WO2000070050A1 - Compositions and methods for the treatment of immune related diseases - Google Patents

Abstract

The present invention relates to a composition containing novel proteins and methods for the diagnosis and treatment of immune related diseases.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE RELATED

DISEASES

Field of the Invention The present invention relates to compositions and methods for the diagnosis and treatment of immune related diseases

Background of the Invention Immune related and inflammatory diseases are the manifestation or consequence of fairly complex, often multiple interconnected biological pathways which in normal physiology are critical to respond to insult or injury, initiate repair from insult or injury, and mount innate and acquired defense against foreign organisms Disease or pathology occurs when these normal physiological pathways cause additional insult or injury either as directly related to the intensity of the response, as a consequence of abnormal regulation or excessive stimulation, as a reaction to self, or as a combination of these

Though the genesis of these diseases often involves multistep pathways and often multiple different biological systems/pathways, intervention at critical points in one or more of these pathways can have an ameliorative or therapeutic effect Therapeutic intervention can occur by either antagonism of a detrimental process/pathway or stimulation of a beneficial process/pathway

Many immune related diseases are known and have been extensively studied Such diseases include immune-mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, etc

T lymphocytes (T cells) are an important component of a mammalian immune response T cells recognise antigens which are associated with a self-molecule encoded by genes withm the major histocompatibihty complex (MHC) The antigen may be displayed together with MHC molecules on the surface of antigen presenting cells, virus infected cells, cancer cells, grafts, etc The T cell system eliminates these altered cells which pose a health threat to the host mammal T cells include helper T cells and cytotoxic

T cells. Helper T cells proliferate extensively following recognition of an antigen-MHC complex on an antigen presenting cell Helper T cells also secrete a variety of cytokines, I e lymphokines, which play a central role in the activation of B cells, cytotoxic T cells and a variety of other cells which participate in the immune response A central event in both humoral and cell mediated immune responses is the activation and clonal expansion of helper T cells Helper T cell activation is initiate by the interaction of the T cell receptor (TCR) - CD3 complex with an antigen-MHC on the surface of an antigen presenting cell This interaction mediates a cascade of biochemical events that induce the resting helper T cell to enter a cell cycle (the Go to Gl transition) and results m the expression of a high affinit) receptor for IL-2 and sometimes IL-4 The activated T cell progresses through the cycle proliferating and differentiating mto memory cells or effector cells

In addition to the signals mediated through the TCR, activation of T cells involves additional costimulation induced b> cytokines released by the antigen presenting cell or through interactions with membrane bound molecules on the antigen presenting cell and the T cell The cytokines IL 1 and IL 6 have been shown to provide a costimulatory signal Also the interaction between the B7 molecule expressed on the surface of an antigen presenting cell and CD28 and CTLA-4 molecules expressed on the T cell surface effect T cell activation Activated T cells express an increased number of cellular adhesion molecules, such as ICAM 1 , integrins, VLA-4, LFA 1 CD56, etc

T-cell proliferation m a mixed lymphocyte culture or mixed l>mphocyte reaction (MLR) is an established indication of the ability of a compound to stimulate the immune system In man) immune responses, inflammatory cells infiltrate the site of injury or infection The migrating cells may be neutrophi c, eosinophi c, monocytic or lymphocytic as can be determined b> histologic examination of the affected tissues Current Protocols in Immunology, ed John E Coligan 1994, John Wiley & Sons, Ine

Immune related diseases can be treated by suppressing the immune response Using neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial m the treatment of immune-mediated and inflammatory diseases Molecules which inhibit the immune response can be utilized

(proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease

Summary of the Invention

A Embodiments

The present invention concerns compositions and methods for the diagnosis and treatment of immune related disease in mammals, including humans The present invention is based on the identification of proteins (including agonist and antagonist antibodies) which either stimulate or inhibit the immune response in mammals Immune related diseases can be treated by suppressing or enhancing the immune response Molecules that enhance the immune response stimulate or potentiate the immune response to an antigen Molecules which stimulate the immune response can be used therapeutically where enhancement of the immune response would be beneficial Such stimulatory molecules can also be inhibited where suppression of the immune response would be of value Neutralizing antibodies are examples of molecules that inhibit molecules having immune stimulatory activity and which would be beneficial in the treatment of immune related and inflammatory diseases Molecules which inhibit the immune response can also be utilized (proteins directly or via the use of antibody agonists) to inhibit the immune response and thus ameliorate immune related disease

Accordingl) , the proteins of the invention encoded by the genes of the invention are useful for the diagnosis and/or treatment (including prevention) of immune related diseases Antibodies which bind to stimulatory proteins are useful to suppress the immune system and the immune response Antibodies which bind to inhibitory proteins are useful to stimulate the immune system and the immune response The proteins and antibodies of the invention are also useful to prepare medicines and medicaments for the treatment of immune related and inflammatory diseases

In one embodiment, the present invention concerns an isolated antibody which binds a PRO200. PR0526 PR0719, PR0725. or PRO 1031 pol> peptide In one aspect, the antibod} mimics the actι\ ιr> of a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide (an agonist antibody) or converseh the antibody inhibits or neutralizes the activity of a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide (an antagonist antibody) In another aspect, the antibody is a monoclonal antibody, which preferably has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues The antibody may be labeled and may be immobilized on a solid support In a further aspect, the antibody is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody In another embodiment, the invention concerns a composition containing a PRO200, PR0526

PR0719, PR0725 , or PRO 1031 polypeptide or an agonist or antagonist antibody which binds the polypeptide in admixture with a carrier or excipient In one aspect, the composition contains a therapeutically effective amount of the peptide or antibody In another aspect, when the composition contains an immune stimulating molecule, the composition is useful for (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) stimulating or enhancing an immune response in a mammal in need thereof, or

(c) increasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen In a further aspect, when the composition contains an immune inhibiting molecule, the composition is useful for: (a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, or (c) decreasing the proliferation of T-lymphocytes in a mammal in need thereof m response to an antigen In another aspect, the composition contains a further active ingredient, which may, for example, be a further antibody or a cytotoxic or chemotherapeutic agent Preferably, the composition is sterile

In another embodiment, the invention concerns the use of the polypeptides and antibodies of the invention to prepare a composition or medicament which has the uses described above In a further embodiment, the invention concerns nucleic acid encoding an antι-PRO200, PR0526

PR0719, PR0725, or PRO1031 antibody, and vectors and recombinant host cells compπsing such nucleic acid. In a still further embodiment, the invention concerns a method for producing such an antibody by culturing a host cell transformed with nucleic acid encoding the antibody under conditions such that the antibody is expressed, and recovering the antibody from the cell culture The invention further concerns antagonists and agonists of a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide that inhibit one or more of the functions or activities of the PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide.

In a further embodiment, the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PRO200, PR0526 PR0719, PR0725. or PRO1031 polypeptides The nucleic acid preferably is DNA, and hybridization preferably occurs under stringent conditions. Such nucleic acid molecules can act as antisense molecules of the amplified genes identified herein, which, in turn, can find use in the modulation of the respective amplified genes, or as antisense primers in amplification reactions Furthermore, such sequences can be used as part of πbozyme and/or triple helix sequence which, in turn, ma> be used in regulation of the amplified genes

In another embodiment, the invention concerns a method for determining the presence of a PRO200, PR0526 PR0719, PR0725, or PRO 1031 pol) peptide comprising exposing a cell suspected of containing the polypeptide to an antι-PRO200, PR0526 PR0719, PR0725, or PRO1031 antibody and determining binding of the antibody to the cell

In yet another embodiment, the present invention concerns a method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encoding a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level in the test sample indicates the presence of immune related disease m the mammal from which the test tissue cells were obtained

In another embodiment, the present invention concerns a method of diagnosing an immune disease m a mammal, comprising (a) contacting an antι-PRO200, PR0526 PR0719, PR0725, or PRO1031 antibody with a test sample of tissue cells obtained from the mammal , and (b) detecting the formation of a complex between the antibody and the PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide in the test sample The detection may be qualitative or quantitative, and may be performed in comparison with monitoring the complex formation in a control sample of known normal tissue cells of the same cell type A larger quantity of complexes formed m the test sample indicates the presence of rumor in the mammal from which the test tissue cells were obtained The antibody preferably carries a detectable label Complex formation can be monitored, for example, by light microscopy, flow cytometry, fluoπmetry, or other techniques known in the art The test sample is usually obtained from an individual suspected of having a deficiency or abnormality of the immune system

In another embodiment, the present invention concerns a diagnostic kit, containing an anti PRO200, PR0526 PR0719, PR0725, or PRO 1031 antibody and a carrier (e g a buffer) in suitable packaging The kit preferably contains instructions for using the antibody to detect the PRO200, PR0526 PR0719, PR0725 , or PRO1031 polypeptide

In a further embodiment, the invention concerns an article of manufacture comprising a container, a label on the container, and a composition comprising an active agent contained within the container, wherein the composition is effective for stimulating or inhibiting an immune response in a mammal, the label on the container indicates that the composition can be used to treat an immune related disease, and the active agent in the composition is an agent stimulating or inhibiting the expression and/or activity of the PRO200, PR0526 PR0719,

PR0725, or PRO1031 polypeptide In a preferred aspect, the active agent is a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide or an antι-PRO200, PR0526 PR0719, PR0725, or PRO1031 antibody

A further embodiment is a method for identifying a compound capable of inhibiting the expression and/or activity of a PRO200, PR0526 PR0719, PR0725, or PRO1031 polypeptide bv contacting a candidate compound with a PRO200 PR0526 PR0719, PR0725 or PRO1031 polypeptide under conditions and for a time sufficient to allow these two components to interact In a specific aspect either the candidate compound or the PRO200 PR0526 PR0719, PR0725 or PRO 1031 polypeptide is immobilized on a solid support In another aspect the non immobilized component carries a detectable label

B Additional Embodiments

In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides Host cell comprising any such vector are also provided By way of example, the host cells may be CHO cells, E coli, or yeast A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture

In other embodiments, the invention provides chimeric molecules comprising any of the herein described polypeptides fused to a heterologous polypeptide or amino acid sequence Example of such chimeric molecules compπse any of the herein described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin In another embodiment, the mvention provides an antibody which specifically binds to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single-chain antibody

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

In other embodiments, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a PRO polypeptide

In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95 % nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a full length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein an extracellular domain ot a transmembrane protein. with or without the signal peptide as disclosed herein or any other specificalh defined fragment of the full length ammo acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a)

In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity alternatively at least about 82% nucleic acid sequence identity , alternative]} at least about 83 % nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence ldentin , alternatively at least about

85 % nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence ldentit} . alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identit} , alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternative!) at least about 94% nucleic acid sequence identity , alternatively at least about 95 % nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule comprising the coding sequence of a full length PRO polypeptide cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane PRO polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a) In a further aspect, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83 % nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity , alternatively at least about 85 % nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87 % nucleic acid sequence identit , alternatively at least about

88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95 % nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97 % nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a) Another aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide which is either transmembrane domain-deleted or transmembrane domam-inactivated, or is complementary to such encoding nucleotide sequence wherein the transmembrane domaιn(s) of such polypeptide are disclosed herein Therefore, soluble extracellular domains of the herein described PRO polypeptides are contemplated

Another embodiment is directed to fragments of a PRO polypeptide coding sequence, or the complement thereof, that may find use as, for example, h)bπdιzatιon probes for encoding fragments of a PRO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-PRO antibody or as antisense oligonucleotide probes Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternativel) at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternativel) at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides m length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein m this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length It is noted that novel fragments of a PRO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the PRO polypeptide encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which PRO polypeptide-encoding nucleotide sequence fragment(s) are novel All of such PRO polypeptide encoding nucleotide sequences are contemplated herein Also contemplated are the PRO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those PRO polypeptide fragments that comprise a binding site for an anti-PRO antibody

In another embodiment, the invention provides isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified

In a certain aspect, the invention concerns an isolated PRO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity , alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83 % ammo acid sequence identity, alternatively at least about 84 % amino acid sequence identity, alternatively at least about 85 % amino acid sequence identity , alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity alternativel) at least about 89% amino acid sequence identity , alternatively at least about 90% amino acid sequence identity , alternativel) at least about 91 % amino acid sequence identity . alternatively at least about 92% ammo acid sequence identity , alternatively at least about 93 % ammo acid sequence identity , alternatively at least about 94% amino acid sequence identity, alternatively at least about 95 % amino acid sequence identity, alternatively at least about 96% ammo acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternativel) at least about 98 % ammo acid sequence identity and alternatively at least about 99 % amino acid sequence identity to a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an ammo acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full length amino acid sequence as disclosed herein In a further aspect, the invention concerns an isolated PRO polypeptide comprising an amino acid sequence having at least about 80% ammo acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83 % amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% ammo acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternativel) at least about

88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% ammo acid sequence identity, alternatively at least about 91 % amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93 % amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95 % amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% ammo acid sequence identity to an ammo acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein In a further aspect, the invention concerns an isolated PRO polypeptide comprising an ammo acid sequence scoring at least about 80% positives, alternatively at least about 81 % positives, alternatively at least about 82% positives, alternatively at least about 83 % positives, alternatively at least about 84% positives, alternatively at least about 85 % positives, alternatively at least about 86% positives, alternatively at least about 87% positives, alternatively at least about 88% positives, alternatively at least about 89% positives, alternatively at least about 90% positives, alternatively at least about 91 % positives, alternatively at least about 92% positives, alternatively at least about 93 % positives, alternatively at least about 94% positives, alternatively at least about 95 % positives, alternatively at least about 96% positives, alternatively at least about 97% positives, alternatively at least about 98% positives and alternatively at least about 99% positives when compared with the amino acid sequence of a PRO polypeptide having a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane protein, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of the full-length amino acid sequence as disclosed herein In a specific aspect, the invention provides an isolated PRO polypeptide without the N terminal signal sequence and/or the initiating methionme and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture

Another aspect the invention provides an isolated PRO polypeptide which is either transmembrane domain-deleted or transmembrane domam-inactivated Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and recovering the PRO polypeptide from the cell culture

In )et another embodiment, the invention concerns agonists and antagonists of a native PRO polypeptide as defined herein In a particular embodiment, the agonist or antagonist is an anti-PRO antibody or a small molecule

In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a PRO polypeptide which comprise contacting the PRO polypeptide with a candidate molecule and monitoring a biological activity mediated by said PRO polypeptide Preferably, the PRO polypeptide is a native PRO polypeptide

In a still further embodiment, the invention concerns a composition of matter comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as herein described, or an anti-PRO antibody, in combination with a carrier Optionally, the carrier is a pharmaceutically acceptable carrier

Another embodiment of the present invention is directed to the use of a PRO polypeptide, or an agonist or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the PRO polypeptide, an agonist or antagonist thereof or an anti-PRO antibody

Brief Description of the Drawings Figure 1 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0526 (UNQ330), wherein the nucleotide sequence (SEQ ID NO 1) is a clone designated herein as "DNA44184 1319" Also presented in bold font and underlined are the positions of the respective start and stop codons

Figure 2 shows the amino acid sequence (SEQ ID NO 2) of a native sequence PR0526 polypeptide as derived from the coding sequence of Figure 1 Also shown are the approximate locations of various other important polypeptide domains if known

Figure 3 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0719 (UNQ387), wherein the nucleotide sequence (SEQ ID NO 3) is a clone designated herein as "DNA49646-1327" Also presented in bold font and underlined are the positions of the respective start and stop codons Figure 4 shows the amino acid sequence (SEQ ID NO 4) of a native sequence PR0719 polypeptide as derived from the coding sequence of Figure 3 Also shown are the approximate locations of \ arious other important polypeptide domains if known

Figure 5 shows the nucleotide sequence of a cDN A containing a nucleotide sequence encoding nativ e sequence PR0725 (UNQ390), wherein the nucleotide sequence (SEQ ID NO 5) is a clone designated herein as "DNA52758 1399" Also presented in bold font and underlined are the positions of the respective start and stop codons

Figure 6 shows the amino acid sequence (SEQ ID NO 6) of a native sequence PR0725 polypeptide as derived from the coding sequence of Figure 5 Also shown are the approximate locations of various other important polypeptide domains if known Figure 7 shows the nucleotide sequence of a cDN A containing a nucleotide sequence encoding native sequence PRO 1031 (UNQ516), wherein the nucleotide sequence (SEQ ID NO 7) is a clone designated herein as "DNA59294-1381 " Also presented in bold font and underlined are the positions of the respective start and stop codons

Figure 8 shows the amino acid sequence (SEQ ID NO 8) of a native sequence PRO1031 polypeptide as derived from the coding sequence of Figure 7 Also shown are the approximate locations of various other important polypeptide domains if known

Figure 9 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PRO200 (UNQ174), wherein the nucleotide sequence (SEQ ID NO 9) is a clone designated herein as "DNA29101-1122" Also presented m bold font and underlined are the positions of the respective start and stop codons

Figure 10 shows the amino acid sequence (SEQ ID NO 10) of a native sequence PRO200 polypeptide as derived from the coding sequence of Figure 9 Also shown are the approximate locations of various other important polypeptide domains if known

Detailed Description of the Preferred Embodiments

I Definitions

The term "immune related disease" means a disease in which a component of the immune system of a mammal causes, mediates or otherwise contributes to a morbidity in the mammal Also included are diseases in which stimulation or intervention of the immune response has an ameliorative effect on progression of the disease Included within this term are immune -mediated inflammatory diseases, non-immune-mediated inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasm, etc

The term "T cell mediated" disease means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity a mammal The T cell mediated disease may be associated with cell mediated effects, lymphokme mediated effects, etc , and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokmes secreted b) T cells

Examples of immune-related and inflammatory diseases some of which are lmune or T cell mediated, which can be treated according to the invention include s) stemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomvositis. pol)in)ositis), S)ogren's syndrome systemic vascuhtis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopema, paroxysmal nocturnal hemoglobinuπa), autoimmune thrombocytopenia (idiopathic thrombocytopemc pu ura. immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis. )uvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes melhtus, immune-mediated renal disease (glomerulonephπtis, tubulomterstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosmg cholangitis. inflammatory bowel disease (ulcerative colitis Crohn's disease), gluten-sensitive enteropathy, and Whipple ' s disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophihc pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft-versus-host-disease Infectious diseases including viral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, and E. herpes, etc , bacterial infections, fungal infections, protozoal infections and parasitic infections.

"Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In treatment of an immune related disease, a therapeutic agent may directly decrease or increase the magnitude of response of a component of the immune response, or render the disease more susceptible to treatment by other therapeutic agents, e g. antibiotics, antifungals, anti-inflammatory agents, chemotherapeutics, etc "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

The "pathology" of an immune related disease includes all phenomena that compromise the well-being of the patient This includes, without limitation, abnormal or uncontrollable cell growth, antibody production, auto-antibody production, complement production and activation, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of any inflammatory or immunological response, infiltration of lnflammatorv cells (neutrophi c, eosinophihc, monocytic, lymphocytic) into tissue spaces, etc "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc Preferably, the mammal is human Administration ' in combination with one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order

"Carriers' as used herein include pharmaceuticall) acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed Often the physiologically acceptable carrier is an aqueous pH buffered solution Examples of physiologicall) acceptable carriers include buffers such as phosphate, citrate, and other organic acids antioxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptide, proteins, such as serum albumin gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrohdone, ammo acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disacchandes, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol. salt forming counterions such as sodium, and/or nomonic surfactants such as

TWEEN™, polyethylene glycol (PEG), and PLURONICS™

The term "cytotoxic agent' as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells The term is intended to include radioactive isotopes (e g 1131 , 1125, Y90 and Rel86), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof

A "chemotherapeutic agent' is a chemical compound useful in the treatment of cancer Examples of chemotherapeutic agents include adπamycin, doxorubicin, epirubicm, 5-fluorouracιl, cytosine arabmoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e g pac taxel (Taxol, Bristol Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, lfosfamide, mitomycm C, mitoxantrone, vincπstine, vinorelbme, carboplatm, temposide, daunomycm, carrmnomycin, ammopteπn, dactinomycin, mitomycms, esperamicins (see U S Pat No 4,675, 187), melphalan and other related nitrogen mustards Also included in this definition are hormonal agents that act to regulate or inhibit hormone action on tumors such as tamoxifen and onapπstone A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially cancer cell overexpressing any of the genes identified herein, either in vitro or in vivo Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells overexpressing such genes in S phase Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest Classical M-phase blockers include the vincas (vincπstine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicm, daunorubicin, etoposide, and bleomycin Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin. methotrexate, 5-fluorouracιl, and ara C Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds Chapter 1 , entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et al (WB Saunders Philadelphia, 1995), especially p

13 The term cvtokme is a generic term for proteins released by one cell population which act on another cell as intercellular mediators Examples ol such cytokines are l) mphokmes, monokmes and traditional polypeptide hormones Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovme growth hormone, parathyroid hormone, thyroxine insulin, pro suhn, relaxin, prorelaxin, glycoprotem hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and lutemizing hormone (LH), hepatic growth factor, fibroblast growth factor, prolactm, placental lactogen tumor necrosis factor and β, mulleπan inhibiting substance mouse gonadotropin-associated peptide, inhibin, activm vascular endothehal growth factor, integrin, thrombopoiet (TPO), nerve growth factors such as NGF β platelet growth factor, transforming growth factors (TGFs) such as TGF- and TGF β, insulin-like growth factor I and -II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon α, β, and γ, colony stimulating factors (CSFs) such as macrophage CSF (M-CSF), granuloc)te macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL 1 , IL-la, IL-2, IL 3, IL-4, IL 5, IL-6, IL 7, IL 8, IL-9, IL 1 1 , IL 12, a tumor necrosis factor such as TNF-cx or TNF β, and other polypeptide factors including LIF and kit ligand (KL) As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culmre and biologically active equivalents of the native sequence cytokines

The terms "PRO polypeptide' and "PRO" as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (I e , PRO/number) refers to specific polypeptide sequences as described herein The terms "PRO/number polypeptide" and "PRO/number" wherein the term ' number' is provided as an actual numerical designation as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein) The

PRO polypeptides described herein may be isolated from a variety of sources such as from human tissue types or from another source, or prepared by recombinant or synthetic methods The term "PRO polypeptide refers to each individual PRO/number polypeptide disclosed herein All disclosures in this specification which refer to the "PRO polypeptide' refer to each of the polypeptides individually as well as jointly For example, descriptions of the preparation of, purification of derivation of, formation of antibodies to or against, administration of, compositions containing, treatment of a disease with, etc , pertain to each PRO polypeptide of the invention individually The term "PRO polypeptide' also includes variants of the PRO/number polypeptides disclosed herein

A ' native sequence PRO polypeptide' comprises a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means The term ' native sequence PRO polypeptide' specifically encompasses naturally occurring truncated or secreted forms of the specific PRO polypeptide (e g , an extracellular domain sequence), naturally-occurring variant forms (e g , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide In various embodiments of the invention, the native sequence PRO polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full length amino acids sequences shown in the accompanying figures Start and stop codons are shown in bold font and underlined in the figures However while the PRO polypeptide disclosed in the accompanying figures are shown to begin with methionme residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionme residues located either upstream or downstream from the ammo acid position 1 in the figures may be employed as the starting ammo acid residue for the PRO polypeptides

The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the PRO polypeptide which is essentially free of the transmembrane and cytoplasmic domains Ordinarily, a PRO polypeptide ECD will have less than 1 % of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0 5 % of such domains It will be understood that any transmembrane domains identified for the PRO polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain The exact boundaries of a transmembrane domain may vary but most likely b) no more than about 5 amino acids at either end of the domain as initially identified herein

Optionally, therefore, an extracellular domain of a PRO polypeptide may contain from about 5 or fewer ammo acids on either side of the transmembrane domain/extracellular domain boundary as identified m the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are comtemplated by the present invention The approximate location of the "signal peptides" of the various PRO polypeptides disclosed herein are shown m the present specification and/or the accompanying figures It is noted, however, that the C- terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e g , Nielsen et al , Prot Eng 10 1-6 (1997) and von

Heinje et al , Nucl Acids Res 14 4683-4690 (1986)) Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species These mature polypeptides, where the signal peptide is cleaved withm no more than about 5 ammo acids on either side of the C terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention

"PRO polypeptide variant" means an active PRO polypeptide as defined above or below having at least about 80% amino acid sequence identity with a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed 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 sequence Ordinarily, a PRO polypeptide variant will have at least about 80% ammo acid sequence identity, alternatively at least about 81 % amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83 % amino acid sequence identity, alternatively at least about 84% ammo acid sequence identity, alternatively at least about 85 % amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% ammo acid sequence identity , alternatively at least about 88% amino acid sequence identity, alternativel) at least about 89% ammo acid sequence identity, alternatively at least about 90% ammo acid sequence identity . alternatively at least about 91 % amino acid sequence identity , alternatively at least about 92 % ammo acid sequence identity, alternatively at least about 93 % ammo acid sequence identity , alternatively at least about 94% amino acid sequence identity, alternatively at least about 95 % amino acid sequence identity, alternatively at least about 96% amino acid sequence identity , alternatively at least about 97 % amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length PRO polypeptide sequence as disclosed herein Ordinaπl) , PRO variant polypeptides are at least about 10 amino acids m length, alternatively at least about 20 ammo acids in length, alternatively at least about 30 amino acids in length, alternatively at least about 40 amino acids in length, alternatively at least about 50 amino acids in length, alternatively at least about 60 amino acids in length, alternatively at least about 70 amino acids in length, alternatively at least about 80 amino acids in length alternatively at least about 90 amino acids in length, alternatively at least about 100 amino acids in length, alternatively at least about 150 amino acids in length, alternatively at least about 200 amino acids in length, alternatively at least about 300 amino acids in length, or more

" Percent ( % ) amino acid sequence identity " with respect to the PRO polypeptide sequences identified herein is defined as the percentage of ammo acid residues in a candidate sequence that are identical with the amino acid residues in the specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity Alignment for purposes of determining percent ammo acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST 2, ALIGN or Mega gn (DNASTAR) software Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN 2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN-2 sequence comparison computer program was authored by Genentech, Ine and the source code shown in Table 1 below has been filed with user documentation in the

U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech, Ine , South San Francisco, California or may be compiled from the source code provided in Table 1 below The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D All sequence comparison parameters are set by the ALIGN 2 program and do not vary

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given ammo acid sequence B (which can alternativ el) be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to with, or against a given ammo acid sequence B) is calculated as follows

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program

ALIGN 2 in that program s alignment of A and B, and where Y is the total number of ammo acid residues in B It will be appreciated that where the length of ammo acid sequence A is not equal to the length of ammo acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the ammo acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO" , wherein "PRO" represents the ammo acid sequence of a hypothetical PRO polypeptide of interest, "Comparison Protein ' represents the amino acid sequence of a polypeptide against which the "PRO' polypeptide of interest is being compared, and "X, "Y' and "Z ' each represent different hypothetical amino acid residues Unless specifically stated otherwise, all % ammo acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN 2 computer program However, % amino acid sequence identity values may also be obtained as described below by using the WU BLAST-2 computer program (Altschul et al , Methods in Enzymology 266 460-480 (1996)) Most of the WU-BLAST 2 search parameters are set to the default values Those not set to default values, I e , the adjustable parameters, are set with the following values overlap span = 1 , overlap fraction = 0 125, word threshold (T) = 1 1 , and scoring matrix = BLOSUM62 When WU BLAST-2 is employed, a % amino acid sequence identity value is determined by dividing (a) the number of matching identical amino acid residues between the ammo acid sequence of the PRO polypeptide of interest having a sequence derived from the native PRO polypeptide and the comparison ammo acid sequence of interest (I e , the sequence against which the PRO polypeptide of interest is being compared which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the total number of ammo acid residues of the PRO polypeptide of interest For example, m the statement "a polypeptide comprising an the amino acid sequence A which has or having at least 80% amino acid sequence identity to the ammo acid sequence B" , the amino acid sequence A is the comparison amino acid sequence of interest and the amino acid sequence B is the amino acid sequence of the PRO polypeptide of interest

Percent amino acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 ( 1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov or otherwise obtained from the National Institute of Health, Bethesda, MD NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand

= all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0 01 , constant for multi-pass = 25 dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62 In situations where NCBI BLAST2 is emplo)ed for amino acid sequence comparisons, the % amino acid sequence identity of a given ammo acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a giv en ammo acid sequence B) is calculated as follows

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of ammo acid sequence B, the % amino acid sequence identity of A to B will not equal the % ammo acid sequence identity of B to A

"PRO variant polynucleotide' or "PRO variant nucleic acid sequence" means a nucleic acid molecule which encodes an active PRO polypeptide as defined below and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein. Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81 % nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83 % nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85 % nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91 % nucleic acid sequence identity, alternatively at least about

92% nucleic acid sequence identity, alternatively at least about 93 % nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95 % nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity with a nucleic acid sequence encodmg a full- length native sequence PRO polypeptide sequence as disclosed herein, a full length native sequence PRO polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length PRO polypeptide sequence as disclosed herein Variants do not encompass the native nucleotide sequence Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides m length, alternatively at least about 60 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 210 nucleotides in length, alternativel) at least about 240 nucleotides in length, alternativel) at least about 270 nucleotides in length, alternativel) at least about 300 nucleotides in length, alternatively at least about 450 nucleotides m length, alternativel) at least about 600 nucleotides in length, alternatively at least about 900 nucleotides in length, or more

"Percent (%) nucleic acid sequence identity' with respect to PRO-encodmg nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides 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 determining percent nucleic acid sequence identity can be achieved in various wa) s that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megahgn (DNASTAR) software For purposes herein, howe er, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN 2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below The ALIGN 2 sequence comparison computer program was authored by Genentech, Ine and the source code shown Table 1 below has been filed with user documentation in the U S Copyright Office, Washington D C , 20559, where it is registered under U S Copyright Registration No TXU510087 The ALIGN-2 program is publicly available through Genentech,

Ine , South San Francisco, California or may be compiled from the source code provided in Table 1 below The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D All sequence comparison parameters are set by the ALIGN 2 program and do not vary

In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D

(which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a hypothetical PRO-encodmg nucleic acid sequence of interest , "Comparison DN A ' represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA ' nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides

Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program However % nucleic acid sequence identity values may also be obtained as described below by using the WU- BLAST-2 computer program (Altschul et al Methods in Enzvmology 266 460-480 ( 1996)) Most of the WU-BLAST-2 search parameters are set to the default values Those not set to default values, I e , the adjustable parameters are set with the following values overlap span = 1 , overlap fraction = 0 125 word threshold (T) = 11 , and scoring matrix = BLOSUM62 When WU-BLAST 2 is employed a % nucleic acid sequence identity value is determined by dividing (a) the number of matching identical nucleotides between the nucleic acid sequence of the PRO polypeptide-encoding nucleic acid molecule of interest having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of interest (I e the sequence against which the PRO polypeptide-encoding nucleic acid molecule of interest is being compared which may be a variant PRO polynucleotide) as determined by WU BLAST-2 by (b) the total number of nucleotides of the PRO polypeptide-encoding nucleic acid molecule of interest For example, in the statement ' an isolated nucleic acid molecule compi ising a nucleic acid sequence A which has or having at least 80% nucleic acid sequence identity to the nucleic acid sequence B", the nucleic acid sequence A is the comparison nucleic acid molecule of interest and the nucleic acid sequence B is the nucleic acid sequence of the PRO polypeptide encoding nucleic acid molecule of interest Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI BLAST2 (Altschul et al , Nucleic Acids Res 25 3389-3402 (1997)) The NCBI-BLAST2 sequence comparison program may be downloaded from http //www ncbi nlm nih gov or otherwise obtained from the National Institute of Health, Bethesda, MD NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi pass e-value = 0 01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62

In situations where NCBI BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D

(which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI BLAST2 m that program s alignment of C and D, and where Z is the total number of nucleotides in D It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C

In other embodiments, PRO variant polynucleotides are nucleic acid molecules that encode an active

PRO polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length PRO polypeptide as disclosed herein PRO variant polypeptides may be those that are encoded by a PRO variant polynucleotide

The term "positives" , in the context of sequence comparison performed as described above, includes residues in the sequences compared that are not identical but have similar properties (e g as a result of conservative substitutions, see Table 6 below) For purposes herein, the % value of positives is determined by dividing (a) the number of amino acid residues scoring a positive value between the PRO polypeptide amino acid sequence of interest having a sequence derived from the native PRO polypeptide sequence and the comparison amino acid sequence of interest (l e , the amino acid sequence against which the PRO polypeptide sequence is being compared) as determined in the BLOSUM62 matrix of WU-BLAST-2 by (b) the total number of amino acid residues of the PRO polypeptide of interest

Unless specifically stated otherwise, the % value of positives is calculated as described in the immediately preceding paragraph However, in the context of the amino acid sequence identity comparisons performed as described for ALIGN-2 and NCBI-BLAST-2 above, includes ammo acid residues in the sequences compared that are not only identical, but also those that have similar properties Amino acid residues that score a positive value to an amino acid residue of interest are those that are either identical to the amino acid residue of interest or are a preferred substitution (as defined in Table 6 below) of the amino acid residue of interest

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the % value of positives of a given ammo acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % positives to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 or NCBI BLAST2 in that program's alignment of A and B, and where Y is the total number of ammo acid residues in B It will be appreciated that where the length of amino acid sequence A is not equal to the length of ammo acid sequence B, the % positives of A to B will not equal the % positives of B to A

"Isolated, " when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-termmal or internal ammo acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the PRO polypeptide natural environment will not be present Ordinarily, however, isolated polypeptide will be prepared by at least one purification step An isolated" PRO polypeptide encoding nucleic acid or other polypeptide encoding nucleic acid is a nucleic acid molecule that is identified and separated trom at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polvpeptide-encodmg nucleic acid An isolated pol) peptιde-encodιng nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells However an isolated polypeptide- encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is m a chromosomal location different from that of natural cells

The term ' control sequences' refers to DNA sequences necessar) tor the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a πbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers

Nucleic acid is ' operably linked' when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a πbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, ' operably linked" 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 accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice

The term "antibody" is used in the broadest sense and specificall) covers, for example, single anti- PRO monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-PRO antibody compositions with polyepitopic specificity, single chain anti-PRO antibodies, and fragments of anti PRO antibodies (see below) The term 'monoclonal antibody' as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, I e , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts

"Stringency" of hybridization reactions is readily determinable by one of ordmary skill in the art, and generally is an empirical calculation dependent upon probe length washing temperature, and salt concentration In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature The higher the degree of desired homology between the probe and hybπdizable sequence, the higher the relative temperature which can be used As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so For additional details and explanation of stringency of hybridization reactions see Ausubel et al Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995)

"Stringent conditions" or "high stringency conditions" . as defined herein, mav be identified by those thaf (1) employ low ionic strength and high temperature for washing, for example 0 015 M sodium chloπde/0.0015 M sodium cιtrate/0 1 % sodium dodec)l sulfate at 50°C. (2) emplov during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0 1 % bovine serum albumm/0 1 % Fιcoll/0 1 % polyvιnylp) rrolιdone/50mM sodium phosphate buffer at pH 6 5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C, or (3) employ 50% formamide, 5 x SSC (0 75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0 1 % SDS. and 10% dextran sulfate at 42°C. with washes at 42°C in 0 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0 1 x SSC containing EDTA at 55 °C

"Moderately stringent conditions" may be identified as described by Sambrook et al , Molecular Cloning. A Laboratory Manual. New ork Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e g., temperature, ionic strength and %SDS) less stringent that those described above An example of moderately stringent conditions is overnight incubation at 37 °C m a solution compπsing. 20% formamide. 5 x SSC (150 mM NaCI, 15 mM tπsodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C The skilled artisan will recognize how to adjust the temperature, ionic strength, etc as necessary to accommodate factors such as probe length and the like The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a PRO polypeptide fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least six ammo acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues)

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e , is "heterologous"), and an immunoglobulin constant domain sequence The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1 , IgG-2, IgG-3. or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM

"Active" or "activity' for the purposes herein refers to form(s) of a PRO polypeptide which retain a biological activity of native or naturally -occurring PRO, wherein "biological' activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring PRO

The term "antagonist" is used in the broadest sense, and includes any molecule that partial]} or full) blocks, inhibits, or neutralizes a biological activity of a native PRO pol) peptide disclosed herein In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native PRO polypeptide disclosed herein Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence v ariants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc Methods for identifying agonists or antagonists of a PRO polypeptide may comprise contacting a PRO polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the PRO polypeptide "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody Examples of antibody fragments include Fab, Fab', F(ab')_:. and Fv fragments, diabodies, linear antibodies (Zapata et al , Protein Eng 8( 10) 1057-1062 [1995]), single chain antibody molecules, and multispecific antibodies formed from antibody fragments

