the ophosphate, 50 mM utrs. 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.

EXAMPLEM Expresaion of PRO Polyoentides in E coli This example illustrates preparation of an unglycosylated form of a desired 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, cli; see Bolivar at al., Gene. 2:95 (1977)) which contains gme tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated 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 tnscri an argU gene.

The ligation mixture is then used to transform a selected E coli stain using the Sambrook et al., siunra. 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 culu 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 hrvested by centrf obtained by the centrifugation can be solubilized using various agents known in the art, an polypeptide can then lie purified using a metal chelating column under conditions that allo protein.

In EP Application No. 99912321.9, PRO polypeptides were expressed in E. coi in a using the following procedure. The DNA encoding the PRO polypeptide was initially ampifi primers. The primers contained restriction enzyme sites which correspond to the restrictie selected expression vector, and other useful sequences providing for efficient and reliable tran purification on a meral chelation column, and protoolytic removal with enterokinase. The PC tagged sequences wern then ligated into an expression vector, which was used to transform a strain 52 (W3110 fuhA(tonA) Ion galE rpoHls(htpRts) clpP(lcTq). Transformants were first mgmil carbenicUllin 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 ciTae.2H20. I 1 yeast extract, 5.36 g Slieffeld hycas SF in 500 mL water, as woll as 110 mM MPOS, pH 7.3, and 7 mM MgSO4) anr grown for approximately 20-30 hours at 30 0 C with shaklng. Samples expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the ccls. C until purification and refolding.

36 ative sequence Po PRO polypeptide by using selected PCR :tion enzyme sites in Ile of a suitable vect r ies for ampicillin aid .The PCR amplifi d which encode for as, polyhis sequenc tional trmninator, a d methods described n id antibiotic resista it and DNA sequencin h supplemented with re. The cells are thn .ation. The cellpell t the solubilized PR) v tight binding of de poly-His tagged fou i, d using selected PC Z a enzyme sites on the laion initiation, rapi R-amplifed poly-H s E. coli host based o ownin LB conain e then diluted 50-10 7g KC, 5.36 gDif 0.55% (wiv) glucos re removed to vert f 11 pellets were lioze 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 etrathionale 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 Ulticentifuge 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 liaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrate column buffer. The column was washed with additional buffer containing 50 mM imidazol grade), pH 7.4. The protein was lduted with buffer containing 250 mM imidazole. Fractions protein were pooled and stored at 4"C. 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 refoldin mM Tris, pH 8-6, 0.3 M NaCI, 2.5 M urea, 5 mM cystelne, 20 mM glycine and 1 m volumes were chosen so that the final protein concentration was between 50 to 100 mnicrogra solution was stirrd gently at 4 0 C for 12-36 hours. The refolding reaction was quenched byt a final concontration oa 0.4% (pH of approximately Before further purification of the pro filtered 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 absorbanee w polyacrylamide gels and fractiuns containing homogeneous refolded protein were pooled- C refolded species of most proteins are eluted at the lowest concentrations oface onitrile sinc, most compact with their hydrophobic inteiors shielded from interaction with the reversed phi species are usually eluied at higher acetonitrile concentrations. In addidon 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 acetont 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 fonnulation buffer and sterile filtered.

EXAMPLE 6: EBxreljpin of PRO Polvpeptides in Mammalian Coll olumes in 7: added to make fin hC. This stcpresuJ entrifuged at 40,0( metal chelate colun ariy. 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 'FA with elution wil ro analyzed on SD nerally, the properl those species are t se resin. Aggregak ded forms of protei rile removed using es, pH 6.8 with 0.1 (Pharmacia) resir This example illustrates preparation of a glycosylated form of a desired PRO polyp expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as t Optionally, the PRO polypptide-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 pRKS-PRO polypcptide.

In one embodiment, the selected host cells may be 293 coils. Human 293 colls grown to confluence in tissue culture plaits in medium such as DMBM supplemented wit 37 ptide by recombina t ze expression vector ion enzymes to allow lok eL al., sa. Th LTCC CCL 1573) ar Sfetal calf scrum an 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 [a 044 optionally, nutrient comrponents and/or antibiotics. About 10 pg pRTK5-PRO polypeptide DN 1 gg DNA encoding the VA RNA gone [Thimmappaya stal., cel, fl:543 (1982)] and dissol Tris-HCI. 0.1 mM EDTA. 0.227 M CaC. 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 to the 293 cells and allowed to settle for about four hours at 37C, aspirated off and 2 ml of20% 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 gCihl "S-cystine and 200 pCilml 25

S-

hour incubation, the cc nditioned medium is collected, concentrated on a spin filter, and loadeA The processed gel may 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 selectcd 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). maximal density in a spinner flask and 700 jg pRK-PRO polypeptide DNA is addc concentrated from tho 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 mediun, and re-introduced into the spinner flak containing dtissue culture insulin and 0.1 pg/ml bovine transferria. After about four days, the conditioned modia is c to Temove colls and debris. The sample containing expressed PRO polypeptide can then be c by any selected inethd, such as dialysis and/or column chromatography.

In another embodiment, PRO polypeptides can be expressed In CHO cells. Theo 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) radiolabel such as 5S -methionine. After determining the presence of PRO polypeptid, the replaced with scrum free medium. Preferably. the cultures re incubated for about 6 days, a medium is havested. The medium containing the expressed PRO polypeptide can then be cc by any aelccted method.

Bpitope-tagged PRO polypeptide may also be expressed in host CHO cells. The subcloned out of the pRK5 vector. The subclone insert can undergo PCR to uAe in frame taz such as apoly-his tag into a aculovirus expression vactor. The poly-his tagged PRO pol be subelrmcnd into a 5V40 driven vector containing a selection marikr such as DHFR for s, Finally, the C1O cells can be transfected (as described above) with the SV40 driven ve performed, as described above, to verify expression. The culture medium containing the "t PRO polypeptide can then be concentrated and purified by any selected method, such as chromatography.

Stable expresaslon in CHO cells was periormed using the following procedure. The 38 is mixed withabt fed in 500 pld of Im M HEPES (pH 73 The precipilate is he culture mediumis e washed with ser roplaced with cule ethionine. After a 2 Sonto a 15% SDSg I.

the presence of PRO m free medium) ad ly using the dext 93 cells are grown to The clls are fis dejtran precipitate is seconds, washed with diumn, 5 jgml bovin nmtrifuged and fillr nccntraied and pufi UCK-PRO polypepti a s described above, ta medium containin a ulture medium may d then theconditiond acentrand and purifici ,O polypeptide may vth a selected epitoa rpcptide insert can th action of stable clms.

tor. Labeling mayb ressed poly-His ta y Nitchelate affity roteins were expresa 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 sequences for the soluble fors extracellul domains) of the respective proteins were fused to an IgG1 constant region sequence containig the hinge. CH2 ai 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 interst 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 ter/enhancer to drii expression of the cDNA of interest and dihydrofolate rtductase (DHFR). DHPR epressiof 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 ivailable transfection reagents Superfect 7 (Quiagen), Dosper 7 or Fugenc 7 (Boehring Mannheim). The cells were grown and described in Lucas er al., supra. Approximately 3 x .0 cells are rlozen an ampule fur further growth and production as describod below.

The ampules containing the plasmid DNA were thawed by placement into war r bath and mixed 1 vortexing. The contents were pipeittd into a centrifuge tube containing 10 mLs of media at d centrifuged at rpm for 5 minutes. The supernalant was aspirated and the cells were rosuspended in 10 mL selective media (0 tm filtered PS20 with 5% 0.2 ptn diafiltered fetal bovine scrum). 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 centriugatl and Tesuspension in production medium. Although any suitable CHO media may be employe, a production medil described In US Patent No. 5,122,469, issued June 16, 1992 was actually used. 3L producti nP spinner is seeded 1.2 x 10 cels/mL. On day 0, the cel number pHwere determined. On day 1, the spinner wa sampled and spargi with filtcred air was commenced. On day 2, the spinner was sampled, the temperature shif d 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 pm 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 l Ni-NTA columnn equilibrated in 20 mM epes, pH 7.4, buffer containing 0.3 M NaCI and 5 m imidazole at a flow rateof 4-5 ml/mln. at 4 0 C. After loading, the column was washed with dditonal equilibrati, buffer and the protein clated with equilibration buffer containing 0.25 M imidazole. The higy purified protein w subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, wi a 25 ml G25 Superfine (Pharmacia) column and stored at Immunoadbhsin (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: SMBI-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 ehId 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 Polyneutides in Yeas The following mrnethod describes recombiant expression of a desired PRO polypel First yeast expression vectors are constructed for intracellular production 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 in together with DNA encoding the ADH2/GAPDH promoter, the yeast alpha-factor secreory and linker sequences (if needed) for expression of the PRO polypeptido.

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 SS-PAGE. followed by s Coormassic Blue stain. I Recombinant PRO polypeptide can subsequently be isolated and purified by remod the fermentation medium by centrifugation and then concentrating the medium using selectc concentrate containing the PRO polypeptide may further be purified using selected column EXAMPLE X- Fxnrelnn of PRO PolvuentidUs In Baculovirus-Infeted Insct Calls bration buffer bef ilecting 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 ~sqoe on plasmids descril s can be analyzed dining of the gls V ng the yeast cells fr l cartridge fillers. I bromatography resi The following method describes recombinant expression of PRO polypeplides i* Baculovirs-lnfec insect cells.

The desired PRO polypeptide is fused upstream of an epitope tag contained with a vector. Such epitope tugs include poly.his tags and inmmunoglobulin tags (like Fe regionm plasmnids may be employed, ncluding plasmids derived from conmnrcially available pla 1 (Novagen). Briefly, the PRO polypeptide or the desired portion of he 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 flaning (selected) restriction en than digested with those selected restriction enzymes and sublonoud into the expression v Recombinant baculovirus is generated by co-irnsfecting the above plasmid and B (Pharmingen) into Spadopterafnrrgiperda cells (ATCC CRL 1711)using lipofectin 1 from GlCO-BRL). Afler 4-5 days of incubation at 28*C, the released viruses ar harves amplifications. Viral infection and protein expression is performed us described by ORO expression vectors: A labhortory Manual, Oxford: Oxford University Press (1994).

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

culoGold" virus IA commercially availa le Bd and used for fter Hley c at., 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. 3_Q175-79 (1993). Briefly, 59 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 KC), 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 (connmaercially available from Qiagen) is prepared with a bad volume of 5 wi of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded mL per minute. The 'olumn is washed to baseline Amn with loading buter, at which poin started. Next the columnis washed with a secondary wash buffer (50 mnM phosphate: 300 I pH which clutes nonspecifically bound protein. After reaching Ano baseline again, th with a 0 to 500 mM faidazole gradient in the secondary wash buffer. One mL fractions are by SDS-PAGE and silver staining or western blot with Ni?-NTA-conjugated to alkaline Fractions containing the elated Hisao-mgged PRO polypeptido are pooled and dialyzed aga Alternatively, purification of the IgG tagged (or Pc tagged) PRO polypeptid ca known chromatography techniques. including for instance, Protein A or protein G column In EP Applic ation No. 99912321.9 PRO polypeptidos 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 (immunoadhs extracllular region was fused to an IgG1 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 subheloned into a baculovirus expression vector (pb.PI.Ig for IgO fuslo 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 polylixiak 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 280.

harvested and subsequently used for the first viral amplification by infecting Sf9 cells in H supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Call days at 280"C. The supernatant was harvested and the expression of the constructs in the vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads tagged proteins or Protein-A Sepharose CL-4B bends (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 wer i 28C. The supernatant was harvested and filtered. Batch binding and SDS-PAGi an necessary, until expression of the spinner culture was confirmed.