Papain digestion ot antibodies produces two identical antigen-binding fragments, called "Fab" fragments, 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')_^ fragment that has two antigen-combining sites and is still capable of cross-linking antigen

"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -bmding site This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer Collectively, the six CDRs confer antigen-binding specificity to the antibody However, even a single variable domain (or half of an Fv comprising onlv three

CDRs specific for an antigen) has the ability to recognize 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 constant domain

(CHI) of the heavy chain Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hmge region. Fab'-SH is the designation herein for Fab' in which the cysteine resιdue(s) of the constant domains bear a free thiol group. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them Other chemical couplings of antibody fragments are also known

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences ot their constant domains Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided mto subclasses (isotypes), e g , IgG l , IgG2, IgG3, IgG4 IgA and IgA2

Antibodies and fragments thereof in this invention also include ' affinity matured' antibodies in which an antibod) is altered to change the amino acid sequence of one or more of the CDR regions and/or the framework regions to alter the affinity of the antibody or fragment thereof for the antigen to which it binds Affinity maturation may result in an increase or in a decrease m the affinity of the matured antibody for the antigen relative to the starting antibod) Typicall) , the starting antibod} will be a humanized, human, chimeric or murine antibody and the affinity matured antibody will have a higher affinity than the starting antibody During the maturation process, one or more of the amino acid residues in the CDRs or in the framework regions are changed to a different residue using any standard method Suitable methods include point mutations using well known cassette mutagenesis methods (Wells et al 1985, Gene, 34 315) or oligonucleotide mediated mutagenesis methods (Zoller et al , 1987 Nucleic Acids Res , 10 6487-6504)

Affinity maturation may also be performed using known selection methods m which man) mutations are produced and mutants having the desired affinity are selected from a pool or library of mutants based on improved affinity for the antigen or ligand Known phage display techniques can be conveniently used in this approach See, for example, U S 5,750,373, U S 5,223,409, etc Human antibodies are also with in the scope of the antibodies of the invention Human antibodies can be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991), Marks et al , J Mol Biol , 222 581 (1991)] The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p 77 (1985) Boerner et al , J Immunol , 147(1) 86-95 (1991), U S 5,750, 373] Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibod) production is observed, which closely resembles that seen in humans m all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U S Patent Nos 5,545 807, 5,545,806, 5,569,825, 5,625, 126, 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al , Bio/Technology

10, 779-783 (1992), Lonberg et al , Nature 368 856 859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al , Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), Lonberg and Huszar, Intern Rev Immunol 13 65 93 (1995)

"Single-chain Fv or "sFv antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain Preferabl) the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds , Sprmger Verlag, New^"York, pp 269-315 (1994)

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H-V_L) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites Diabodies are described more fully in, for example, EP 404,097, WO 93/1 1 161 , and Hol nger et al , Proc Natl Acad Sci USA, 90 6444-6448 (1993)

An "isolated' antibody is one which has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural env lronment are materials which would interfere with diagnostic or therapeutic uses for the antibody , and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes In preferred embodiments, the antibody will be purified (1) to greater than 95 % by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-termmal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stam Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present Ordinarily, however, isolated antibody will be prepared by at least one purification step

The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibod) The label may be detectable by itself (e g radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysacchaπdes (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate, in others it is a purification column (e.g., an affinity chromatography column) This term also includes a discontinuous solid phase of discrete particles, such as those described in U S Patent No 4,275, 149

A "liposome" is a small vesicle composed of various types of lipids, phosphohpids and/or surfactant which is useful for delivery of a drug (such as a PRO polypeptide or antibody thereto) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes

A "small molecule" is defined herein to have a molecular weight below about 500 Daltons

Table 1

/*

* C-C increased from 12 to 15

* Z is average of EQ

* B is average of ND

* match with stop is _M: stop-stop = 0; J (joker) match = 0

*/

#define M /* value of a match with a stop */ int day[26][26] = {

/* A^" B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/

/* A */ 2, 0,-2.0.0.-4. l.-l.-l.0.-1.-2.-1. O. M.1.0.-2.1, 1.0.0.-6.0.-3, 0}. /*B */ 0.3.-4.3, 2,-5.0.1.-2, 0, 0.-3.-2.2. M.-l.1.0.0.0, 0.-2.-5.0.-3, 1}, l*Q*l -2.-4.15.-5.-5,-4,-3,-3,-2.0.-5,-6.-5,-4,_M.-3,-5.-4.0.-2.0.-2.-8.0.0,-5}.

I*Ό */ 0.3,-5.4.3,-6, 1.1.-2, 0.0.-4,-3, 2,_M.-1.2.-1.0.0.0.-2,-7.0.-4, 2}.

/*E */ 0.2,-5.3.4.-5, 0.1.-2, 0, 0.-3,-2, l._M.-l.2,-1, 0.0.0.-2.-7.0.-4.3}. /* p */ -4.-5.-4.-6.-5.9.-5.-2.1, 0.-5.2.0.-4,_M.-5.-5.-4.-3.-3.0,-1, 0, 0.7.-5}.

/*G*/ 1.0.-3.1.0,-5, 5.-2.-3.0.-2.-4.-3.0. M.-l. -1.-3.1.0, 0.-1.-7.0.-5.0}. /*H */ -1, 1,-3, 1, 1,-2.-2, 6.-2, 0.0.-2,-2.2._M.0, 3.2.-1.-1.0,-2.-3, 0.0, 2},

1*1*1 -1.-2.-2.-2.-2.1.-3.-2.5.0.-2.2.2,-2._M.-2.-2.-2.-l.0, 0.4.-5.0.-1,-2}.

/* ] */ 0.0.0.0.0.0.0.0.0, 0.0.0.0.0,_M.0.0.0.0.0.0.0.0.0.0.0}, ι*κ*ι -1.0.-5.0, 0.-5.-2.0.-2.0.5.-3.0. l._M.-l.1.3.0.0.0.-2.-3.0.-4.0}. l*L*/ -2.-3.-6.-4,-3, 2.-4.-2.2.0.-3.6, 4,-3._M.-3.-2.-3.-3.-l.0.2.-2.0.-1.-2}. /*M*/ -1,-2.-5.-3,-2.0,-3.-2.2, 0.0, 4, 6.-2._M,-2.-l, 0.-2.-1, 0.2.-4.0,-2,-1}. /*N*/ 0.2.-4.2, 1,-4, 0.2.-2, 0.1,-3.-2, 2,_M,-1.1, 0.1.0.0.-2.-4.0,-2, 1}. 1*0*1 {_M , _M , _M . _M , _M , _M , _M , _M , _M , _M . M . M , M M 0,_M,_M, M,_M._M._M,_M,_M,_M,_M,_M},

/* p *j 1.-1,-3.-1,-1,-5,-1.0,-2.0,-l,-3,-2,-l._M.6, 0.0, 1.0, 0.-1,-6, 0.-5, 0}.

/*Q*/ 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0.4, l.-l.-l.0,-2,-5, 0,-4, 3}, /*R */^■2, 0.-4,-1.-1,-4,-3.2,-2, 0.3.-3, 0, 0,_M.0.1.6.0.-1, 0.-2, 2, 0.-4.0}. /*S */ 1.0, 0, 0.0,-3, 1.-1,-1.0, 0.-3.-2. l. M. l.-l, 0, 2.1.0,-1,-2.0,-3, 0}, /* Υ */ 1, 0,-2.0, 0,-3, 0,-1, 0, 0.0.-1,-1, O. M.0,-1.-1.1.3.0.0.-5, 0.-3, 0},

/*u*/ 0, 0.0.0, 0, 0, 0.0, 0, 0, 0.0.0, O. M.0.0.0, 0.0.0.0.0.0.0.0}.

/* v */ 0,-2,-2,-2,-2,-1,-1.-2.4, 0,-2.2, 2.-2,_M,-l.-2,-2.-l.0.0, 4.-6, 0.-2,-2},

/*w*/ -6.-5.-8,-7,-7, 0,-7,-3,-5, 0,-3.-2,-4,-4,_M,-6.-5, 2.-2.-5.0.-6.17.0.0,-6}. ι*x*ι 0, 0.0, 0.0, 0, 0.0.0, 0, 0.0.0.0,_M.0, 0.0, 0, 0.0.0.0.0.0, 0},

/* Y*/ -3,-3.0,-4,-4, 7,-5.0,-1.0.-4.-l.-2,-2,_M,-5.-4.-4.-3.-3.0.-2.0.0.10.-4}. l*_z*ι 0.1.-5.2, 3,-5, 0.2.-2.0, 0.-2,-1, l. M.0.3.0.0.0.0.-2,-6.0.-4, 4}

};

Table 1 (conf)

/* */

#include < stdιo.h > #include < ctype.h >

#defϊne MAXJMP 16 /* max jumps in a diag */

#define MAXGAP 24 /* don't continue to penalize gaps larger than this */

#defϊne JMPS 1024 /* max jmps in an path */

#define MX 4 /* save if there's at least MX-1 bases since last jmp */

#defϊne DMAT 3 /* value of matching bases */

#define DMIS 0 /* penalty for mismatched bases */

#define DINSO 8 /* penalty tor a gap */

#define DINS 1 1 /* penalty per base */

#define PINSO 8 /* penalty for a gap */

#defϊne PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP]. /* size of jmp (neg tor dely) *^■■.I unsigned short xfMAXJMP], /* base no of jmp in seq x */

}; '* limits seq to 2^*16 -1 struct diag { iπt score; I* score at last jmp */ long offset, /* offset of prev block */ short ljmp: /* current jmp index */ struct jmp jp; /* list of jmps */

}; struct path { int spc; /* number of leading spaces */ short n[JMPS]: /* size of jmp (gap) */ int x[JMPS]: /* loc of jmp (last elem before gap) */

}; char *ofιle: /* output file name */ char *namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for err msgs */ char *seqx[2], /* seqs: getseqs() */ int dmax. /* best diag: nw() */ int dmaxO, /* final diag */ int dna; /* set if dna: maιn() */ int endgaps: /* set if penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int lenO, lenl ; /* seq lens */ int ngapx. ngapy; /* total size of gaps */ int smax: /* max score: nw() */ int *xbm; /* bitmap tor matching */ long offset; /* current offset in jmp file */ struct diag *dx; /* holds diagonals */ struct path PPf2]; /* holds path tor seqs */ char *callocQ. *malloc(), *ιndex(). *strcpy(): char *getseq(). *g_calloc(). Table 1 (conf)

/* Needleman-Wunsch alignment program

* usage: progs filel file2

* where file 1 and fιle2 are two dna or two protein sequences

* The sequences can be in uppei- or lower-case an may contain ambiguity

* Any lines beginning with '.'. '>' or '< ' are ignored

* Max file length is 65535 (limited by unsigned short x in the jmp struct)

* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA

* Output is m the file "align. out"

* The program may create a tmp file in /tmp to hold info about traceback

* Original version developed under BSD 4.3 on a vax 8650 */

^include "nw.h" ^include "day.h" static _dbval[26] = {

1.14.2,13.0.0.4.11.0,0.12,0.3.15.0,0.0.5.6.8.8.7.9.0.10.0

static _pbval[26] = {

1.2|(1< <('D^,-'A'))|(1< <('N'-'A')).4.8.16.32.64. 128.256. OxFFFFFFF, 1<<10.1<<11.1< < 12.1<<13.1< < 14. 1<<15, 1<<16.1<<17, 1<<18, 1< < 19.1< <20.1< <21.1< <22. 1<<23, 1<<24, 1<<25|(1<<('E'-'A'))|(1< <('Q'-'A'))

}^;

int ac; char *avf],

{ prog = av[0]; if (ac ! = 3) { fpπntf(stderr. "usage: %s filel file2\n", prog): fpπntf(stderr. "where filel and file2 are two dna or two protein sequences. \n"); fpπntf(stderr."The sequences can be in upper- or lower-case\n"). fpπntf(stderr."Any lines beginning with ';' or ' < ' are ιgnored\n"). fpπntf(stderr. "Output is in the file \"ahgn.out\"\n"); exιt(l), } namex[0] = av[l]; namex[lj = av[2]; seqx[0] = getseq(namex[0], &len0); seqx[l] = getseq(namex[l]. &lenl): xbm = (dna)? dbval : _pbval, endgaps = 0; /* 1 to penalize endgaps */ ofile = "ahgn.out"; /* output file */ nw(): /* fill in the matrix, get the possible jmps */ readjmpsO; /* get the actual jmps */ printQ; /* print stats, alignment */ cleanup(O); /* unlink any tmp files */ Table 1 (co⁰)

/* do the alignment return best score maιn() * dna values in Fitch and Smith PNAS 80 1382 1386 1983

* pro PAM 250 values

* When scores are equal we preter mismatches to any gap prefer

* a new gap to extending an ongoing gap and preter a gap in seqv

* to a gap m seq y */ nw() nw

{ char *px, *py /* seqs and ptrs */ int *ndely *del) /* keep track ot del> */ int ndelx, delx /* keep track of delx * int *tmp /* for swapping rowO rowl *7 int mis, /* score for each type */ int insO, insi . /* insertion penalties */ register id. /* diagonal index */ register¹J /* jmp index */ register *col0, *col l /* score for curr last row */ register xx, yy /* index into seqs */ dx = (struct diag *)g_calloc( to get diags lenO + lenl + 1 sizeoftstruct diag)) ndely = (int *)g_calloc( to get ndely' , lenl + 1 sizeof(int)) dely = (int *)g_calloc( to get del) ' , lenl -¹- 1 sizeof(int)) colO = (int *)g_calloc("to get colO ', lenl + 1 si?eof(int)) col l = (int *)g_calloc("to get coll ' , lenl + 1 , sizeof(int)) insO = (dna)'' DINS0 PINSO, insi = (dna^ DINSl PINS1 , smax = -10000 if (endgaps) { for (col0[0] = dely[0] = -insO, yy = 1 , yy < = lenl yy + + ) { col0[yy] = dely[yy] = col0[yy-l] - insi ndely[yy] = yy, } col0[0] = 0 /* Waterman Bull Math Biol 84 */

} else for (yy = 1 , yy < = lenl yy+ +) dely[yy] = -insO,

/* fill in match matrix */ for (px = seqx[0], xx = 1 , xx < = lenO, px+ + xx + +) { /* initialize first entry in col

*/ if (endgaps) { if (xx = = 1) coll [0] = delx = (msO + insl ) else coll [0] = delx = col0[0] - insi ndelx = xx

} else { coi i[θ] = o delx = -insO ndelx = 0 } Table 1 (conf)

...n for (p = seqx[lj \} = 1. y> < = lenl py++ >'} + +){

mis += _day[*px-'A'][*py-'A']

/* update penalty for del in x seq 10 * ta\or new del over ongong del

* ignore MAXGAP if weighting endgaps */ if (endgaps | | ndelyfyy] < MAXGAP) { if (col0[yy] - insO > = dely[yy]) { 15 dely[yy] = col0[yy] - (insO + insI). ndely[yy] = 1 } else { dely[yyj -= insi, ndely[yy] + +

20 }

} else { if (col0[yy] - (insO + insI) > = dely[yy]) { delylyy] = col0[yy] - (insO + insI), ndelylyy] = 1. 25 } else ndelylyy] + + ,

}

/* update penalty for del in y seq, 30 * favor new del over ongong del

*/ if (endgaps | | ndelx < MAXGAP) { if (coll[yy-l] - msO > = delx) { delx = coll[yy-l] - (insO+insl), 35 ndelx = 1,

} else { delx -= insi, ndelx + + ,

> 40 } else { if (coll[yy-l] - (insO + insl) > = delx) { delx = col 1 [yy-lj - (insO + insl), ndelx = 1,

} else 45 ndelx+ + ,

}

/* pick the maximum score, we're favoring * mis over any del and delx over dely

50 */

55

60 Table 1 (conf)

...nv, id = xx y + lenl 1 if (mis > = delx && mis > = del>[yy])

5 else if (delx >= dely[y>]) { coll[y ] = delx lj = dx[ιd] ljmp if (dxfid] jp n[0] && ('dna j | (ndelx > = MAXJMP && xx > dx[ιdj jp x|ιj] + MX) | | mis > dx[ιd] score + DINSO)) { 10 dx[ιd] ιjmp+ + if(++ιj >= MAXJMP) { wπtejmps(ιd) IJ = dx[ιd] ljmp = 0 dx[ιd) offset = offset 15 offset += si7eof(struct jmp) + sιzeof(offset)

}

} dx[ιd] jp n[ιj] = ndelx dx[ιd] jp x[ιj] = xx

20 dxfid] score = delx

} else { collfyyj = dely[y>]

25 if (dxfid] jp n[0] && ('dna | | (ndely[yy] > = MAXJMP

&&xx > dx[ιd]jp x[ιj] + MX) | | mis > dxfid] score + DINSO)) { dxfid] ιjmρ+ + if (++ιj >= MAXJMP) { wπtejmps(ιd) 30 ij = dx[ιd] ljmp = 0 dx[ιd] offset = offset offset + = sιzeof(struct jmp) + sιzeof(offset)

}

JJ d^}x[ιd] jp n[ιjj = ndely[yy] dxfid] jp x[ιj] = xx dxfid] score = dely[yy]

} if (xx = = lenO && yy < lenl) { 40 /* last col

*/ if (endgaps) coll[yy] = ιnsO+ιnsl*(lenl-yy)

} } } 0 if (endgaps && xx < lenO) coll[yy 1] -= ιnsO + ιnsl*(lenO-xx) if (coll [yy 1] > smax) { smax = coll [yy-1] dmax = id 5 } tmp = colO colO = coll coll = tmp

}

(\oid) free((char *)ndely) (void) free((char *)dely) 0 (void) free((char *)col0)

(void) free((char *)coll) } Table 1 (conf)

* pπntO — only routine visible outside this module

* * static:

* getmatO — trace back best path, count matches: pπntO

* pr_align() -- print alignment of described in array pf]: pπnt()

* dumpblockO -- dump a block of lines with numbers, stars pr_ahgn()

* numsO — put out a number line: dumpblockO * puthneO — put out a line (name, [mini], seq. [num]): dumpblockO

* stars() - -put a line of stars- dumpblockO

* stπpnameO — strip any path and prefix from a seqname */ ^include ' nw.h"

#define S PC 3 #define P LINE 256 /* maximum output line */ ^define P SPC 3 /* space between name or num and seq */ extern _day[26][26], int olen. /* set output line length */

FILE *fx. /* output file */ ppππnnttOo print { int lx, ly. firstgap, lastgap, /* overlap */ if ((fx = fopen(ofile. "w")) = = 0) { fpπntf(stderr, " %s: can't write %s\n" , prog, ofile); cleanup(l ),

} fpπntf(fx. " < first sequence: %s (length = %d)\n" , namexfO], lenO), fpπntf(fx. " < second sequence- %s (length = %d)\n". namexf l], lenl ). olen = 60, lx = lenO; ly = lenl ; firstgap = lastgap = 0; if (dmax < lenl - 1) { /* leading gap in x */ pp[0].spc = firstgap = lenl - dmax - 1. ly - = ppfOj.spc.