The conditioned medium from the ftranfcted 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 Nit A washed with 25 L nto the column at fraction collection is I NaCI. 10% Glycer l, column is developed olleefrd and analyzed hosphatase (Qiage).

ast loading buffer.

n be performed using .hromatography.

in baculovirus infetd 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-transf d BRL). pb.PH.IgG Gd #1393 (Pharminge wn in Hink's TNM-F The supernatant s ik's TNM-FH mdLm s were incubated fo 3 aculovirus expression )IAGEN) for histidine lowed by SDS-PA ig.

md) of Sf9 calls grown ncubaled for 3 days at ysis was repeated, as utrifuigation to ra c 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 fihtors. For the poly-His tagged constructs, the potein constct we purified using a Ni-NTA coluimn (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 rVmin. at 40C. Af loading, the column wus washed with additional equilibration buffer and the protein eluted 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 stot at Immunoadhetin (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 (Pharmacia) which had an equilibrated in mM Na phosphate buffer, pH 6.8. After loading, the column was washed extensively with equ ibration buffer hef< 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 dusalted it storage buffer as described above for the poly-His tagged proteins. The homogeneity of thd proteins was verif by SDS polyacrylamide gel (PEO) eloctrophoresis and N-rerminal amino acid sequencing Edman degradatio In BP Application No. 99912321.9 PRO polypeptides were succestally expressed n baculovirus infect insect cells. While the expression was actually perfrned in a 0.5-2 L scale, it can be re ily scaled up for lar 8 L) preparations.

For expression in baculovirus-infected Hi5 insct cells, the PRO polypeptide-ccoding DNA may amplified with suitable systems, such as Pfu (Stratagene), or fused upstream of an ope tag contained w a baculovirus expressLon vector. Such epitope tags include poly-his tags and immunoglobul Eags (like Pc regk of gO). A variety of plasmids may be employed, including plasmaids dorived from comna ally available plasm such as pVLl393 (Nowagen). Briefly, the PRO polypeptide or the desired partion of the RO polypoptide (st as the sequence encoding the erncellulur 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) restrction 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.PI.Hlis) tag dovustream the NAME sequenc. Preferably, the vctor con ruc is sequenced confirmation.

IS cells air grown to a confluency of 50% under the conditions of, 270C, no C02. NO pac/strep. For e.

150 mm plate, 30 Sg 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 sentsh and in a separ tube, 100 ul of CellFcctin (CellFECTIN (GibcoBRL #10362-010) (vortexed 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 DNAICeIFECTIN 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 m temperature. 1 DNAIColFECTIN mix is then aspirated, and the cells are washed once with Ex-Coll to remove excess ClUlFECI ml of fresh Hz-Coll media is added and the cells are incubated for 3 days at 280C. The hupernatait 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 supemrnatent to 25 mL of Ni-NTA bonds (QIAGEN) for histidine agg&ed proteins Protein-A Sepharo CL-4B beads (Pharmcia) for IgG tagged proteins followed by SDS-PAGE analysis comparing to a knoN 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 copeentratitn oft5 mM. The conditioned media is pumped onto a 6 mi Ni-NTA clumn equilibrated mM Hepes, pH 7, buffer containing 0.3 M NC and 5 mM imidazole at a flow rate of 4- ml/min. 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 (Phamnislia) column and stan at Inuoadhein (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 (Phaurnacia) which had been .quilibrated in 20 ni Na phosphate buffer. pH 6.8. Alter 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 fractio: 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: mpanAtion of Antibodies that Bind to PRO Polucotides 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. an Imurnogens that may be employed include purified PRO polypeptide, fus the PRO polypeptide. and cells expressing recombinant PRO polypeptide on the cell sur immunogen can be mode by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO polypeptide immunogca a Pround's adjuvant and injected subcutaneously or intraperitoneally in an amount from Alternatively, the immanogen is emulsilied in MPL-TDM adjuvant (Rihi Irnmunochemical Re and iiected into the animal's hind foot pads. The immunized mice are then boosted 10 additional immunogen emulsifled in the selected adjuvant. 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 atibodi a final Intravenous injection of PRO polypeplide. Three to four days later, the mice are s 43 ally bind to a PR scribed, 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

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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 myclox cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate 1ybridoma cells whi can then be plated in 96 well tissue cultore plate; containing HAT (bypoxanthine, aminoperin, and thymidin medium to inhibit proliferation of non-afused 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 polypeptide is within the skill in the art.

I

The positive hybridoma cells can be injected iniraperltoneally into syngenceic Bali cc ito 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 chrornatograply based upon binding of antibody to protein A or protein G can be wployed.

EXAMpE 20: hinwric PRO PolvuMtides PRO polypptides may be expressed as chimeric proteins with one or more additions polypeptide domai added to facilitate protein purification. Such purification facilitating domains include, but at not limited to, mer 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.) betweena the purification domain and the PR polypeptide scqunce may be useful to facilitate expression of DNA encoding the PRO pol peptide.

EXAMPE 11J: Purification of PRO Pnlvnwutides 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 immunoafinity columa is construhcted by covalently coupling the anti-PRO polypeptide a tibody to an activaL chromatographic rosin.

Polyclonal inunoglobulins am prepared from imnneB sara either by precipitadtion ivith ammonium sulfi or by purification on immobilized Protein A (Pharmacia LKB Biotecbnology, Piscataway, Likewi4 monoclonal antibodies amr prepared from moie ascites fluid by ammonium sulFate precipita on or chromatograp on immobilized Protein A. Partially purified inmunoglobulin 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 manufacturr's instructions.

Such an imumnoafliniy column is utilized in the purification of PRO polypptide py preparing a frnatii 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 se rnce 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-comaaining preparation is passed over the immunoaff nity column, and te column is washed under conditions that allow the preferential absorbance of PRO polype tide high io ic strength buffers in the ipesence of detergent). Thn, th column is eluted under conditions that dirupt antibody/P O polypeplide binding a low pH buffer such as approximately pH 2-3. or a high concenati n of a chaorope suh as urea or thiocyanate ion), and PRO polypeptide is collected.

EXAMPLE 12: Djrn Screcninn 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 li free in solution, affixed to a solid support, borne on a cell surface, or located inracellular y.

One method of drug screening utilizes ukaryotic or prokaryotic host cells which are st bly transformed w th recorabinant nucleic atids expressing the PRO polypoptido or fragmen. Drugs are screened a ainst such transform id cells in competitive binding assays. Such cells, either in viable or fixed form, can be use for standard bindi g assays. One may measure, for example, the formation of complexes betwoen PRO polypep de or a tfagment aid the agent being tested. Alternatively, one can examine the diminution in complex forma ion between the F0 polypeptide 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 O polypeptide or fragment thereof and assaying for the presence of a complex between te agent and the P.O polypeptide or fragment, or (ii) for tIh presence of a complex between the PRO polypepide c r fragmnt and the c 1l, 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 amount of frue or uncomplexed label is a measure of the ability of the particular agent to bind to PO polypeptide or to interfere with the PRO polypeptide/cell complex.

Another telmnique for drug screening provides high throughput screening for cornm ounds having suita l binding affinity to a polypeptde 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 coml nounds are reacted w th PRO polypeptide and washed. Bound PRO polypeptide is detected by methods well known i the art- Purified P O polypeptide can also be coated directly onto plates for use in the aforementioned drug s rening techniques. In addition, non-neutralizing antibodies can be used to capture the puptide and immobilize it an the solid support.

This invention also contemplates the use of competitive drug screening assay in which nculralizi 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 nce of any peptide which shares one or more antigenic determinants with PRO polypeptide.

EXAMPLE 13: Ratmal Drug Desi-n COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 li Ill I II lil III nli nl 19/09 2003 18:39 FAX 61 3 92438333 GRIFFITH BACK 1052 The goal of rational drug design is to produce structural analogs of biologically divc polypeptide interest a PRO polypoptido) or of small molecules with which they ipteract, ag nists. antagonists, inhibitors. Any of thcse examples can be used to fashion drugs which are more active or sta le forms of the PR polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo Hodgsc Bio/Technoloev. 9; 19-21 (1991)).

In one appioach, the three-dimensional structur of the PRO polypeptie, or of an PR polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeli ig or, most typical] by a combination of the two approaches- Both the shape and charges of the PRO polypeptid must be ascertain, to elucidate the structure and to determine active site(s) of the molecule. Less often, useful I iformation regardij the structure of the PRO polypeptide may be gained by modeling based on the structure of ho ologous proteins.

both cases, relevant structural information is used to design analogous PRO polypeptide-like n oleculcs or to idenl efficient inhibitors. Useful examples of rational drug design may include molecules which h re improved activi or stability as shown by Braxton and Wells, Biochmis~y 13:7796-7801 (1992) or which act inhibitors, agonis or antagonists of native peptides as shown by Athauda et aL, J. Biochem.. 13:742-746 (19 3).

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

By virtue of ihe present invention, sufficient amounts of the PRO polypeptide ma] be made available perform such analytical studics as X-ray crystallography. In addition, knowledge of the PRO olypeptide amino sa sequence provided herein will provide guidance to those employing computer modeling tec miques in place of in addition to x-ray crystallography.

EXAMPLE 14: Gone Amplification This example shows that genes encoding various PRO polypeptides are amplified i the genome ofcerta human cancers. Amplification is associated with overexpression of the gene product. in cating that the PR polypeptide is a useful target for therapeutic intervention in certain cancers such as colon. l ng and other cancer Therapcutic agent numy take the form of antagonists of PRO polypeptide-encoding genes, for example, murin human chimeric, huminized or human antibodies against the PRO polypeptide.

The starting material for the screen was genomic DNA isolated from a variety anccrs. The DNA quantitated precisely, fluoromerically. As a negative control, DNA was isolated from he cells of ten norm healthy individuals which was pooled and used as assay controls for the gene copy in health individuals (NorHu The 5' nuclwse assay (for example, TaqMan and real-time quantitative PCR (fc er xample, ABI Pri 7700 Sequence Detecton System M (Perkin Elmer, Applied Biosystems Division, Foster City, were used i find genes potentially amplified in certain cancers. The results were used to determine whet e the DNA oncodir 46- COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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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 approximalely a 2-fold ar plification relative normal, two units cornesponds to 4-fold, 3 units to 8-fold and so on. Quantitation was obtai ed using primers a a Taqman T 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 spliced out introns prcferred for the primer derivation, 3=-untranslatcd region.

The 5' nuclease assay reaction is a fluorescent PCRbased technique which makes us of the 5' exonudcl 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 oligonucleotide, or probe is designed to det nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymec enzyme, and is laboled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced enessi 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 ii template-dependent manner. The resultant probe fragments disassociate In solution, and si nal from the releat reporter dye is free frnm the quenching offect of the second fluorophore. One molecule of rportor dye is liberal for each new mokcult 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 tie ABI Prism 77001 Sequence Detection. The system consists of a thcrmocyler, laser, charge-coupled devi c (CCD) camera a computer. The sysute amplifies samples in a 96-well format on a thormocycler. During amp iticaion, 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 intrument and for analyzing the data.

NucIclse assay data are initially expressed as Ct, or ththreshold cycle. This is dofined as the cyckl which the reporter signal accumulates above the background level of fluorescence. The Ct values are used quantitative measurement of the relative number of starting copies of a particular target seuence in a nucleic a sample.

EXAMPLE 15: I .r 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 usefl for example, to identify si es of gene expressii analyze the tissue dislribution of transcription, identify and localize viral infction, follow ch nges in specific mR synthesis and aid in ch rumosome mapping.