} else if (dmax > lenl - 1) { /* leading gap in y */ ppf l].spc = firstgap = dmax - (lenl - 1 ); lx -= pp[ l].spc;

} if (dmaxO < lenO - 1) { /* trailing gap in x */ lastgap = lenO - dmaxO -1 , lx -= lastgap, } else if (dmaxO > lenO - 1) { /* trailing gap in y */ lastgap = dmaxO - (lenO - 1); ly - = lastgap.

} getmat(lx. ly. firstgap. lastgap), pr ahgnO; Table 1 (conf)

/*

* trace back the best path, count matches */ static getmat(lx. 1 . firstgap lastgap) g^βtmat int lx. ly, 1* ' core" (minus endgaps) */ int firstgap lastgap. /" leading trailing overlap */

char outx[32], double pet. register nO. nl . register char *pO, *pl

/* £ ;et total matches, score

*/

pO = seqxfO] + pp[ l].spc

Pl = seqxf l ] + ppfO] spc, nO^■■ = ppfl] spc + 1 nl = ppfO] spc + 1. nm = 0 while ( *pO && *pl ) {

} else { if (xbm[*pO-'A']&xbm[*pl-'A']) nm+ + ,

/* pet homology

* if penalizing endgaps. base is the shorter seq

* else, knock oft overhangs and take shorter core */ if (endgaps) lx = (lenO < lenl) ' lenO lenl , else lx = (lx < ly)> lx ly pel = 100 *(double)nm/(double)lx. fpπntf(fx, "\n"), tpπntf(fx. " < %d match%s in an overlap of %d % 21 percent sιmιlaπty\n' nm. (nm = = 1 )'' es" . lx, pet), Table 1 (conf) fpπntf(fλ " < gaps m first sequence %d . gapx) .getmat if (gapx) {

(void) spπntf(outλ, " ( %d %s%s)' ngapx (dna) ' ' base' residue' (ngapx = = 1)' "s ) fpπntf(fx," %s' outx). fpπntf(fx. ", gaps in second sequence %d gapy) if (gapy) {

(void) spπntf(outx. " ( %d %s%s)", ngapy, (dna) ' "base" ' residue" (ngapy 1 )' s"), fpπntf(fx. " %s". outx).

} if (dna) fpπntf(fx,

"\n < score %d (match = %d mismatch = %d gap penalty = %d + %d per base)\n" , smax. DMAT DMIS. DINS0. DINS1). else fpπntf(fx.

"\n < score %d (Dayhoff PAM 250 matπx, gap penalty = %d + %d per resιdue)\n" smax, PINS0 PINS1 ) if (endgaps) tpπntf(fx

' < endgaps penalized left endgap %d %s%s, right endgap %d %s%s\n' . firstgap, (dna)⁹ ' base" ' residue", (firstgap = = 1) ' " " "s' , lastgap. (dna)⁹ "base" "residue" , (lastgap = = 1)' " " "s"). else fpπntf(fx " < endgaps not penahzed\n"),

static nm, /* matches m core — tor checking */ static lmax, /* lengths of stripped file names */ static ιj[2], /* jmp index for a path */ static nc[2]. /* number at start of current line */ static m[2]. /* current elem number — for gapping */ static sιz[2], static char *ps[2], /* ptr to current element */ static char *po[2]. /* ptr to next output char slot */ static char oouutt[[22]][[]P_LINE] /* output line */ static char star[P E], /* set by stars() */

/*

* print alignment ot descπbed in struct path ppf] */ static pr ahgn() pr align

{ int nn, /* char count */ int more, register 1, for (l = 0, lmax = 0. l < 2, ι + + ) { nn = stπpname(namex[ι]), if (nn > lmax) lmax = nn. nc[ι] = 1. m[ι] = 1.

ps[ι] = seqxfi] pofi] = outfi] } Table 1 (conf) for (nn = nm = 0 more = 1 more ) { .pr align for ( l = more = 0 l < 2 I + + ) {

* do we have more of this sequence' */ if(»*ps[ι]) continue more + + if (ppfl] spc) { /* leading space */

else if (sιz[ι]) { /* in a gap */

*po[ι] + + = sιz[ι]

} else { /* we re putting a seq element */ *po[ι] = *ps[ι] if (ιslower(*ps[ι]))

*ps[ι] = toupper(*ps[ιJ) po[ι]++, ps[ιj + +

/*

* are we at next gap for this seq' */ if(nι[ι] ==pp[ι]x[ιj[ι]]){

* we need to merge all gaps

* at this location */ sιz[ι] = ppfi] n[ιj[ι] + +] while (nifi] == ppfi] xfijfi]]) sιz[ι] += ppfi] n[ιj[ι]++]

} m[ι] + + ,

}

} if (+ +nn == olen | | 'more && nn) { dumpblockO for(ι = 0 l < 2 ι++) po[ι] = outfi], nn = 0 } }

* dump a block of lines including numbers stars pr_ahgn()

*/ static dumpblockO dumpblock

{ register 1, for (l = 0 l < 2 ι++) *po[ι] = \0 Table 1 (conf)

...dumpblock

(void) putc( \n'. fx) ford = 0 i < 2. ι++){ if ("outfi] && (*out[ι] '= ' || *(po[ι])^')){

nums(ι) if (l == 0&& *out[l]) stars(). putlιne(ι). if d == 0&& *out[l]) tpπntt(fx. star) if(ι == 1) nums(ι)

/*

* put out a number line dumpblockO

*/ static nums(ιx) nums int ix /* index in out[] holding seq line */

{ char nl efP LINE], register i. J, register char *pn, *px, *py, for (pn = nline.1 = 0, l < lmax + P SPC, ι++, pn++)

*pn = '¹ for (l = nc[ιx], p> ' = outfix]. *py, py+ + , pn++) { if (*py = = = " II *py ==^'-^■)

*pn = ' ', else { if (ι% 10 = = 0 || (l = = 1 && ncfix] '^{■ ■} D){ j = (i < 0)⁹ -i i for (px = pn, j. j /= 10, px-)

*px =j%10 + '0', if d < 0)

*px = '-'.

} else

*pn ι++.

}

}

*pn = '\0'. nc[ιx] = l, for (pn = nline. *pn, pn+ +) (void) putc(*pn. fx), (void) putc('\n', fx).

!*

* put out a line (name, [num]. seq. [num]) dumpblockO */ static putlιne(ιx) putline

Table 1 (conf)

...putline register char *px for (px = namexfix] l = 0. *px && *px PX++ !++)

/* these count from 1

* nif] is current element (from 1)

* ncf] is number at start of current line */ for (px = out[ιx]. *px. px++)

(void) putc(*px&0x7F. fx). (void) putc('\n', fx).

* put a line of stars (seqs always in outfO], outfi]) dumpblockO

*/ static starso stars int l, register char *p0, *pl. ex, *px, if C*out[0] | | (*out[0] == ' ' && *(po[0]) = = '^•) '*out[l] | | (*out[l] == ' '&& *(po[l]) == '^•)) return, px = star, for (l = lmax + P SPC, I, ι~)

*px++ = ' ', for (pO = out[0], pl = outfi], *p0 && *pl. p0+ + . pl + +) { if (ιsalpha(*p0) && ιsalpha(*pl)) { if (xbm[*pO-'A']&xbm[*pl-'A']) { ex = '*'. nm+ +,

} else if ('dna && _day[*pO-'A'][*pl-'A ] > 0) ex = ' ', else ex = ' ',

} else ex = ' ',

*px+ + = ex.

}

*px++ = '\n', *px = '\0' Table 1 (conf)

/*

* strip path or piefix from pn. return len pr_ahgn()

*/ static stπpname(pn) Stripname char *pn. /* file name (may be path) "7

{ register char *px. *p> . py = 0. for (px = pn: *px. px + + ) if (*px = = '/') p> = px + 1. if (py) (void) strcpytpn. py). return(strlen(pn)).

Table 1 (cont')

/*

* cleanupO - cleanup any tmp file

* getseqO — read in seq set dna len maxlen

* g_calloc() — calloc() with error checkin

* readjmpsO — get the good jmps. from tmp file it necessary

* writejmpsO — write a filled arra> ot jmps to a tmp file nw()

^include ' nw h'

#include < sys/file h > char *jname = "/tmp/homgXXXXXX" /* tmp file for jmps */ FILE *fj, int cleanupO, /* cleanup tmp file */ long lseek().

/*

* remove any tmp file if we blow

*/ cleanup(ι) cleanup int { if(j⁾

(void) unlinkdname) exιt(ι).

* read, return ptr to seq, set dna, len, maxlen

* skip lines starting with ','. '<', or '>'

* seq in upper or lower case

*/ char * getseq(file, len) getseq char *fιle, /* file name */ int *len, /* seq len */

{ char lιne[1024] *pseq. register char *px, *py. int natgc. tlen

FILE *fp, if ((fp = fopen(fιle."r")) = = 0) { fpπntf(stdeπ,"%s can't read %s\n", prog. file). exιt(l),

} tlen = natgc = 0. while (fgetsdine.1024, fp)) { if (*hne == ',' | | *lιne == '<' | | *lιne == '>') continue. for (px = line, *px '= '\n',px++) if (ιsupper(*px) | | ιslower(*px)) tlen+ + ,

} if ((pseq = malloc((unsigned)(tlen + 6))) == 0) { fpπntt(stderr "%s mallocO failed to get %d bytes for %s\n , prog, tlen+6. file). exιt(l)

} pseq[0] = pseqfl] = pseq|2] = pseq[3] = '\0 Table 1 (cont')

.getseq py = pseq + 4 *len = tlen rewmd(fp) while (tgetsOine.1024. fp)) { if ("line = = ' ' | | *lme = = ' < ' | | *lιne = = ' > ) continue. for (px = line. *px '= '\n . px + + ) { if (ιsupper(*px))

*py+ + *px else if (ιslower(*px))

*py+ + = toupper(*px). if (ιndex( ΑTGCU".*(py-l))) natgc + + ,

}

}

*py++ = '\0 ,

*py = 0

(void) fclose(fp) dna = natgc > (tlen/3). return(pseq+4)

char * g_calloc(msg, nx, sz) g_calloc char *msg, /* program, calling routine */ int nx, sz, /* number and size of elements */

{ char *px *calloc(), if ((px = calloc((unsigned)nx. (unsigned)sz)) = = 0) { if (*msg) { fpπntf(stderr, "%s g_calloc() failed %s (n=%d sz=%d)\n", prog, msg, nx, sz). exιt(l), } } return(px).

* get final jmps from dx[] or tmp file, set ppf], reset dmax maιn()

*/ readjmpsO readjmps { int fd = -1,

register I, j. xx. if(fj){

(void) fclose(fj), if ((td = openOname. O RDONLY, 0)) < 0) { fpπntf(stderr, "%s can't open() %s\n'. prog, jname) cleanup(l). } } for (l = iO = ii =0. dmaxO = dmax. xx = lenO.. ι+ +) { while (1) { for (j = dx|dmax] ijmp. j > = 0 && dxldmax] ip xfj] > = xx, j— ) Table 1 (conf)

...readjmps if < 0 &.& dxldmax] offset && tj) {

(void) lseek(rd dxldmax] offset, 0) (void) read(td (char *)&dx[dmax] jp sizeof( struct jmp)) (void) readdd (char *)&dx|dmax] oftset sizeof(dx|dmax] oftset)) dxldmax] ijmp = MAXJMP 1

} else break } if (I > = JMPS) { fpπntt( stderr %s too many gaps m alignmenΛn prog) cleanupO) } if > =

if (siz < 0) { /* gap in second seq */

/* id = xx >> + lenl 1 */ ppfl] x[ιl] = xx - dmax + lenl - 1 gapy++, ngapy - = siz, /* ignore MAXGAP when doing endgaps */ siz = (-siz < MAXGAP | | endgaps)⁹ -siz MAXGAP ιl + + , } else if (siz > 0) { /* gap in first seq */

gapx + + , ngapx + = siz

/* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP | | endgaps)' siz MAXGAP ι0+ + } } else break } /* reverse the order of jmps

*/ ford = 0, iO j < iO, j++ iO- ){ i = ppfO] nfj], pp[0] n[j] = ppfO] nfiO], pp[0] nfiO] = i i = ppfO] xfj], pp[0] x[j] = pp[0] xfiO], pp[0] xfiO] = i } ford = 0 ii- j < ιl,j++ ιl-){ i = PPfl] nfj], ppfl] nfj] = pp[l] n[U], pp[l] n[ιl] = i i = PPfl] Lj] PPll] xϋl = PPfl] x[ιl], PPfl] x[ιl] = i } if (td > = 0)

(void) close(td) if(j){

(void) unlinkdname) fj =0 offset = 0

} } Table 1 (conf)

/*

* write a filled jmp struct oftset of the prev one (it any) nw()

*/ wπtejmps(ιx) writejmpS

{ char *mktemp() if ('fj) { if (mktempdname) < 0) { tpπntf(stderr " s can t mktempO % s\n . prog, j name), cleanup(l), if ((fi = fopendname. "w")) = = 0) { fpπntf(stderr. " %s can't write %s\n' . prog jname) exιt(l) } } (void) fwπte((char *)&dx[ιx] jp. sizeof( struct jmp), 1 , fj)

(void) fwπte((char *)&dx[ιx] oftset sizeof(dx|ιx] offset). 1 , fj )

Table 2

PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)

Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)

% amino acid sequence identity =

(the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =

5 divided by 15 = 33.3 %

Table 3

PRO XXXXXXXXXX (Length = 10 ammo acids)

Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)

% amino acid sequence identity =

(the number of identically matching ammo acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) =

5 divided by 10 = 50%

Table 4

PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)

% nucleic acid sequence identity =

(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =

6 divided by 14 = 42.9%

Table 5

PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)

Comparison DNA NNNNLLLVV (Length = 9 nucleotides)

% nucleic acid sequence identity =

(the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) =

4 divided by 12 = 33.3 %

II Compositions and Methods of the Invention

1 Preparation of the polypeptides of the inv ention

The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO polypeptides In particular cDNAs encoding various PRO polypeptides have been identified and isolated, as disclosed in further detail in the Examples below It is noted that proteins produced in separate expression rounds may be given different PRO numbers but the UNQ number is unique for any given DNA and the encoded protein, and will not be changed However, for sake of simplicity, in the present specification the protein encoded by the full length native nucleic acid molecules disclosed herein as well as all further native homologues and variants included in the foregoing definition of PRO, will be referred to as "PRO' or "PRO/number , regardless of their origin or mode of preparation

As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill For the PRO polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time

A PRO Polypeptides

The description below relates primarily to production of the PRO polypeptide of the invention by culturing cells transformed or transfected with a vector containing nucleic acid which encodes of the PRO polypeptide of the invention It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare of the PRO polypeptide of the invention For instance, the polypeptide sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e g , Stewart et al , Solid-Phase Peptide Synthesis W H Freeman Co , San Francisco, CA (1969), Merπfield, J Am Chem Soc , 85 2149-2154 (1963)] In \ttio protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of the PRO polypeptide of the invention may be chemically synthesized separately and combmed usmg chemical or enzymatic methods to produce the full length polypeptide

B PRO Polypeptide Variants

In addition to the full length native sequence PRO polypeptides described herein, it is contemplated that PRO variants can be prepared PRO variants can be prepared by introducing appropriate nucleotide changes into the PRO DNA, and/or by synthesis of the desired PRO polypeptide Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the PRO, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics Variations m the native full-length sequence PRO or in various domains of the PRO described herein, can be made, for example, using any of the techniques and guidelines tor 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 that results in a change in the amino acid sequence of the PRO as compared with the native sequence PRO Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more ot the domains of the PRO

Guidance m determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO with that of homologous known protein molecules and minimizing the number of ammo acid sequence changes made in regions of high homology Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucme with a serine, l e , conservative ammo acid replacements Insertions or deletions may optionally be in the range of about 1 to 5 amino acids The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence PRO polypeptide fragments are provided herein Such fragments may be truncated at the N- terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native protein Certain fragments lack amino acid residues that are not essential for a desired biological activity of the PRO polypeptide

PRO fragments may be prepared by any of a number ot conventional techniques Desired peptide fragments may be chemically synthesized An alternative approach involves generating PRO fragments by enzymatic digestion, e g , by treating the protein with an enzyme known to cleav e proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR) Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5 and 3' primers in the PCR Preferably, PRO polypeptide fragments share at least one biological and/or immunological activ ity with the native PRO polypeptide disclosed herein

In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened Table 6

Original Exemplar} Preferred Residue Substitutions Substitutions

Ala (A) v al, leu; lie val Arg (R) lys; gin; asn lys Asn (N) gin; his; lys; arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gin; lys; arg arg He (I) leu; val; met; ala; phe, norleucine leu

Leu (L) norleucme, lie; val, met; ala; phe lie

Lys (K) arg; gin; asn arg

Phe (F) leu; val; lie; ala, tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) lie; leu; met; phe; ala; norleucine leu

Substantial modifications in function or lmmunological identity of the PRO polypeptide are accomplished by selecting substimtions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain Naturally occurring residues are divided into groups based on common side-chain properties. (1) hydrophobic- norleucine, met, ala, val, leu, lie; (2) neutral hydrophilic: cys, ser, thr,

(3) acidic: asp, glu,

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class Such substituted residues also may be introduced into the conservative substimtion sites or, moie preferably, into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such as ohgonucleotide-mediated (site- directed) mutagenesis, alanine scanning, and PCR mutagenesis Site-directed mutagenesis [Carter et al., Nucl Acids Res.. 13-4331 (1986), Zoller et al., Nucl. Acids Res. , J0:6487 (1987)], cassette mutagenesis

[Wells et al. , Gene. 34 315 (1985)], restriction selection mutagenesis [Wells et al , Philos Trans R. Soc London SerA 317 415 (1986)] or other known techniques can be performed on the cloned DNA to produce the PRO v ariant DNA