In situ hybridization was performed following at optmized version of the protoco by Lu and Gillett, C Mioan 1:169-176 (1994), using PCR-generated "P-labelod riboprobes. Briefly, fnrmalin-fi cd, parafin-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 antiset riboprobe was generated from aPCR product and hybridied at 55C overnight. The slide were dipped in Koc NTB2 nuclear track emulsion 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 Il 054 P-Riboprobe svnthrsla p (125 mCi) of "P-UTP (Amersham BF 1002, SA<2000 Cil/mnol) were speed v' containing dried 3P-UTT, the following ingredients wre added: gl Sx transcription buffer pI DT' (100 mM) l NTP mix (2.5 mM 10 each of lo mM GTP. CTP ATP +10 plH2O) pL UTP (50 pM) pIl Rnasin p DNA template (I g) p1 liO p RNA polymerase (for PCR products T3 AS. T7 S, usually) The tubes were incubated at 371C 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 pipotted 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 of the probe or 5 pl of RNA Mirk IL loading buffer. After heating on a 95C heal 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 3 3 P-Hybridizatio A. ftatmocnt of frown sections The slides ware removed from the freeer, placed on aluminiumn trays and thawed minutes. The trays were placed in 55 0 C incubator for five minutes o reduce condn sation for 10 minutes in 4% paraformaidehyde on Ice in the tfume hood, and washed in 0.5 x SSC temperature (25 mil 20 a SSC 975 mi SQ IO). After deproteination in 0.5 pt/ 1 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 paraffin-mbcdded sctio The slides were deparaffinized, placed in SQ H2O, and rinsed twice in 2 x SSC a minutes each time. The sections were deproteinated in 20 tg/ml proteinass K (500 pa RNasc-free RNase buffer, 37 0 C 15 minutes) human embryo, or 8 x protoinase K (100 pn1 37C, 30 minutes) forrmalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were p above.

C. Pruhybridizaio i The slides were laid our in a plastic box lined with Box buffer (4 x SSC, 50% for a 48 c dried. To each tube )lowcd by incubati and the mixture a n unit, and spun usi rogramrn 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 wmperawre: The slides wereo hb or 5 minutes, at ro se K for 10 minutes ms were washed in ethanol, 2 minu room temperature. 1 f 10 mg/lmi 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.

7 5 g Dextran Sulfate 6 SQ vorto d and healed in the microwave for 2 minutes with the cap loosened. After cooling on ice, 18.15 ml formanmide, 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. WIidixation 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 coaled on ice, and 48 p hybridization buffer were added per slide. After vortexig, 50 p] p nmx 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/mi), The slides were washed 2 x 10 minutLe with 2 x SSC, EDTA at rqom temperamrO. 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 nucleAdrides 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 EXMjE 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 liumati 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 Adeno(a (61WC43) [CT3J Human colon AdenoCa CSRCC744) (CT8] Human colon AdonoCa (SRCC74S) [C710] Human colon AdenoCa (SRCC74S) [Cfl2] MO, R Humanj colon AdenoCa (SRC0747) (C11 pMO, Humnan colon AdcnoCa (SRCC74S) [CTIS] Ml R Human colon AdanoCa CSRCC749) [CT 16] pMQ Human uolon AdenoCa (SRCC2750) [Ori?] Human colon AdenoCa (SRCC75 1) (all3 I Ilman colon AdenoCa (SP.CC752) [(a43 Human colon AdenoCa (SRCO7S3) (015] Human colon AdenoCa (5RC07S4) (CTGJ Human colon AdenoCa (SRCC75S) (CI Human colon AdcnoCa (81100756) Human colon AdenoCa (SRCC7S7) [Te III Hluman colon AdenoCa (SRCC7SS) [Cal8 B] MO, Other Stu Dukes tlage IT Sfl

TI

T3 T2

TI

T2 T2

TI

722 T2

TI

T21 T2 T21 T21 T2 T3

TI

D pT4 NO a p12 B T3 A pT2 NO B3 n NO OB pT3 pNO 1) T4 N2 B p173 pNO Cl p173 pNl MO.0,11 B p 1 B p12 02 C1 l pMO, RO B3PT 0I A p22 03 D p1T4 B n2 NO B 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, noamal human blood. 'Pe isolation was perfbrmed using purification kit, buffer set and protme and all from Qulagen, according to thiJ manutlcture' instructions and the description below.

Cell culture ly. is: Ceills were wa* hod and trypsinized at a concentrati 'on of 735 x 10 per Up9 and pelleted b$1 ocatrifuging a: 1000 rpm for 5 minutias at 40(2, followed by washing again with 1/2 volumo of PBS recenfligatiou. The pallets wore washed -A third time, the suspended cells collected and washed 2x with PBS. The cells were then suspended into 10 ml PBS. Buffer Cl was equrilibratcd at VC.2 Qiagen protcaze #1!15 wa lluted into 6.25 ml cold ddH 2 O to a finalI concentration of 20 mg/mI and equilibrated at 4*C 0m2 Onl 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 10/09 2003 18:40 FAX 61 3 9243833 GRIFFITH HACK a057 Buffr- CI (10 ml, 4QC) and ddH2O (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 wcre pelleted by certrifuging 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 ddlhO, 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 suspcnded 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 wer delar incubating additional 30-60 minu es, pelloting at 3000 x g for 10 min., 4"C).

Solid hiuan mor 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 ddHO to a final concentration of20 mg/ml and stored at 4°C. G2 buffer ml) was prepared by diluting DNAse A to a final conentration of 200 mganl (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. B3 twen samples, the polytron was cleaned by spinning at 2 x 30 seconds each in 2L ddH20, followed by G2 bufer (50 ml). If tissue was still present on the generator tip, the apparatus was disassembled and cleaned.

Qidagen prot ase (preparcd 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., 4C).