Scanning amino acid analysis can also be employed to identify one or more ammo acids along a contiguous sequence Among the preferred scanning ammo acids are relativ el) small neutral amino acids Such amino acids include alanine, glycine, serine, and cysteine Alanme is typically a preferred scanning amino acid among this group because it eliminates the side chain beyond the beta-carbon and is less likely to alter the main chain conformation of the variant [Cunningham and Wells, Science. 244 1081 1085 (1989)] Alanine is also typically preferred because it is the most common ammo acid Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W H Freeman & Co , N Y ), Chothia, J Mol Biol , 150 1 (1976)] If alanine substitution does not yield adequate amounts of variant, an lsoteπc amino acid can be used

C Modifications of PRO Covalent modifications of PRO are included within the scope of this invention One type of covalent modification includes reacting targeted ammo acid residues of a PRO polypeptide with an organic deπvatizing agent that is capable of reacting with selected side chains or the N or C- terminal residues of the PRO

Deπvatization with bifunctional agents is useful, for instance, for crosslmking PRO to a water-insoluble support matrix or surface for use m the method for purifying anti PRO antibodies, and vice-versa Commonly used crosslmking agents include, e g , l , l-bιs(dιazoacetyl) 2-phenylethane, glutaraldehyde, N hydroxysuccimmide esters, for example, esters with 4-azιdosalιcyhc acid, homobifunctional lmidoesters, including disuccimmidyl esters such as 3 , 3 ' dιthιobιs(succιnιmιdylpropιonate) bifunctional maleimides such as bιs-N-maleιmιdo-l ,8-octane and agents such as methyl-3-[(p-azιdophenyl)dιthιo]propιoιmιdate

Other modifications include deamidation of glutammyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the ammo groups of lysine, arginine, and histidine side chains [T E Creighton, Proteins Structure and Molecular Properties W H Freeman & Co ,

San Francisco, pp 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group

Another type of covalent modification of the PRO polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide ' Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence PRO (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence PRO In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present

Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence The alteration mav be made for example, by the addition of or substitution b , one or more serine or threonine residues to the native sequence PRO (for O linked glycosylation sites) The PRO ammo acid sequence may optionally be altered through changes at the DNA lev el. particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids

Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is b> chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 1 1 September 1987, and in Aplm and Wπston, CRC Cπt Rev Biochem , pp 259-306 (1981)

Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemicall} or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs 259 52 ( 1987) and by Edge et al , Anal Biochem . 1 18 131 (1981) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo glycosidases as described by Thotakura et al , Meth Enzymol , 138 350 (1987) Another type of covalent modification of PRO comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e g , polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth m U S Patent Nos 4,640,835, 4,496,689, 4,301 , 144, 4,670,417, 4,791 , 192 or 4, 179,337

The PRO of the present invention may also be modified in a way to form a chimeric molecule comprising PRO fused to another, heterologous polypeptide or amino acid sequence

In one embodiment, such a chimeric molecule comprises a fusion of the PRO with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally placed at the amino or carboxyl- terminus of the PRO The presence of such epitope-tagged forms of the PRO can be detected using an antibody against the tag polypeptide Also, provision of the epitope tag enables the PRO to be readily purified by affimty purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag Various tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidine (poly-his) or poly-histidme-glycine (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol . 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E 10 antibodies thereto [Evan et al , Molecular and Cellular Biology. 5 3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody

[Paborsky et al , Protein Engineering, 3(6) 547-553 ( 1990)] Other tag polypeptides include the Flag -peptide [Hopp et al . BioTechnologv, 6 1204-1210 (1988)] the KT3 epitope peptide [Martin et al , Science, 255 192 194 ( 1992)] , an α-tubu n epitope peptide [Skinner et al , J Biol Chem , 266 15163-15166 (1991 )1, and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA. 87 6393-6397 (1990)] In an alternative embodiment, the chimeric molecule may comprise a fusion of the PRO with an immunoglobulin or a particular region of an immunoglobulin For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule The lg fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a PRO polypeptide in place of at least one variable region withm an lg molecule In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI CH2 and CH3 regions of an IgGl molecule For the production of immunoglobulin fusions see also US Patent No 5 428, 130 issued June 27, 1995

D Preparation of PRO The description below relates primarily to production of PRO by culturing cells transformed or transfected with a vector containing PRO nucleic acid It is, of course, contemplated that alternati e methods, which are well known in the art, may be employed to prepare PRO For instance, the PRO sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e g , Stewart et al , Solid-Phase Peptide Synthesis, W H Freeman Co , San Francisco, CA (1969), Merπfield, J Am Chem Soc . 85 2149-2154 (1963)] In protein synthesis may be performed using manual techniques or by automation Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer s instructions Various portions of the PRO may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO

1 Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared from tissue believed to possess the PRO mRNA and to express it at a detectable level Accordingly, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples The PRO- encoding gene may also be obtained from a genomic library or by known synthetic procedures (e g automated nucleic acid synthesis)

Libraries can be screened with probes (such as antibodies to the PRO or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it Screening the cDN A or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spring Harbor Laborator) Press, 1989) An alternative means to isolate the gene encoding PRO is to use PCR methodology [Sambrook et al , supra, Dieffenbach et al , PCR Primer A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]

The Examples below describe techniques for screening a cDNA library The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened Methods of labeling are well known m the art, and include the use of radiolabels like P labeled ATP, biotinylation or enzyme labeling Hybridization conditions including moderate stringency and high stringency, are provided in Sambrook et al , supra Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and a\ ailable in public databases such as GenBank or other private sequence databases Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full length sequence can be determined using methods known in the art and as described herein

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using con entional primer extension procedures as described m Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse transcribed into cDN A

2 Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for

PRO production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences The culmre conditions, such as media temperature, pH and the like, can be selected by the skilled artisan without undue experimentation In general principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach M Butler, ed (IRL

Press, 1991) and Sambrook et al , supra

Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCL, CaP0₄, liposome mediated and electroporation Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells The calcium treatment employing calcium chloride, as described in Sambrook et al , supra, or electroporation is generally used for prokaryotes Infection with Agrobactenutn tumefaciens is used for transformation of certain plant cells, as described by Shaw et al , Gene. 23 315 (1983) and WO 89/05859 published 29 June 1989 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology. 52 456 457 (1978) can be employed General aspects of mammalian cell host system transfections have been described in U S Patent No 4,399,216 Transformations into yeast are typically carried out according to the method of Van Solingen et al , J Bact . 130 946 (1977) and Hsiao et al , Proc Natl Acad Sci (USA). 76 3829 (1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells , or polycations , e g , polybrene, polyornithme, may also be used For various techniques for transforming mammalian cells, see Keown et al , Methods in Enzymology, 185 527 537 (1990) and Mansour et al , Nature 336 348 352

(1988)

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells Suitable prokaryotes include but are not limited to eubacteπa such as Gram negative or Gram-positive organisms for example Enterobacteriaceae such as E coli Various E coli strains are publicly available, such as E coli K12 strain MM294 (ATCC 31 ,446), E coli X1776 (ATCC

31 ,537), E coh strain W31 10 (ATCC 27,325) and K5 772 (ATCC 53,635) Other suitable prokaryotic host cells include Enterobacteriaceae such as Escheiichta, e g E coli, Entetobacter Erwtnia Klebsiella Proteus. Salmonella, e g Salmonella nphimunum, Serratia, e g , Serratia marcescans, and Shigella, as well as Bacilli such as B subtilis and B lichenifonms (e g . β lichenifonms 41 P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P aeruginosa, and Streptotm ces These examples are illustrative rather than limiting Strain W31 10 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations Preferably , the host cell secretes minimal amounts of proteolytic enzymes For example, strain W31 10 may be modified to effect a genetic mutation m the genes encoding proteins endogenous to the host, with examples of such hosts including E colt W31 10 strain 1A2, which has the complete genotype tonA , E coli W31 10 strain 9E4, which has the complete genotype tonA ptr3, E coli W31 10 strain 27C7 (ATCC 55 244) which has the complete genotype tonA ptr3 phoA El 5 (argF lac) 169 degP ompT kan^r, E coli W31 10 strain 37D6, which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT rbs7ιl\ G kan^r E coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation, and an E coli strain having mutant periplasmic protease disclosed in U S Patent No 4,946,783 issued 7 August 1990 Alternatively, in vitro methods of cloning, e g , PCR or other nucleic acid polymerase reactions are suitable

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO-encoding vectors Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism Others include Schizosaccharomyces pombe (Beach and Nurse, Nature. 290 140 [1981], EP 139,383 published 2 May 1985), Kluyveromyces hosts (U S Patent No 4,943,529, Fleer et al , Bio/Technology. 9 968-975 (1991)) such as, e g , K lactts (MW98-8C, CBS683, CBS4574, Louvencourt et al , J Bacteriol , 154(2) 737-742 [1983]), K fragilis (ATCC 12,424), K bulgancus (ATCC 16,045), K mckeramii (ATCC 24, 178), K waltn (ATCC 56,500), K drosophilarum (ATCC 36,906, Van den Berg et al , Bio/Technology. 8 135 (1990)), K thermotolerans , and K marxianus, varrowia (EP 402,226), Pichia pastons (EP 183,070, Sreekrishna et al , J Basic Microbiol . 28 265-278 [1988]), Candida, Tnchoderma reesia (EP 244,234), Neurospora crassa (Case et al , Proc Natl Acad Sci USA. 76-5259-5263 [1979]), Schwanniomvces such as Schwannwmyces occidentahs (EP 394,538 published 31 October 1990), and filamentous fungi such as, e g , Neurospora, Penicillium Tolvpocladium (WO 91/00357 published 10 January 1991), and Aspergύlus hosts such as A mdulans (Ballance et al , Biochem Biophys Res Commun . 1 12 284-289 [1983], Tilburn et al , Gene, 26 205 221 [1983], Yelton et al , Proc Natl Acad Sci USA. 81 1470-1474 [1984]) and A niger (Kelly and Hynes, EMBO J . 4 475-479 [1985]) Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,

Torulopsis, and Rhodotorula A list of specific species that are exemplary of this class of yeasts may be found in C Anthony, The Biochemistry of Methylotrophs, 269 (1982)

Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells

More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al , J Gen Virol . 36 59 (1977)), Chinese hamster ovaπ cells/-DHFR (CHO, Urlaub and Chasm, Proc Natl Acad Sci USA. 77 4216 (1980)), mouse sertoli cells (TM4, Mather, Biol Reprod , 23 243 251 (1980)), human lung cells (W138 ATCC CCL 75). human liver cells (Hep G2, HB 8065), and mouse mammary tumor (MMT 060562, ATCC CCL51) The selection of the appropriate host cell is deemed to be within the skill in the art

3 Selection and Use of a Rephcable Vector

The nucleic acid (e g , cDNA or genomic DNA) encoding PRO may be inserted into a rephcable vector for cloning (amplification of the DNA) or for expression Various vectors are publicly available The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures In general, DNA is inserted into an appropriate restriction endonuclease sιte(s) using techniques known in the art Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan

The PRO may be produced recombmantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide In general, the signal sequence may be a component of the vector, or it may be a part of the PRO encoding DNA that is inserted into the vector The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders For yeast secretion the signal sequence may be, e g , the yeast mvertase leader, alpha factor leader (including Saccharomyces and Kl xveromyces α-factor leaders, the latter described in U S Patent No 5,010, 182), or acid phosphatase leader, the C albicans glucoamylase leader (EP 362, 179 published 4 April 1 90), or the signal described in WO 90/13646 published 15 November 1990 In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders

Both expression and clomng vectors contain a nucleic acid sequence that enables the 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 from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells

Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e g , ampicilhn, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e g , the gene encoding D-alanine racemase for Bacilli An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or thymidine kinase An appropriate host cell when wild type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA, 77 4216 (1980) A suitable selection gene for use in yeast is the trp\ gene present in the yeast plasmid YRp7 [Stinchcomb et al , Nature, 282 39 (1979), Kingsman et al , Gene, 7 141 (1979) Tschemper et al , Gene,

10 157 (1980)] The trp\ gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No 44076 or PEP4-1 [Jones, Genetics, 85 12 (1977)]

Expression and cloning vectors usually contain a promoter operably linked to the PRO-encoding nucleic acid sequence to direct mRNA synthesis Promoters recognized by a variety of potential host cells are well known Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al , Nature, 275 615 (1978), Goeddel et al Nature, 281 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res , 8 4057 (1980), EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al , Proc Natl Acad Sci USA, 80 21 -25 ( 1983)] Promoters for use in bacterial systems also will contain a Sh e-Dalgarno (S D ) sequence operably linked to the DNA encoding PRO

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al , J Biol Chem , 255 2073 (1980)] or other glycolytic enzymes [Hess et al , J Adv Enzyme Reg . 7 149 (1968), Holland, Biochemistry. 17 4900 (1978)], such as enolase, glyceraldehyde-3 phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, tπosephosphate isomerase, phosphoglucose isomerase, and glucokinase

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further descπbed in EP 73,657

PRO transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211 ,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e g , the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems

Transcription of a DNA encoding the PRO by higher eukaryotes may be increased by inserting an enhancer sequence into the vector Enhancers are cis acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotem, and insulin) Typicalh however, one will use an enhancer from a eukaryotic cell virus Examples include the SV40 enhancer on the late side of the replication origin (bp 100 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin and adenovirus enhancers The enhancer may be spliced into the vector at a position 5 or 3 to the PRO coding sequence, but is preferably located at a site 5 from the promoter Expression vectors used m eukaryotic host cells (yeast, fungi, insect, plant, animal human, or nucleated cells from other multicellular organisms) w ill also contain sequences necessary for the termination of transcription and for stabilizing the mRNA Such sequences are commonly available from the 5' and, occasionally 3 ' , untranslated regions of eukaryotic or viral DNAs or cDNAs These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO Still other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO in recombinant vertebrate cell culture are described in Gethmg et al , Nature 293 620 625 (1981), Mantei et al , Nature. 281 40-46 ( 1979), EP 1 17,060, and EP 1 17,058

4 Detecting Gene Expression Gene expression may be measured in a sample directly, for example, by conv entional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Natl Acad Sci USA. 77 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA protein duplexes The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected

Gene expression, alternatively, may be measured by lmmunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culmre or body fluids, to quantitate directly the expression of 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 prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to PRO DNA and encoding a specific antibody epitope

5 Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host cell lysates If membrane- bound, it can be released from the membrane using a suitable detergent solution (e g Triton X 100) or by enzymatic cleavage Cells employed in expression of PRO can be disrupted by various physical or chemical means, such as freeze-thaw cycling, somcation, mechanical disruption, or cell lysing agents

It may be desired to purify PRO from recombinant cell proteins or polypeptides The following procedures are exemplary of suitable purification procedures by fractionation on an ιon-e\change column, ethanol precipitation reverse phase HPLC, chromatography on silica or on a cation exchange resm such as DEAE, chromato focusing, SDS PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, protein A Sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of the PRO Various methods of protein purification may be employed and such methods are known m the art and described for example in Deutscher, Methods in Enzvmologv. 182 (1990). Scopes, Protein Purification Principles and Practice, Springer Verlag, New York (1982) The purification step(s) selected will depend, for example, on the nature of the production process used and the particular PRO produced

E Tissue Distribution The location of tissues expressing the PRO polypeptides of the invention can be identified by determining mRNA expression in various human tissues The location of such genes provides information about which tissues are most likely to be affected by the stimulating and inhibiting activities of the PRO polypeptides of the invention The location of a gene in a specific tissue also provides sample tissue for the activity blocking assays discussed below As noted before, gene expression in various tissues may be measured by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc Natl Acad Sci USA. 77 5201-5205 [1980]), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes

Gene expression in various tissues, alternatively, may be measured by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of 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 prepared against a native sequence of a PRO polypeptide of the invention or against a synthetic peptide based on the DNA sequences encoding the PRO polypeptide of the invention or against an exogenous sequence fused to a DNA encoding a PRO polypeptide of the invention and encodmg a specific antibody epitope General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided below

F Antibody Binding Studies

The activity of the polypeptides of the invention can be further verified by antibody binding studies, in which the ability of anti PRO antibodies to inhibit the effect of the PRO polypeptides on tissue cells is tested Exemplary antibodies include polyclonal, monoclonal, humanized bispecific, and heteroconjugate antibodies, the preparation of which will be described hereinbelow

Antibody binding studies may be carried out in any known assav method, such as competitive binding assays, direct and indirect sandwich assay s, and immunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Ine , 1987)

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of antibody The amount of target protein in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies preferably are lnsolubihzed before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibodv binds to the analyte, thus forming an insoluble three-part complex See, e g , US Pat No 4,376, 110 The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assa> ) For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme

For immunohistochemistry, the tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin, for example

G Cell Based Assays

Cell-based assays and animal models for immune related diseases can be used to further understand the relationship between the genes and polypeptides identified herein and the development and pathogenesis of immune related disease

In a different approach, cells of a cell type known to be involved in a particular immune related disease are transfected with the cDNAs described herein, and the ability of these cDNAs to stimulate or inhibit immune function is analyzed Suitable cells can be transfected with the desired gene, and monitored for immune function activity Such transfected cell lines can then be used to test the ability of poly- or monoclonal antibodies or antibody compositions to inhibit or stimulate immune function, for example to modulate T-cell proliferation or inflammatory cell infiltration Cells transfected with the coding sequences of the genes identified herein can further be used to identify drug candidates for the treatment of immune related diseases

In addition, primary cultures derived from transgenic animals (as described below) can be used in the cell-based assays herein, although stable cell lines are preferred Techniques to derive continuous cell lines from transgenic animals are well known in the art (see, e g Small et al , Mol Cell Biol 5, 642-648 [1985])

One suitable cell based assay is the mixed lymphocyte reaction (MLR) Current Protocols in

Immunology, unit 3 12, edited by JE Cohgan, AM Kruisbeek, DH Marghes, EM Shevach W Strober National Insitutes of Health, Published by John Wiley & Sons, Ine In this assay, the ability of a test compound to stimulate or inhibit the proliferation of activated T cells is assayed A suspension of responder

T cells is culmred with allogeneic stimulator cells and the proliferation of T cells is measured by uptake of tπtiated thymidine This assay is a general measure of T cell reactivity Since the majority of T cells respond to and produce IL-2 upon activation, differences m responsiveness in this assay m part reflect differences in IL-2 production by the responding cells The MLR results can be verified by a standard lymphokme (IL 2) detection assay Current Protocols in Immunology, above, 3 15, 6 3