Hmnan 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 rm/ml and stored at 4C., 02 buffer was prepared by diluting RNAec A to a final concentration of200 e/ml from 100 mg/ml stock. The blood (10 ml) was placed into a 50 ml 4onical tube and ml CI buffer and 30 ml ddH20 (both previously equilibrated to 4°C) were added, and the co rponents mixod 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 vonexing, followed by vigorous vortcxing for 30 seconds. Quiagen protase was added (200 QI) and incubated at 50°C for 60 minutes. The incubation and centrifugation was repeated until the lysatcs were clear incubating additional 30-60 minutes, pclleting at 3000 x g for 10 min, 4"C.

Purfication ocleared lysates: Islaion of Manomi DNA: Gcnomic DNA was equilibrated (1 sample per maxi tip preparation) with 10 ml Q T buffer, QP elution buffer wAs equiFibrated at 50C. The samples were vortexed for 30 seconds, thm loaded onto 51 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 1 19/09 2003 18:41 FAX 61 3 92438333 GRIFFITH HACK I058 equilibrated tips and drained by gravity. The lips were washed with 2 x 15 mi QC buffer. The DI into 30 ml silanized, auioclaved 30 ml Corex tubes with 15 mil QP buffer (50 0 Isopropanol added to each sample, the tubes covered with parafin and mixed by repeated inversion u precipitated. Samples Aere pelleted by centrifugation in the SS-34 rotor at 15,000 rpm for 10 n The pellet location was marked, the supernMltat discarded, and 10 ml 70% ethanfol (4 0 C) was ac were pelleted again by cOntrifugation on the SS-34 rotor at 10,000 rpm for 10 minutes at 4 1 location was marked and the superntant discarded. The tubes were then placed on their side in and dried 10 minutes at 3 7C, taking care not to overdry the samples.

After drying, the pellets were dissolved into 1.0 ml TB (pH 8-5) and placed at 50"C Samples were held overnight at 4 0 C as dissolution continued. The DNA solution was then transf tubes with a 26 gauge needle on a tuberculin syringe. The transfer was repeated 5x in order to s Samples were then placed at 50OC for 1-2 hours.

Quantitation ofeenomic DNA and preparation for gnte amplificatioan assay The DNA levels in each tube were quantified by standard Au, A=o spcetrophotonm dilution (5 1 DNA 95 O1 ddH 2 O) using the 0.1 ml quartz uvtts In the Beokan DU640 spec A2/A2N ratios were in ihe range of 1.8-1,9. Each DNA samples was then diluted further to app ng/ml in TE (pH If the original material was highly concentrated (about 700 ng/l), thi placed at 50'C for several hours until resuspcnded.

Fluoromtric DNA quantitation was then performcd on the dilintd material (20-600 n4 manufacturer's guidelines as modified below. This was accomplished by allowing a Hooffer Dy fluoromeer to warm-up for about 15 minutes. The Hoechst dye working solution (#H33258, 1 within 12 hours of use) was diluted into 100 ml 1 x TNE bffer. A 2 ml cuvette was filled with solution, placed into the machine, and the machine was zeroed. pGEM 3Zf(+) (2 01, lot #3 added to 2 ml of fluoromcter solution and calibrated at 200 units. An additional 2 c1I ofpOWE was then teated and the reading confirmed at 400 10 units. Each sample was then read aS l When 3 samples were It und to be within 10% of each other, their average was taken and this val the quantifl ation value.

The fluorometricly determined concentration was then used to dilute each sample ddH 2 O. This was done simultaneously on all template samples for a single TaqMan plate .cnough material to run 500-1000 assays. The samples were tested in triplicate with Taqman" probe both B-actin and GAPDH on a single plate with normal human DNA and no-templatc diluted samples were ua:d provided that the CT value of normal human DNA subtracted from tt 1 Ct.' The diluted, lot-qualified genomic DNA was stored in 1.0 ml aliquots at -80C. Aliqw1 subsequently to be used in the gene amplification assay were stored at4*C. Bach 1 ml aliquot is plates or 64 tests.

Gene anplificaton assay: The PRO polypeptlde compounds of the invention were screened in the following pri i the resulting ACt values greater than or equal to 1.0 are reported in Table 9 below.

52 RA was eluted 10.5 ml) was til the DNA inutes at 4"C.

dcd. Samples The pellet a drying rack Or 1-2 hours.

rrd to 1.5 ml lear the DNA.

try on a 1:20 rophotometer.

ximately 200 material was 'ml) using the IA Quant 200 I 01, prepared e fluorometer 3851026) was 3Zf(+) DNA it in triplicat.

ae was used as 10 ng/l in say, and with primers and controls. The DNA was s which were nough for 8-9 ry tumors and COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19

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Tdlc ACtv___ in hfg ld coln p~imuytimO 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 .35 '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 1.21 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 LT9 1.24 1- 1.1 1.04 1~.10 Z 174 L39 1.62 1- 1,99 3-56 1.I ~24 .1.14 1.11 159 I I ~3 14 1.-6 I1.6 1.1 1.46 3-2 1.2 1.4- 1 M- 4 -2 1.1 11.16 1L.29 115 1 2- 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 tFL A ri 4 I a 1sI I jII__ In n oe I-.I 1 1~ I- I -f q I l()I I i -s I

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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 cior '4 c SI I I I I I I I I I I Bs;I I i~ S( I N- N N c II i N ci N N -I ~a2 Ii r UN 0: :9 II a _f 'F W i t I 1.1 1 I I i I I 11 I I I e~ I I i i i i r i O ~ri I 2 1 COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:4BI& I 3 92438333 GRIFFITH BACK I 82

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__19/QL2903 18:42 FAX 61 3 92438333 GRIFFITH HACK Il 03I it I~a I 1 I II I I~b 1 I I I I i CC a N (I nnIn a l I I I I I I I I I1 1 I I i I I I I I I I I I I II L~ 1 1 1i i I I I I I I I I U~9 111 1I±fliZ I I I I I I I I 1 I I I Mi I I I II I l! COMS ID No: SMBI-00423805 Received by iP Australia: Time 18:46 Date 2003-09-19