A proliferative T cell response in an MLR assay may be due to direct mitogenic properties of an assayed molecule or to external antigen induced activation Additional verification of the T cell stimulatory activity of the polypeptides of the invention can be obtained by a costimulation assay T cell activation requires an antigen specific signal mediated through the T-cell receptor (TCR) and a costimulatory signal mediated through a second ligand binding interaction, for example, the B7(CD80. CD86)/CD28 binding interaction CD28 crosslmking increases lymphokme secretion by activated T cells T cell activation has both negative and positive controls through the binding of ligands which have a negative or positive effect CD28 and CTLA-4 are related glycoprote s in the lg superfamily which bind to B7 CD28 binding to B7 has a positive costimulation effect of T cell activation, conversely, CTLA 4 binding to B7 has a negative T cell deactivating effect Chambers and Allison, Curr Opin Immunol (1997) 9 396 Schwartz, Cell (1992)

71 1065, Linsey, and Ledbetter, Annu Rev Immunol (1993) 1 1 191 , June et al, Immunol Today (1994) 15 321 , Jenkins, Immunity (1994) 1 405 In a costimulation assay, the PRO polypeptides of the invention are assayed for T cell costimulatory or inhibitory activity

PRO polypeptides of the invention, as well as other compounds of the invention, which are stimulators (costimulators) of T cell proliferation and agonists , e g agonist antibodies, thereto as determined by MLR and costimulation assays, for example, are useful m treating immune related diseases characterized by poor, suboptimal or inadequate immune function These diseases are treated by stimulating the proliferation and activation of T cells (and T cell mediated immunity) and enhancing the immune response in a mammal through administration of a stimulatory compound, such as the stimulating polypeptides of the invention The stimulating polypeptide may, for example, be a PRO polypeptide or an agonist antibody therefor Direct use of a stimulating compound as in the invention has been validated in experiments with

4-1BB glycoprotein, a member of the tumor necrosis factor receptor family, which binds to a ligand (4- 1 BBL) expressed on primed T cells and signals T cell activation and growth Alderson et al , J Immunol (1994) 24 2219

The use of an agonist stimulating compound has also been validated experimentally Activation of 4- IBB by treatment with an agonist antι-4-lBB antibody enhances eradication of tumors Hellstrom and

Hellstrom, Cπt Rev Immunol (1998) 18 1 Immunoadjuvant therapy for treatment of tumors, descπbed m more detail below, is another example of the use of the stimulating compounds of the invention

An immune stimulating or enhancing effect can also be achieved by antagonizing or blocking the activity of a protein which has been found to be inhibiting in the MLR assay Negating the inhibitory activity of the compound produces a net stimulatory effect Suitable antagonists/blocking compounds are antibodies or fragments thereof which recognize and bind to the inhibitory protein, thereby blocking the effective interaction of the protein with its receptor and inhibiting signaling through the receptor This effect has been validated in experiments using antι-CTLA-4 antibodies which enhance T cell proliferation, presumably by removal of the inhibitory signal caused by CTLA 4 binding Walunas et al Immunity (1994) 1 405

On the other hand, PRO polypeptides of the invention, as well as other compounds of the invention, which are direct inhibitors of T cell proliferation/activation and/or lymphokme secretion, can be directly used to suppress the immune response These compounds are useful to reduce the degree of the immune response and to treat immune related diseases characterized by a hyperactive, superoptimal, or autoimmune response

This use of the compounds of the invention has been validated by the experiments described above m which CTLA-4 binding to receptor B7 deactivates T cells The direct inhibitory compounds of the invention function in an analogous manner

Alternatively, compounds, e g antibodies, which bind to stimulating PRO polypeptides of the invention and block the stimulating effect of these molecules produce a net inhibitory effect and can be used to suppress the T cell mediated immune response by inhibiting T cell proliferation/activation and/or lymphokme secretion Blocking the stimulating effect of the PRO polypeptides suppresses the immune response of the mammal This use has been validated in experiments using an antι-IL2 antibody In these experiments, the antibody binds to IL2 and blocks binding of IL2 to its receptor thereby achieving a T cell inhibitory effect

H Animal Models

The results of the cell based in vitro assays can be further verified using in vivo ammal models and assays for T-cell function A variety of well known animal models can be used to further understand the role of the genes identified herein in the development and pathogenesis of immune related disease, and to test the efficacy of candidate therapeutic agents, including antibodies, and other antagomsts of the native polypeptides, including small molecule antagonists The in vivo nature of such models makes them predictive of responses in human patients Animal models of immune related diseases include both non-recombinant and recombinant (transgenic) animals Non-recombinant animal models include, for example, rodent, e g , murine models Such models can be generated by introducing cells into syngeneic mice using standard techniques, e g subcutaneous injection, tail vein injection, spleen implantation, intraperitoneal implantation, implantation under the renal capsule, etc

Graft-versus-host disease occurs when immunocompetent cells are transplanted into immunosuppressed or tolerant patients The donor cells recognize and respond to host antigens The response can vary from life threatening severe inflammation to mild cases of diarrhea and weight loss

Graft-versus-host disease models provide a means of assessing T cell reactivity against MHC antigens and minor transplant antigens A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4 3

An animal model for skin allograft rejection is a means of testing the ability of T cells to mediate in vivo tissue destruction and a measure of their role in transplant reiection The most common and accepted models use murine tail-skin grafts Repeated experiments have shown that skin allograft rejection is mediated by T cells, helper T cells and killer-effector T cells, and not antibodies Auchincloss and Sachs, Fundamental Immunology. 2nd ed W E Paul ed , Raven Press, NY, 1989, 889 992 A suitable procedure is described m detail in Current Protocols in Immunology . above, unit 4 4 Other transplant reiection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe et al , Transplantation (1994) 58 23 and Tinubu et al J Immunol (1994) 4330 4338

Animal models for delayed type hypersensitivity provides an assay of cell mediated immune function as well Delayed type hypersensitivity reactions are a T cell mediated in \ ι\ o immune response characterized by inflammation which does not reach a peak until after a period of time has elapsed after challenge with an antigen These reactions also occur in tissue specific autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE, a model for MS) A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4 5 EAE is a T cell mediated autoimmune disease characterized by T cell and mononuclear cell inflammation and subsequent demyelination of axons m the central nervous system EAE is generally considered to be a relevant animal model for MS in humans Bolton. Multiple Sclerosis (1995) 1 143 Both acute and relapsing remitting models have been developed The compounds of the invention can be tested for T cell stimulatory or inhibitory activity against immune mediated dem> ehnating disease using the protocol described in Current Protocols in Immunology, above, units 15 1 and 15 2 See also the models for myelin disease in which ohgodendrocytes or Schwann cells are grafted into the central nervous system as descπbed in Duncan et al, Molec Med Today (1997) 554-561

Contact hypersensitivity is a simple delayed type hypersensitivity in vivo assay of cell mediated immune function In this procedure, cutaneous exposure to exogenous haptens which gives rise to a delayed type hypersensitivity reaction which is measured and quantitated Contact sensitivity involves an initial sensitizing phase followed by an ehcitation phase The ehcitation phase occurs when the T lymphocytes encounter an antigen to which they have had previous contact Swelling and inflammation occur, making this an excellent model of human allergic contact dermatitis A suitable procedure is described in detail in Current Protocols in Immunology, Eds J E Cologan, AM Kruisbeek, DH Marguhes, EM Shevach and Strober, John Wiley & Sons, Ine , 1994, unit 4 2 See also Grabbe and Schwarz, Immun Today

19(1) 37-44 (1998)

An animal model for arthritis is collagen-induced arthritis This model shares clinical, histological and lmmunological characteristics of human autoimmune rheumatoid arthritis and is an acceptable model for human autoimmune arthritis Mouse and rat models are characterized by synovitis, erosion of cartilage and subchondral bone The PRO polypeptides and compounds of the invention can be tested for activity against autoimmune arthritis using the protocols described in Current Protocols in Immunology, above, units 15 5 See also the model using a monoclonal antibody to CD 18 and VLA-4 integrins described in Issekutz et al , Immunology (1996) 88 569

A model of asthma has been described in which antigen-induced airway hyper-reactivity , pulmonary eosinophilia and inflammation are induced by sensitizing an animal with ovalbumin and then challenging the animal with the same protein delivered by aerosol Several animal models (guinea pig, rat, non human primate) show symptoms similar to atopic asthma in humans upon challenge with aerosol antigens Murine models have many of the features of human asthma Suitable procedures to test the PRO polypeptides and compounds of the invention for activ ιr> and effectiveness in the treatment of asthma are described b> Wolyniec et al, Am J Respir Cell Mol Biol (1998) 18 777 and the references cited therein

Additionally , the PRO polypeptides and compounds of the invention can be tested on animal models for psoriasis like diseases Evidence suggests a T cell pathogenesis for psoriasis The compounds of the invention can be tested in the scid/scid mouse model described by Schon et al, Nat Med (1997) 3 183, m which the mice demonstrate histopathologic skin lesions resembling psoriasis Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff et al, Am J Path (1995) 146 580

Recombinant (transgenic) animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic animals Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e g baboons, chimpanzees and monkeys Techniques known in the art to introduce a transgene into such animals include pronucleic microinjection (Hoppe and Wanger, U S Patent No 4,873, 191), retrovirus mediated gene transfer into germ lines (e g , Van der Putten et al , Proc Natl Acad Sci USA 82, 6148 615 [1985]), gene targeting in embryonic stem cells (Thompson et al , Cell 56, 313-321 [1989]), electroporation of embryos

(Lo, Mol Cel Biol 3. 1803-1814 [1983]), sperm-mediated gene transfer (Lavitrano et al , CeH 57, 717-73 [1989]) For review, see, for example, U S Patent No 4,736,866

For the purpose of the present invention, transgenic animals include those that carry the transgene only m part of their cells ("mosaic animals") The transgene can be integrated either as a single transgene, or in concatamers, e g , head-to-head or head-to-tail tandems Selective introduction of a transgene into a particular cell type is also possible by following, for example, the technique of Lasko et al , Proc Natl Acad Sci USA 89, 6232-636 (1992)

The expression of the transgene m transgenic animals can be monitored by standard techniques For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry

The animals may be further examined for signs of immune disease pathology, for example by histological examination to determine infiltration of immune cells into specific tissues Blocking experiments can also be performed in which the transgenic animals are treated with the compounds of the invention to determine the extent of the T cell proliferation stimulation or inhibition of the compounds In these experiments, blocking antibodies which bind to the PRO polypeptide of the invention, prepared as described above, are administered to the animal and the effect on immune function is determined

Alternatively, "knock out" animals can be constructed which have a defective or altered gene encoding a polypeptide identified herein, as a result of homologous recombination between the endogenous gene encoding the polypeptide and altered genomic DNA encoding the same polypeptide introduced into an embryonic cell of the animal For example, cDNA encoding a particular polypeptide can be used to clone genomic DNA encoding that polypeptide in accordance with established techniques A portion of the genomic DNA encoding a particular polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration Typicall , several kilobases of unaltered flanking DNA (both at the 5 and 3 ends) are included in the vector [see e g , Thomas and Capecchi, Cell, 51 503 (1987) for a description of homologous recombination vectors] The vector is introduced mto an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed with the endogenous DNA are selected [see e g , Li et al , Cell, 69 915

(1992)] The selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, Teratocarcmomas and Embryonic Stem Cells A Practical Approach, Robertson, ed (IRL, Oxford, 1987), pp 113 152] A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a ' knock out' animal Progeny harboring the homologously recombmed DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombmed DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the polypeptide

I Immunoadiuvant Therapy

In one embodiment, the lmmunostimulating PRO polypeptides and compounds of the invention can be used in immunoadjuvant therapy for the treatment of tumors (cancer) It is now well established that T cells recognize human tumor specific antigens One group of tumor antigens, encoded by the MAGE, BAGE and GAGE families of genes, are silent in all adult normal tissues, but are expressed in significant amounts in tumors, such as melanomas, lung tumors, head and neck tumors, and bladder carcinomas DeSmet et al, (1996) Proc Natl Acad Sci USA. 93 7149 It has been shown that costimulation of T cells induces tumor regression and an antitumor response both in vitro and in vι\o Melero et al, Nature Medicine (1997) 3 682, Kwon et al, Proc Natl Acad Sci USA (1997) 94 8099, Lynch et al, Nature Medicine (1997) 3 625, Finn and Lotze. J Immunol (1998) 21 114 The stimulatory PRO polypeptides and compounds of the invention can be administered as adjuvants, alone or together with a growth regulating agent, cytotoxic agent or chemotherapeutic agent, to stimulate T cell proliferation/activation and an antitumor response to tumor antigens The growth regulating, cytotoxic, or chemotherapeutic agent may be administered in conventional amounts using known administration regimes lmmunostimulating activity by the PRO polypeptides and compounds of the invention allows reduced amounts of the growth regulating, cytotoxic, or chemotherapeutic agents thereby potentially lowering the toxicity to the patient

J Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compounds that bind to or complex with the polypeptides encoded by the genes identified herein or a biologically active fragment thereof, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins Such screening assays will include assays amenable to high throughput screening of chemical libraries making them particularly suitable for identifying small molecule drug candidates Small molecules contemplated include synthetic organic or inorganic compounds, including peptides preferably soluble peptides, (poly)peptιde immunoglobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single chain antibodies a i ldiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art All assays are common in that they call for contacting the drug candidate with a polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture In a particular embodiment, the polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e g on a microtiter plate by covalent or non-covalent attachments Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the polypeptide and drying Alternatively, an immobilized antibody, e g a monoclonal antibody, specific for the polypeptide to be immobilized can be used to anchor it to a solid surface The assay is performed by adding the non immobilized component, which may be labeled by a detectable label, to the immobilized component, e g the coated surface containing the anchored component When the reaction is complete, the non-reacted components are removed, e g by washing, and complexes anchored on the solid surface are detected When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred Where the originally non immobilized component does not carry a label, complexing can be detected, for example by using a labelled antibody specifically binding the immobilized complex

If the candidate compound interacts with but does not bind to a particular protein encoded by a gene identified herein, its interaction with that protein can be assayed by methods well known for detecting protein-protem interactions Such assays include traditional approaches, such as, cross-linking, co-immunoprecipitation, and co purification through gradients or chromatographic columns In addition, protein protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers [Fields and Song, Nature (London) 340, 245-246 (1989), Chien et al , Proc Natl Acad Sci USA 88, 9578-9582 (1991)] as disclosed by Chevray and Nathans rProc Natl Acad Sci USA 89, 5789-5793 (1991)] Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, while the other one functioning as the transcription activation domain The yeast expression system described in the foregoing publications (generally referred to as the two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one m which the target protein is fused to the DNA-bmding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain The expression of a GALl-lacZ reporter gene under control of a GAL4-actιvated promoter depends on reconstitution of GAL4 activity via protein-protem interaction Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase A complete kit (MATCHMAKER™) for identifying protein-protem interactions between two specific proteins using the two hybrid technique is commercially available from Clontech This system can also be extended to map protein domains involved m specific protein interactions as well as to pinpoint ammo acid residues that are crucial for these interactions

In order to find compounds that interfere with the interaction of a gene identified herein and other intra- or extracellular components can be tested, a reaction mixture is usually prepared containing the product of the gene and the intra or extracellular component under conditions and for a time allowing for the interaction and binding of the two products To test the ability of a test compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound In addition, a placebo may be added to a third reaction mixture, to serve as positive control The binding (complex formation) between the test compound and the intra or extracellular component present in the mixture is monitored as described above

The formation of a complex in the control reactιon(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner

K Compositions and Methods for the Treatment of Immune Related Diseases

The compositions useful m the treatment of immune related diseases include, without limitation, proteins, antibodies, small orgamc molecules, peptides, phosphopeptides, antisense and πbozyme molecules, triple helix molecules, etc that inhibit or stimulate immune function, for example, T cell proliferation/activation, lymphokme release, or immune cell infiltration For example, antisense RNA and RNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation When antisense DNA is used, oligodeoxyπbonucleotides derived from the translation initiation site, e g between about -10 and + 10 positions of the target gene nucleotide sequence, are preferred

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage Specific πbozyme cleavage sites within a potential RNA target can be identified by known techniques For further details see, e g Rossi, Current Biology 4, 469-471 (1994), and PCT publication No WO 97/33551 (published September 18, 1997)

Nucleic acid molecules in triple helix formation used to inhibit transcription should be smgle-stranded and composed of deoxynucleotides The base composition of these oligonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of puπnes or pyπmidines on one strand of a duplex For further details see, e g PCT publication No WO 97/33551 , supra

These molecules can be identified by any or any combination of the screening assays discussed above and/or by any other screening techniques well known for those skilled in the art L Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies Exemplary antibodies include polyclonal monoclonal, humanized, bispecific, and heterocon]ugate antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibodv tragments which may inhibit (antagonists) or stimulate (agonists) T cell proliferation, eosmophil infiltration, etc

1 Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or mtrapeπtoneal injections

The immunizing agent may include the PRO polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be lmmunogenic in the mammal being immunized Examples of such lmmunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumm, bovine thyroglobulm, and soybean trypsm inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM ad]uvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate) The immunization protocol may be selected by one skilled m the art without undue experimentation.

2 Monoclonal Antibodies The anti-PRO antibodies may, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybπdoma methods, such as those described by Kohler and Milstem, Nature. 256:495 (1975) In a hybπdoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable ot producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro The immunizing agent will typically include the PRO polypeptide or a fusion protein thereof

Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybπdoma cell [Goding, Monoclonal Antibodies Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed The hybπdoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoπbosyl transferase (HGPRT or HPRT), the culture medium for the hybπdomas typically will include hypoxanthine. aminopteπn, and thymidme ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells Preferred immortalized cell lines are those that fuse efficiently , support stable high le el expression of antibody by the selected antibody producing cells and are sensitive to a medium such as HAT medium More preferred immortalized cell lines are murine m eloma lines, which can be obtained for instance, from the Salk Institute Cell Distribution Center San Diego, California and the American T pe Culture Collection Manassas Virginia Human myeloma and mouse human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J Immunol , 133 3001 (1984), Brodeur et al

Monoclonal Antibody Production Techniques and Applications Marcel Dekker, Ine , New York, (1987) pp 51-63]

The culmre medium m which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against PRO Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in

binding assay, such as radioimmunoassay (RIA) or enzyme linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art The binding affinity of the monoclonal antibody can for example, be determined b> the Scatchard analysis of Munson and Pollard, Anal Biochem . 107 220 (1980)

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra] Suitable culture media for this purpose include for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culmre medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite 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 be readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybridoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells The DNA also may be modified for example, by substituting the coding sequence for human heavy and light chain constant domains m place of the homologous murine sequences [U S Patent No 4,816,567, Morrison et al , supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non immunoglobulin polypeptide Such a non immunoglobulin polypeptide can be substituted for the constant domains ot an antibody of the invention, or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to create a chimeric bivalent antibody

The antibodies may be monovalent antibodies Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heavv chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking Alternatively the relevant cysteine residues are substituted with another ammo acid residue or are deleted so as to prevent crosslmking

In \ιtro methods are also suitable for preparing monovalent antibodies Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art

3 Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' , F(ab'), or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR ot a non human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences In general, the humanized antibody will compπse substantially all of at least one, and typically two, variable domams, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that ofa human immunoglobulin [Jones et al , Nature. 321 522 525 (1986), Riechmann et al , Nature, 332 323 329 (1988), and Presta, Curr Op Struct Biol , 2 593 596 (1992)]

Methods for humanizing non-human antibodies are well known in the art Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non human These non-human amino acid residues are often referred to as ' import' residues, which are typically taken from an "import' variable domain Humamzation can be essentially performed following the method of Winter and co-workers [Jones et al , Nature, 321 522-525 (1986), Riechmann et al , Nature, 332 323 327 (1988), Verhoeyen 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 (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol , 227 381 (1991 ), Marks et al , J Mol Biol . 222 581 (1991)] The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy Alan R Liss, p 77 (1985) and Boerner et al J Immunol 147(1 ) 86 95 ( 1991)] Similarly human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g mice in which the endogenous immunoglobulin genes hav e been partially or completely inactivated Upon challenge human antibod) production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly , and antibody repertoire This approach is described, for example, in U S Patent

Nos 5,545,807, 5,545,806, 5,569,825, 5,625, 126, 5,633 425, 5,661 ,016 and in the following scientific publications Marks et al , Bio/Technology 10, 779 783 (1992), Lonberg et al Nature 368 856-859 (1994), Morrison. Nature 368. 812 13 (1994) Fishwild ef a/ , Nature Biotechnolog^y 14 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), Lonberg and Huszar, Intern Rev Immunol L3 65-93 (1995) The antibodies may also be affinity mamred using known selection and/or mutagenesis methods as described above Preferred affinity mamred antibodies have an affinity which is five times more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the mamred antibody is prepared

4 Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens In the present case, one of the binding specificities is for the PRO, the other one is for any other antigen, and preferably for a cell surface protein or receptor or receptor subunit Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities [Milste and Cuello, Nature. 305 537- 539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of ten different antibody molecules of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed m WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J . K) 3655-3659 (1991)

Antibody variable domains with the desired binding specificities (antibody antigen combining sites) can be fused to immunoglobulin constant domain sequences The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hmge, CH2 and CH3 regions

It is preferred to have the first heavy chain constant region (CH 1 ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism For further details of generating bispecific antibodies see, for example, Suresh et al , Methods in Enzymology, 121 210 (1986)

According to another approach described in WO 96/2701 1 , the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture The preferred interface comprises at least a part of the CH3 region of an antibody constant domain In this method one or more small ammo acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e g tyrosine or tryptophan) Compensatory "cavities' of identical or similar size to the large side cham(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e g alanine or threonme) This provides a mechanism for increasing the yield of the heterodimer over other unwanted end products such as homodimers

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e g F(ab')ι bispecific antibodies) Techniques for generating bispecific antibodies from antibody fragments have been described in the literature For example bispecific antibodies can be prepared can be prepared using chemical linkage Brennan et al , Science 229 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'), fragments These fragments are reduced in the presence of the dithiol complexing agent sodium arsemte to stabilize vicinal dithiols and prevent intermolecular disulfide formation The Fab' fragments generated are then converted to thiomtrobenzoate (TNB) derivatives One of the Fab' -TNB derivatives is then reconverted to the Fab' thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab' TNB derivative to form the bispecific antibody The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes

Fab' fragments may be directly recovered from E coli and chemically coupled to form bispecific antibodies Shalaby et al , J Exp Med 175 217 225 (1992) describe the production of a fully humanized bispecific antibody F(ab'), molecule Each Fab' fragment was separately secreted from E coli and subjected to directed chemical coupling in vitro to form the bispecific antibody The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets

Various technique for making and isolating bispecific antibody fragments directly from recombinant cell culmre have also been described For example, bispecific antibodies have been produced using leucine zippers Kostelny et al , J Immunol 148(5) 1547 1553 (1992) The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab portions of two different antibodies by gene fusion The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers This method can also be utilized for the production of antibody homodimers The "diabody " technology described by Holhnger et al , Proc Natl Acad Sci USA 90 6444 6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments The fragments comprise a heavy chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain Accordingly, the V„ and V_I domains of one fragment are forced to pair with the complementary V, and V„ domains of another fragment thereby forming two antigen-binding sites Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported See, Gruber et al , J Immunol 152 5368 (1994)

Antibodies with more than two valencies are contemplated For example, tπspecific antibodies can be prepared Tutt et al , J Immunol 147 60 (1991 ) Exemplary bispecific antibodies may bind to two different epitopes on a given PRO polypeptide herein Alternatively , an anti-PRO polypeptide arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e g CD2 CD3, CD28, or B7) or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular PRO poly peptide Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a particular PRO polypeptide These antibodies possess a PRO-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA. or TETA Another bispecific antibody of interest binds the PRO polypeptide and further binds tissue factor (TF)

5 Heteroconiugate Antibodies

Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676 980], and for treatment of HIV infection [WO 91 /00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry including those involving crosshnking agents For example, immunotoxms may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include lminothiolate and methyl-4- mercaptobutyπmidate and those disclosed, for example, in U S Patent No 4,676,980

6 Effector Function Engineering

It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e g , the effectiveness of the antibody in treating cancer For example, cysteine resιdue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region The homodimeric antibody thus generated may have improved mtemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) See Caron et al ,

J Exp Med . 176 1191-1195 (1992) and Shopes. J Immunol . 148 2918-2922 (1992) Homodimeric antibodies with enhanced anti tumor activity may also be prepared using heterobifunctional cross linkers as described in Wolff et al Cancer Research. 53 2560-2565 (1993) Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities See Stevenson et al , Anti Cancer Drug Design. 3 219-230 (1989)

7 Immunoconiugates

The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e g , an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i e , a radioconjugate)

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain nonbmdmg active fragments of diphtheria toxm exotoxin A chain (from Pseudomonas aeruginosa). πcin A chain, abπn A chain modeccin A chain, alpha-sarcm Aleuntes fordu proteins, dianthm proteins PhMolaca americana proteins (PAPI PAPII. and PAP S), momordica charantia inhibitor curcin. crotm, sapaonaria officinahs inhibitor, gelonm, mitogellin. restrictocin, phenomycm, enomycm and the tπcothecenes A variety of radionuc des are available for the production of radioconjugated antibodies Examples include²¹²Bι,^nιI, ' ''In, ^Y, and¹⁸⁶Re Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succimmidyl 3 (2-pyπdyldιthιol) propionate (SPDP), lm othiolane (IT), bifunctional derivatives of lmidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccimmidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p azidobenzoy 1) hexanediamine), bis diazomum derivatives (such as bιs-(p-dιazomumbenzoyl)- ethylenediamine), dnsocyanates (such as tolyene 2,6-dnsocyanate), and bis-active fluorine compounds (such as l ,5-dιfluoro-2,4 dimtrobenzene) For example, a πcm mimunotoxm can be prepared as described in Vitetta et al , Science. 238 1098 (1987) Carbon- 14-labeled 1 ιsothιocyanatobenzyl-3-methyldιethylene tπammepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody See W094/ 1 1026 In another embodiment, the antibody may be conjugated to a ' receptor" (such streptav idin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e g , avidin) that is conjugated to a cytotoxic agent (e g , a radionucleotide)

8 Immunol lposomes

The antibodies disclosed herein may also be formulated as immunoliposomes Liposomes containing the antibody are prepared by methods known in the art, such as described m Epstein et al , Proc Natl Acad Sci USA. 82 3688 (1985), Hwang et al , Proc Natl Acad Sci USA. 77 4030 (1980), and U S Pat Nos 4,485,045 and 4,544,545 Liposomes with enhanced circulation time are disclosed in U.S Patent No 5,013,556

Particularly useful liposomes can be generated by the reverse-phase evaporation method ith a lipid composition comprising phosphatidylchohne, cholesterol, and PEG derivatized phosphatidylethanolamine (PEG-PE) Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al , J Biol Chem . 257 286-288 (1982) via a disulfide-mterchange reaction A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome See Gabizon et al , J National Cancer Inst . 81(19) 1484 (1989)

M Pharmaceutical Compositions The active molecules of the invention. PRO polypeptides and antibodies, as well as other molecules identified by the screening assays disclosed above, can be administered for the treatment of immune related diseases, m the form of pharmaceutical compositions Therapeutic formulations of the active molecule, preferably a PRO pol) peptide or anti-PRO antibody of the invention are prepared for storage by mixing the active molecule hav mg the desired degree of purity with optional pharmaceutically acceptable carriers excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed [1980]), in the form of lyophilized formulations or aqueous solutions Acceptable carriers excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate citrate, and other organic acids, antioxidants including ascorbic acid and methionme, preservatives (such as octadecyldimethvlbenzyl ammonium chloride, hexamethomum chloride benzalkomum chloride benzethomum chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcmol cyclohexanol, 3 pentanol, and m cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvmylpyrrolidone, amino acids such as glycme, glutamine, asparagine, histidme, arginine, or lysine, monosaccharides, disacchandes, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counter ions such as sodium, metal complexes (e g Zn protein complexes), and/or non ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG)

Compounds identified by the screening assays of the present inv ention can be formulated in an analogous manner, using standard techniques well known in the art

Lipofections or liposomes can also be used to deliver the polypeptide, antibody, or an antibody fragment, into cells Where antibody fragments are used, the smallest inhibitory fragment which specifically binds to the binding domain of the target protein is preferred For example, based upon the variable region sequences of an antibody, peptide molecules can be designed which retain the ability to bind the target protein sequence Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, e g Marasco et al , Proc Natl Acad Sci USA 90, 7889-7893 [1993])

The formulation herein may also contain more than one active polypeptide or compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent Such molecules are suitably present in combination in amounts that are effective for the purpose intended

The active molecules may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by mterfacial polymerization, for example hydroxymethylcellulose or gelatm-microcapsules and poly-(methylmethacylate) microcapsules respecm ely , in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions nano-particles and nanocapsules) or in macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed (1980) The formulations to be used for in vivo administration must be sterile This is readily accomplished by filtration through sterile filtration membranes Sustained-release preparations may be prepared Suitable examples of sustained-release preparations include semipermeable matrices of solid hy drophobic polymers containing the antibody, which matrices are in the form of shaped articles, e g films, or microcapsules Examples of sustamed-release matrices include polyesters, hydrogels (for example. poly(2-hydroxyethyl-methacrylate), or poly(vmy alcohol)). polylactides (U.S. Pat No 3,773,919), copolymers of L-glutamic acid and g ethyl-L-glutamate, non-degradable ethylene-vmyl acetate, degradable lactic acid glycolic acid copolymers such as the LUPRON DEPOT™

(injectable microspheres composed of lactic acid-glycohc acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyπc acid While polymers such as ethylene-vmyl acetate and lactic acid-glycohc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moismre at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity Rational strategies can be devised for stabilization depending on the mechanism involved For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-di sulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

N Methods of Treatment

It is contemplated that the PRO polypeptides, antibodies and other active compounds of the present invention may be used to treat various immune related diseases and conditions, such as T cell mediated diseases, including those characterized by infiltration of inflammatory cells mto a tissue, stimulation of T-cell proliferation, inhibition of T-cell proliferation, increased or decreased vascular permeability or the inhibition thereof

Exemplary conditions or disorders to be treated with the PRO polypeptides, antibodies and other compounds of the invention, include, but are not limited to systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vascuhtis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuπa), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune -mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mel tus, immune-mediated renal disease (glomerulonephπtis, tubulomterstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis: Crohn's disease) , gluten-sensitive enteropathy , and Whipple ' s disease , autoimmune or immune-mediated skin diseases including bullous skm diseases, erythema multiforme and contact dermatitis psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophihc pneumonias, idiopathic pulmonar) fibrosis and hypersensitivity pneumonitis. transplantation associated diseases including graft rejection and graft -versus-host-disease

In systemic lupus erythematosus, the central mediator of disease is the production of auto-reactive antibodies to self proteins/tissues and the subsequent generation of immune-mediated inflammation Though T lymphocytes have not been shown to be directly involved m tissue damage, T lymphocytes are required for the development of auto-reactive antibodies The genesis of the disease is thus T lymphocyte dependent Multiple organs and systems are affected clinically including kidney, lung, musculoskeletal system, mucocutaneous, eye, central nervous system, cardiovascular system, gastrointestinal tract, bone marrow and blood Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease that mainly involves the synovial membrane of multiple joints with resultant injury to the articular cartilage The pathogenesis is T lymphocyte dependent and is associated with the production of rheumatoid factors, auto-antibodies directed against self IgG, with the resultant formation of immune complexes that attain high levels in ]omt fluid and blood These complexes in the joint may induce the marked infiltrate of lymphocytes and monocytes into the synovium and subsequent marked synovial changes, the joint space/fluid if infiltrated by similar cells with the addition of numerous neutrophils Tissues affected are primarily the joints, often m symmetrical pattern. However, extra-articular disease also occurs in two major forms. One form is the development of extra-articular lesions with ongoing progressive joint disease and typical lesions of pulmonary fibrosis, vascuhtis, and cutaneous ulcers The second form of extra-articular disease is the so called Felty ' s syndrome which occurs late in the R A disease course , sometimes after joint disease has become quiescent, and involves the presence of neutropenia, thrombocytopenia and splenomegaly. This can be accompanied by vascuhtis in multiple organs with formations of infarcts, skin ulcers and gangrene Patients often also develop rheumatoid nodules in the subcutis tissue overlying affected joints, the nodules late stage have necrotic centers surrounded by a mixed inflammatory cell infiltrate Other manifestations which can occur in RA include pericarditis, pleuπtis, coronary arteπtis, intestitial pneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, and rhematoid nodules

Juvenile chronic arthritis is a chronic idiopathic inflammatory disease which begins often at less than 16 years of age Its phenotype has some similarities to RA, some patients which are rhematoid factor positive are classified as juvenile rheumatoid arthritis The disease is sub-classified mto three major categories: pauciarticular, polyaπicular, and systemic The arthritis can be severe and is typically destructive and leads to joint ankylosis and retarded growth Other manifestations can include chronic anterior uveitis and systemic amyloidosis

Spondyloarthropathies are a group of disorders with some common clinical features and the common association with the expression of HLA-B27 gene product The disorders include, ankylosing sponyhtis. Reiter's syndrome (reactive arthritis), arthritis associated with inflammatory bowel disease, spondylitis associated with psoriasis, juvenile onset spondyloarthropathy and undifferentiated spondyloarthropathy Distinguishing features include sacroileitis with or without spondylitis; inflammatory asymmetric arthritis, association with HLA B27 (a serologically defined allele of the HLA B locus of class I MHC ) ocular inflammation, and absence of autoantibodies associated with other rheumatoid disease The cell most implicated as key to induction of the disease is the CD8⁺ T lymphocyte, a cell which targets antigen presented by class I MHC molecules CD8" T cells may react against the class I MHC allele HLA B27 as if it were a foreign peptide expressed by MHC class I molecules It has been hypothesized that an epitope of HLA-B27 may mimic a bacterial or other microbial antigenic epitope and thus induce a CD8^" T cells response

Systemic sclerosis (scleroderma) has an unknown etiology A hallmark of the disease is induration of the skm, likely this is induced by an active inflammatory process Scleroderma can be localized or systemic, vascular lesions are common and endothelial cell injury in the microvasculature is an early and important event in the development of systemic sclerosis, the vascular injury may be immune mediated An immunologic basis is implied by the presence of mononuclear cell infiltrates in the cutaneous lesions and the presence of anti nuclear antibodies many patients ICAM 1 is often upregulated on the cell surface of fϊbroblasts in skin lesions suggesting that T cell interaction with these cells may have a role in the pathogenesis of the disease Other organs involved include the gastrointestinal tract smooth muscle atrophy and fibrosis resulting in abnormal peπstalsis/motihty, kidney concentric subendothehal intimal proliferation affecting small arcuate and interlobular arteries with resultant reduced renal cortical blood flow, results in proteinuπa, azotemia and hypertension, skeletal muscle atrophy, interstitial fibrosis, inflammation, lung interstitial pneumonitis and interstitial fibrosis, and heart contraction band necrosis, scarπng/fibrosis

Idiopathic inflammatory myopathies including dermatomyositis, polymyositis and others are disorders of chronic muscle inflammation of unknown etiology resulting m muscle weakness Muscle injury/inflammation is often symmetric and progressive Autoantibodies are associated with most forms These myositis-specific autoantibodies are directed against and inhibit the function of components, proteins and RNA's, involved in protein synthesis

Sjogren's syndrome is due to immune-mediated inflammation and subsequent functional destruction of the tear glands and salivary glands The disease can be associated with or accompanied by inflammatory connective tissue diseases The disease is associated with autoantibody production agamst Ro and La antigens, both of which are small RNA-protein complexes Lesions result in keratoconjunctivitis sicca, xerostomia, with other manifestations or associations including bilary cirrhosis, peripheral or sensory neuropathy, and palpable purpura Systemic vascuhtis are diseases in which the primary lesion is inflammation and subsequent damage to blood vessels which results ischemia/necrosis/degeneration to tissues supplied by the affected vessels and eventual end organ dysfunction in some cases Vascuhtides can also occur as a secondary lesion or sequelae to other immune-inflammatory mediated diseases such as rheumatoid arthritis, systemic sclerosis, etc , particularly in diseases also associated with the formation of immune complexes Diseases in the primary systemic vascuhtis group include systemic necrotizing vascuhtis polyarteritis nodosa allergic angntis and granulomatosis, polyangntis, Wegener's granulomatosis, lymphomatoid granulomatosis, and giant cell arteπtis Miscellaneous vascuhtides include mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease) isolated CNS vascuhtis, Behet s disease thromboangntis obhterans (Buerger s disease) and cutaneous necrotizmg venuhtis The pathogenic mechanism of most of the types of vascuhtis listed is believed to be primarily due to the deposition of immunoglobulin complexes in the vessel wall and subsequent induction of an inflammatory response either via ADCC complement activ ation, or both