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19/09 2003 18:42 FAX 61 3 92438333 GRIFFITH HACK 0064 Deposit of Material The following materials have been deposited with the American Type Culture Cowt Drive, Rockvile, MD, USA (ATCC): ction, 12301 Parklawn MNAteial A C PDe. No. Deposit Dat DNA50988-1326 ATCC 209814 April 28, 1998 This deposit were made under the provisions of the Budapest Treaty on the Intern the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations Treaty). This assures maintenance of a viable culture of the deposit for 30 years from thi deposits will be made available by ATCC under the terms of the Budapest Treaty. and s between Geneulch, Inc. and ATCC, which assures permanent and unrestricted availabilit culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon layin any U.S. or foreign patent application, whichever comes first, and assures avaijability determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto accO and the Commissioner's rules pursuant therto (including 37 CFR 1.14 with particular rzf The assigne of the present application has agreed that if a culture of the material or be lost or destroyed when cultivated under suitable conditions, the materials will be notification with anolher of the same. Availability of the deposited material is not to be c practice the invention in contravention of the rights granted under the authority of any gov with its patent laws.

The foregoing written pecification is considered to be sufficient to enable one s the invention. The present invention is not to be limited in scope by the construct depos embodiment is intended as a single illustration of certain aspects of the invention and functionally equivalact are within the scope of this invention. The deposit of material herei admision that the witten description herein contained is inadequate to enable the praci invention, including die best mode thereof, nor is it to be construed as limiting the scope of illustrations thai it represents. Indeed, various modifications of the invention in additi described herein will become apparent to those skilled in the art ifom the foregoing descri; scope of the appended claims.

The entire disclosure in the complete specification of our Australian Patent Applic 30721/99 is by this crois-reference incorporated into the present specification.

58 ional Rcognitior thercunder (Budap date of deposit. 1 bject to an agreem of the progeny of Sopen to the public bf the progeny to rding to 35 USC 1 rence to 886 00 63 on deposit should promptly replaced instrud as a license ernment in accordai ed in the ar to pract d, since the deposil ry constructs that i does not constitute e of any aspect of I a claims to the sped n to those shown a tion and fall within lion No.

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

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Claims (8)

1. An isolated nucleic acid molecule encoding a polypeptide compri' acid sequence having at least 80% sequence identity to the amino acid se Figure 169 (SEQ ID) NO: 420).

2. The isolated nucleic acid molecule of claim 1 encoding a polypep an amino acid sequence having at least 85% sequence identity to the ami shown in Figure 169 (SEQ ID NO: 420).

3. The isolated nucleic acid molecule of claim 2 encoding a polypep an amino acid sequence having at least 90% sequence identity to the am shown in Figure 169 (SEQ ID NO: 420).

4. The isolated nucleic acid molecule of claim 3 encoding a polyper the amino acid sequence shown in Figure 169 (SEQ ID NO: 420). The isolated nucleic acid molecule of claim 1 comprising the full sequence from within the nucleotido sequence shown in Figure 168 (SE

6. The isolated nucleic acid molecule of claim 1 which comprises th coding sequence of the DNA deposited under accession number ATCC

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 linke sequences recognized by a host cell transformed with the vector. 9, A host cell comprising the vector of claim 7 or claim 8. iing an amino quence shown ii tide comprisin no acid sequent tide comprisin; no acid sequen tide comprisin .length coding ID NO: 419). e full-length

109814. 1 to control The host cell of claim 9 which is a CHO cell, an E. coli cell or a ,east cell. 11. A process for producing a polypeptide comprising culturing the 1 9 of clam 10 under conditions suitable for expression of said polypeptid said polypeptide. 12. Isolated polypeptide comprising an amino acid sequence having sequence identity to the amino acid sequence shown in Figure 169 (SE( 13. The isolated polypeptido of claim 12 comprising an amino acid s least 85% sequeno: identity to the amino acid sequence shown in Figux NO: 420). 59 lost cell of clai e, and recoveriu it least SID NO: 420). equence having S169 (SEQ ID COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 19/09 2003 18:43 FAX 61 3 92438333 GRIFFITH HACK 066 14. The isolated polypeptide of claim 13 comprising an amino acid seq least 90% sequence identity to the amino acid sequence shown in Figure 1 NO: 420). The isolated polypeptide of claim 12 comprising the amino acid sec in Figure 169 (SEQ ID NO: 420). 16. The isolated polypeptide of claim 12 comprising the amino acid sec by the DNA deposited under accession number ATCC 209814, 17. A chimeric molecule comprising a polypeptide according any one o 16, fused to a hcterologous amino acid sequence. 18. The chimeric molecule of claim 17 wherein said heterologous amin, sequence is an epitope sequence. 19.. The chimeric nlolecule of claim 17 wherein said heterologous amino sequence is an Fc region of an immunoglobulin. An antibody which specifically binds to a polypeptide according to claims 12 to 16. 21. The antibody orclaim 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 clair admixture with a phannaceutically acceptable carrier. Dated this 19th day of September 2003 ENENTECH.INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Aus ralia cnce having a 59 (SEQ ID uence shown uence encoded f claims 12 to acid acid my one of is 21 to 24, in COMS ID No: SMBI-00423805 Received by IP Australia: Time 18:46 Date 2003-09-19 I 19/09 2003 18:44 FAX 61 3 92438333 GRIFFITH HACK I1008 FIGURE 1 GTGGAGGCCG7CCACGATGGCGGGGCCCGACGGAGCCCGAGACGGGTTCGGCUACCC 5000C TGTGCGCGCAGCG7(GGCCACCGCACCTACGCGCGCCGCTGGGTGTTCCTG42TCGCGATCA CTO CTCAACTGCTCCAACGCCACGCIJGTGGCTCAGCTTTGCACCTTGCTG4 GTCATTGCT -AGGA CTTGGTCCTGTCCATGGAGCAGATCAACTGGCTGTCACTGGTCTACCTCGtrGGTATCCAC COAT TTCCCGTGGCGGCCATCTCCATCCTGGACTCCGTCGGGCTCCOTGCGGCG# CCATCCTGG -TOCO TGGCTG AACTTTlGCCGGGAGTGTGCTACGCATGGTGCCCTGCATGTTGTE'rGGGACCC ACCC ATTTQCCPTCCT'CATGGGTGG CCAGAGCCTCTQ TGCCCTTGCCCAGAGCC CGG3TCATCTT ,TCTC CAGCCAACCTGCCTGCCTTGTGGQNICCCAGAGCACCAGCGAGCCACGGCC.hACAPS CTCGC'CACO CATTACCTTTGCTCQTGTTATCCATCCTGCTGCGTGCT CTTGCACCA COATTACCTCATGCTCCTGTGGATCCATGCTGCNTCCCCTCGTGCTFCTCGACCA TCTGCCTGTGGCAGAGTGTGCCCCCCACCCCGCCCTCTGCCGGGGCTGCC EGCTCCACCTCAGAG AAGTTCCTGGATGG GCTCAAGCTGCAGCTCATGTGGAACAAGGCCTATGThACCTGGCTCTGTG CTTGGGGGGAATQATCGGGATCTCTGCCACCTTCTCAGCCCTCCTGGAGCKGTCCTCTG IGCAA GCGGCCACTCCAGTGOOTTTTCCGGCCTCTGTGGCGCTCTCTTCAT.CACGHTTGGGATCC 5000 GCACTGGCTCTCCGCCCCTATGTGGACCGGACCAAGCACTTCACTGAGGC .ACCAAGAT OGCT GTGCCTGTTCTCTCTGOCCTGCGTGCCCrL'TGCCCTGGTGTCCCAGCTGC OGCGACAGALCCCTTG CCCTGGCTGCCACCTGCTCGCTGCTCGGGCTGTTTGGCTTCTCGTGGG -CCGTGGCOCr GOAG TTG GCGTCGACPGTTCCTTCCCCGTGGGGGAGGGGOCTGCCACAC.GCAT ATCTTTG.TGCT050 GCAGGCCGAOGO-*AATrACTCATCATGCTGGCAATGACGGCACTGACTGTGC ACOCTCGGA OST CCTTGTCCACCTGCCAGCAGGGGGAGGATCCACLWGACTGOACAGTGTCT TGCTGCTGAT 000 GGCCTGTGCACCT2TCTTCAGCTGCATCCTGGCGGTCTTCT'200ACACCCCATACCGSCGCCTOCA GGCCGAGTCTOC450AOCCCCCCTCCACCCGTAACGCCGTOGGGCGGCGCA OCTCAGGOCCO TO TGGACCGAGGGC4G'ACCAGGAAGGGCTGGGG'TCCTGGGGCCCAGCACGGCG OTCGASTG :Cs GCGAGGGGGCCTCGCTAGAGGACCCCAGAGG7GCCCGGGAGCCCCCACCC GOCTOCCACC AGO GACTCCCCGTOCGCAAOGCCCAGCAGCCACCGACGCGCCCTCCCGCCCC OCAGACTC SCA GGGTCCA&GCGTCCAGTTTATTGACCCGCGGTCTCACTCCTCCTT CCCTCCCCG TO ATCACGTAO;CTG-AGCGCCTTGTAGTCCAGGTTGCCCGCCACATCGATGG rCGAACTGGA CAT CTGGTCCACCTGCGGGCG3GGGGCGAAAGOGCTCCTTGCGGGCTCCGGGA 3GAATTACAAG 000 CACCTQAAAA COMS ID No: SMBI-00423805 Received by IP Australia: Time (1-tm) 18:46 Date 2003-09-19 1 LflL2pOS 18:44 FAX 01 3 92438333 GRIFFITH HACK 106 FIGURE 2 >.c/usr/seqdb2/sst/DNA/DlaBSQcs.miflsS.DNASOS 8 8 ><subunit 1 of 1, 560 aa, 1 stop >cMW: 50427, p1: 6.86, x(Sg/rr) 2 MAGPTEAE1 TGLAEPRAICAQRGHRkTYARRWVFLAISLLNCSNATLWLSF EQ IN L SIVYLVVS TPF VAAIWILDS VGLRAATfLJGAWLNAGSVLRMV LGVYTIPAQVVCLLSTICLWESVPPTPPSAGAASSTSEKFLDGLKLQIJJM 01 SASFSAIJLEQILCASGHS SGFSCLCGALYITFGILGALALQPYVDRTK ACPAVQQQLLACLGFFVPAEAESPGG ILAV PPSTKNAVGGAfSGPGVDRQGAGRAGVIGPSATPCTRGALEDPRP1 GPATDAPSRPG.'RLAGRVQASR1'IDPAGSHSSFSSPWVIT Imortant features: Potential Tranammnhrane donains a amino acids 30-50, 61-79, 98-112, 126-146, 169-182, 268, 280-300, 318-337, 341-357, 375-387, 420-441 N-glyotylttiol sits. amino acids 40-43 and 43-46 clycosaminoglycan attachment Site. amino acids 468-471 L.PVADVIAEDI ?CKWGTQNPI .SPVLV1CKGEI nCAYVILALVCJ {FTEATKIGL( kIATGMIFVLG ,PTETPYRRLQI ZSPHPACfl'W 1 vLSM 'AFL14 )IPLM IGGMIC :LFSL AEGI ESGB ?PEAQ 201-215, 248 COMS ID No: SMBI-00423805 Received by IP Australia: Time (1-tm) 18:46 Date 2003-09-19 I