Sarcoidosis is a condition of unknown etiology which is characterized by the presence of epithehoid granulomas in nearly any tissue in the body, involvement of the lung is most common The pathogenesis involves the persistence of activated macrophages and lymphoid cells at sites of the disease with subsequent chronic sequelae resultant from the release of locally and systemically active products released by these cell types

Autoimmune hemolytic anemia including autoimmune hemolytic anemia, immune pancytopenia, and paroxysmal noctural hemoglobinuπa is a result of production of antibodies that react with antigens expressed on the surface of red blood cells (and in some cases other blood cells including platelets as well) and is a reflection of the removal of those antibody coated cells via complement mediated lysis and/or

ADCC/Fc receptor-mediated mechanisms

In autoimmune thrombocytopenia including thrombocytopenic purpura, and immune-mediated thrombocytopenia m other clinical settings, platelet destruction/removal occurs as a result of either antibody or complement attaching to platelets and subsequent removal by complement lysis, ADCC or FC-receptor mediated mechanisms

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, and atrophic thyroiditis, are the result of an autoimmune response against thyroid antigens with production of antibodies that react with proteins present in and often specific for the thyroid gland Experimental models exist including spontaneous models rats (BUF and BB rats) and chickens (obese chicken strain), inducible models immunization of animals with either thyroglobulm, thyroid microsomal antigen (thyroid peroxidase)

Type I diabetes melhtus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islet β cells, this destruction is mediated by auto-antibodies and auto-reactive T cells Antibodies to insulin or the insulin receptor can also produce the phenotype of insulin-non-responsiveness

Immune mediated renal diseases, including glomerulonephπtis and tubulointerstmal nephritis, are the result of antibody or T lymphocyte mediated injury to renal tissue either directly as a result of the production of autoreactive antibodies or T cells against renal antigens or indirectly as a result of the deposition of antibodies and/or immune complexes in the kidney that are reactive against other, non-renal antigens Thus other immune mediated diseases that result in the formation of immune-complexes can also induce immune mediated renal disease as an indirect sequelae Both direct and indirect immune mechanisms result in inflammatory response that produces/induces lesion development in renal tissues with resultant organ function impairment and in some cases progression to renal failure Both humoral and cellular immune mechanisms can be involved in the pathogenesis of lesions Demyelinating diseases of the central and peripheral nervous systems including Multiple Sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barr syndrome, and Chronic Inflammatory Demyelinating Polyneuropathy, are believed to have an autoimmune basis and result in nerve demyehnation as a result of damage caused to ohgodendrocytes or to myelm directly In MS there is evidence to suggest that disease induction and progression is dependent on T lymphocytes Multiple Sclerosis is a demyelinating disease that is T lymphocyte-dependent and has either a relapsing remitting course or a chronic progressive course The etiolog) is unknown however, viral infections, genetic predisposition, environment, and autoimmunity all contribute Lesions contain infiltrates of predominantly T lymphocyte mediated, microghal cells and infiltrating macrophages, CD4⁺T lymphocytes are the predominant cell type at lesions The mechanism of ohgodendrocyte cell death and subsequent demyehnation is not known but is likely T lymphocyte driven

Inflammatory and Fibrotic Lung Disease, including Eosinophihc Pneumonias, Idiopathic Pulmonar) Fibrosis, and Hypersensitivity Pneumonitis may involve a deregulated immune-inflammatory response Inhibition of that response would be of therapeutic benefit

Autoimmune or Immune-mediated Skm Disease including Bullous Skm Diseases, Erythema Multiforme, and Contact Dermatitis are mediated by auto-antibodies, the genesis of which is T lymphocyte-dependent

Psoriasis is a T lymphocyte-mediated inflammatory disease Lesions contain infiltrates of T lymphocytes, macrophages and antigen processing cells, and some neutrophils

Allergic diseases, including asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity; and urticaria are T lymphocyte dependent These diseases are predominantly mediated by T lymphocyte induced inflammation, IgE mediated-inflammation or a combination of both

Transplantation associated diseases, including Graft reiection and Graft- Versus-Host-Disease (GVHD) are T lymphocyte-dependent, inhibition of T lymphocyte function is ameliorative

Other diseases in which intervention of the immune and/or inflammatory response have benefit are infectious disease including but not limited to viral infection (including but not limited to AIDS, hepatitis A, B, C, D, Eand herpes) bacterial infection, fungal infections, and protozoal and parasitic infections (molecules (or derivatives/agonists) which stimulate the MLR can be utilized therapeutically to enhance the immune response to infectious agents) , diseases of immunodeficiency (molecules/deπvatives/agonists) which stimulate the MLR can be utilized therapeutically to enhance the immune response for conditions of inherited, acquired, infectious induced (as in HIV infection), or latrogemc (I e as from chemotherapy) immunodeficiency), and neoplasia

It has been demonstrated that some human cancer patients develop an antibody and/or T lymphocyte response to antigens on neoplastic cells It has also been shown in animal models of neoplasia that enhancement of the immune response can result m rejection or regression of that particular neoplasm Molecules that enhance the T lymphocyte response in the MLR have utility in vivo in enhancing the immune response against neoplasia Molecules which enhance the T lymphocyte prohferative response in the MLR (or small molecule agonists or antibodies that affected the same receptor m an agonistic fashion) can be used therapeutically to treat cancer Molecules that inhibit the lymphocyte response in the MLR also function in vivo during neoplasia to suppress the immune response to a neoplasm, such molecules can either be expressed by the neoplastic cells themselves or their expression can be induced b) the neoplasm in other cells Antagonism of such inhibitory molecules (either with antibody small molecule antagonists or other measn) enhances immune-mediated tumor rejection

Additionally , inhibition of molecules with promflammator) properties ma) have therapeutic benefit in reperfusion injur) , stroke myocardial infarction, atherosclerosis, acute lung miur) , hemorrhagic shock, burn, sepsis/septic shock, acute tubular necrosis, endometπosis, degenerative joint disease and pancreatis The PRO polypeptides and compounds of the present invention, e g PRO polypeptides or antibodies, are administered to a mammal, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, lntracerobrospmal, subcutaneous, intra articular, intrasynovial, mtrathecal, oral, topical, or inhalation (intranasal, intrapulmonary) routes Intravenous or inhaled administration of polypeptides and antibodies is preferred

In immunoadjuvant therapy, other therapeutic regimens, such administration of an anti-cancer agent, may be combined with the administration of the proteins, antibodies or compounds of the instant invention For example, the patient to be treated with a the immunoadjuvant of the invention may also receive an anti-cancer agent (chemotherapeutic agent) or radiation therapy Preparation and dosmg schedules for such chemotherapeutic agents may be used according to manufacturers instructions or as determined empirically by the skilled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed , M C Perry, Williams & Wilkins, Baltimore, MD (1992) The chemotherapeutic agent may precede, or follow administration of the immunoadjuvant or may be given simultaneously therewith Additionally, an anti-oestrogen compound such as tamoxifen or an anti -progesterone such as onapπstone (see, EP 616812) may be given in dosages known for such molecules

It may be desirable to also administer antibodies against other immune disease associated or tumor associated antigens, such as antibodies which bind to CD20, CD1 la, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothehal factor (VEGF) Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be coadmmistered to the patient Sometimes, it may be beneficial to also administer one or more cytokines to the patient In one embodiment, the polypeptides of the invention are coadmmistered with a growth inhibitory agent For example, the growth inhibitory agent may be administered first, followed by a PRO polypeptide of the invention However, simultaneous administration or administration first is also contemplated Suitable dosages for the growth inhibitory agent are those presently used and may be lowered due to the combined action (synergy) of the growth inhibitory agent and the PRO polypeptide of the invention

For the treatment or reduction in the severity of immune related disease, the appropriate dosage of an a PRO polypeptide or compound of the invention will depend on the type of disease to be treated, as defined above, the seventy and course of the disease whether the agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the compound, and the discretion of the attending physician The compound is suitably administered to the patient at one time or over a series of treatments For example, depending on the type and seventy of the disease, about 1 μg/kg to 15 mg/kg (e g 0 l-20mg/kg) of polypeptide or antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion A typical dail) dosage might range from about 1 μg/kg to 100 mg/kg or more depending on the factors mentioned above For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs However, other dosage regimens may be useful The progress of this therapy is easily monitored by conventional techniques and assays

O Articles of Manufacture

In another embodiment of the invention, an article of manufacture containing materials useful for the diagnosis or treatment of the disorders described above is provided The article of manufacture comprises a container and a label Suitable containers include, for example, bottles, vials, syringes, and test tubes The containers may be formed from a variety of materials such as glass or plastic The container holds a composition which is effective for diagnosing or treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) The active agent the composition is usually a PRO polypeptide or an antibody of the invention The label on, or associated with, the container indicates that the composition is used for diagnosing or treating the condition of choice The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use

P Diagnosis and Prognosis of Immune Related Disease

Cell surface proteins, such as proteins which are overexpressed in certain immune related diseases, are excellent targets for drug candidates or disease treatment The same proteins along with secreted proteins encoded by the genes amplified m immune related disease states find additional use in the diagnosis and prognosis of these diseases For example, antibodies directed against the protein products of genes amplified in multiple sclerosis, rheumatoid arthritis, or another immune related disease, can be used as diagnostics or prognostics For example, antibodies, including antibody fragments, can be used to qualitative!) or quantitatively detect the expression of proteins encoded by amplified or overexpressed genes (' marker gene products") The antibody preferably is equipped with a detectable, e g fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluoπmetry, or other techniques known m the art These techniques are particularly suitable, if the overexpressed gene encodes a cell surface protein Such binding assays are performed essentially as decπbed above

In situ detection of antibody binding to the marker gene products can be performed, for example, by lmmunofluorescence or immunoelectron microscopy For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it preferabh by overlaying the antibody on a biological sample This procedure also allows for determining the distribution ot the marker gene product in the tissue examined It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection

The following examples are offered for illustrative purposes onl , and are not intended to limit the scope of the present invention m any way All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety

Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and m the following textbooks Sambrook et al , Molecular Cloning A Laboratory Manual. Cold Spring Harbor Press N Y , 1989, Ausubel et al , Current Protocols in Molecular Biology. Green Publishing Associates and Wiley Interscience N Y , 1989, Innis et al , PCR Protocols A Guide to Methods and Applications Academic Press, Ine , N Y , 1990, Harlow et al , Antibodies A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, 1988, Gait,

Oligonucleotide Synthesis. IRL Press, Oxford, 1984, Freshney, Animal Cell Culture. 1987, Cohgan et al , Current Protocols m Immunology. 1991

EXAMPLES Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA

EXAMPLE 1 Isolation of cDNA clones Encoding Human PRQ526. PRQ719. PRQ725. PRO1031 or

PRO200

A Isolation of cDNA clones Encoding Human PRQ526

Extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g GenBank) and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzymology 266 460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence Those comparisons resulting m a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington. Seattle, Washington) An initial consensus DNA sequence was assembled relativ e to other EST sequences using phrap In addition, the initial consensus DN A sequence was extended using repeated cycles of BLAST and phrap to extend the initial consensus sequence as far as possible using the sources of EST sequences discussed above

Based on the final consensus sequence obtained, oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest and 2) for use as probes to isolate a clone of the full-length coding sequence for PR0526 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100 1000 bp in length The probe sequences are typically 40 55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1 5kbp In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs

A pair of PCR primers (forward and reverse) were synthesized forward PCR primer 5' TGGCTGCCCTGCAGTACCTCTACC-3 (SEQ ID NO 1 1), and reverse PCR primer 5' CCCTGCAGGTCATTGGCAGCTAGG 3 (SEQ ID NO 12)

Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus sequence which had the following nucleotide sequence hybridization probe

5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC 3 ' (SEQ ID NO 13) In order to screen several libraries for a source of a full-length clone, DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PR0526 gene using the probe oligonucleotide and one of the PCR primers RNA for construction of the cDNA libraries was isolated from human fetal liver tissue (LIB228) The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)) in the unique Xhol and Notl sites DNA sequencing of the clones isolated as described abo\ e gave the full length DNA sequence for

PR0526 [herein designated as DNA44184-1319] (SEQ ID NO 1 ) and the derived protein sequence for PR0526

The entire nucleotide sequence of DNA44184-1319 is shown in Figure 1 (SEQ ID NO 1 ) Clone

DNA44184 1319 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 514-516 and ending at the stop codon at nucleotide positions 1933 1935 (Figure 1 ) The predicted polypeptide precursor is 473 amino acids long (Figure 2) The full length PR0526 protein shown in Figure 2 has an estimated molecular weight of about 50,708 daltons and a pl of about 9 28 Clone DNA44184-1319 has been deposited with the ATCC on March 26. 1998 It is understood that the clone contains the actual sequence, whereas the sequences presented herein are representative based on current sequencing techniques

Analysis of the amino acid sequence of the full-length PR0526 polypeptide suggests that portions of it possess significant homology to the leucine repeat rich proteins including ALS, SLIT, carboxypeptidase and platelet glycoprotein V thereby indicating that PR0526 is a novel protein which is involved in protein-protem interactions Still analyzing SEQ ID NO.2, the signal peptide sequence is at about amino acids 1-26 A leucine zipper pattern is at about ammo acids 135-156 A glycosaminoglycan attachment is at about amino acids 436-439 N-glycosy ation sites are at about ammo acids 82-85, 179-182, 237-240 and 423-426. A von Willebrand factor (VWF) type C domaιn(s) is found at about amino acids 411-425 The skilled artisan can understand which nucleotides correspond to these ammo acids based on the sequences provided herein

B Isolation of cDNA clones Encoding Human PRQ719

The extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzvmologv 266 460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled into consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)

A consensus DNA sequence was assembled relative to other EST sequences using phrap This consensus sequence is herein designated DNA44851 Based on the DNA44851 consensus sequence, oligonucleotides were synthesized 1) to identify by PCR a cDNA library that contained the sequence of interest, and 2) for use as probes to isolate a clone of the full-length coding sequence for PR071 Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length. The probe sequences are typically 40-55 bp in length In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNA from the libraries was screened by PCR amplification, as per Ausubel et al , Current Protocols in Molecular Biology, with the PCR primer pair A positive library was then used to isolate clones encoding the gene of interest using the probe oligonucleotide and one of the primer pairs

A pair of PCR primers (forward and reverse) were synthesized forward PCR nnmer. 5'-GTGAGCATGAGCGAGCCGTCCAC-3 ' (SEQ ID NO- 14) reverse PCR nπmer 5 '-GCTATTACAACGGTTCTTGCGGCAGC-3 ' (SEQ ID NO- 15) Additionally , a synthetic oligonucleotide hybridization probe was constructed from the consensus DN A44851 sequence which had the following nucleotide sequence hybridization probe 5'-TTGACTCTCTGGTGAATCAGGACAAGCCGAGTTTTGCCTTCCAG 3 (SEQ ID NO 16)

In order to screen several libraries for a source of a full-length clone DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PR0719 gene using the probe oligonucleotide and one of the PCR primers

RNA for construction of the cDNA libraries was isolated from human placenta tissue (LIB90) The cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commercially available reagents such as those from Invitrogen, San Diego, CA The cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD, pRK5B is a precursor of pRK5D that does not contain the Sfil site see, Holmes et al , Science.

253 1278-1280 (1991)) in the unique Xhol and Notl sites

DNA sequencing of the clones isolated as described above gave the full length DNA sequence for PR0719 [herein designated as DNA49646-1327] (SEQ ID NO 3) and the derived protein sequence for

PR0719

The entire nucleotide sequence of DNA49646-1327 is shown in Figure 3 (SEQ ID NO 3) Clone DNA49646-1327 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 223-225 and ending at the stop codon at nucleotide positions 1285-1287 (Figure 3) The predicted polypeptide precursor is 354 amino acids long (Figure 4) The full-length PR0719 protein shown in Figure 4 has an estimated molecular weight of about 39,362 daltons and a pi of about 8 35 Analysis of the full length PR 0719 sequence evidences the presence of a signal peptide from about amino acid 1 to about ammo acid 16, a hpase associated serine containing active site at about amino acid 163 to about ammo acid 172, and two potential N-glycosylation sites from about amino acid 80 to about ammo acid 83 and about amino acid 136 to about amino acid 139 Clone DNA49646-1327 has been deposited with ATCC on March

26, 1998 and is assigned ATCC deposit no 209705

Analysis of the amino acid sequence of the full-length PR0719 polypeptide suggests that it possesses significant sequence similarity to the lipoprotein hpase H protein, thereby indicating that PR0719 may be a novel lipoprotein hpase homolog More specifically, an analysis of the Dayhoff database (version 35 45 SwissProt 35) evidenced significant homology between the PR0719 amino acid sequence and the following

Dayhoff sequences, LIPL_HUMAN, LIPH HUMAN, D83548 1 , A24059 1 , P R30740, D88666 1 , A43357, A46696, B43357 and A49488

C Isolation of cDNA clones Encoding Human PRQ725 1 Preparation of oligo dT primed cDNA library mRNA was isolated from human fetal bram tissue using reagents and protocols from Invitrogen, San Diego, CA (Fast Track 2) This RNA was used to generate an oligo dT primed cDNA library m the vector pRK5D using reagents and protocols from Life Technologies Gaithersburg MD (Super Script Plasmid System) In this procedure, the double stranded cDNA was sized to greater than 1000 bp and the Sall/Notl nkered cDNA was cloned mto Xhol/Notl cleaved v ector pRK5D is a cloning vector that has an sp6 transcription initiation site followed by an Sfil restriction enzyme site preceding the Xhol/Notl cDNA cloning sites

2 Preparation of random primed cDNA library

A secondary cDNA library was generated order to preferentially represent the 5' ends of the primary cDNA clones Sp6 RNA was generated from the primary library and this RNA was used to generate a random primed cDNA library in the vector pSST-AMY 0 using reagents and protocols from Life Technologies (Super Script Plasmid System, referenced above) In this procedure the double stranded cDNA was sized to 500-1000 bp, hnkered with blunt to Notl adaptors, cleaved with Sfil, and cloned into Sfil/Notl cleaved vector pSST AMY 0 is a cloning vector that has a yeast alcohol dehv drogenase promoter preceding the cDNA cloning sites and the mouse amylase sequence (the mature sequence without the secretion signal) followed by the yeast alcohol dehydrogenase terminator, after the cloning sues Thus, cDNAs cloned into this vector that are fused in frame with amylase sequence will lead to the secretion of amylase from appropriately transfected yeast colonies

3 Transformation and Detection

DNA from the library described above was chilled on ice to which was added electrocompetent DH10B bacteria (Life Technologies, 20 ml) The bacteria and vector mixture was then electroporated as recommended by the manufacturer Subsequently, SOC media (Life Technologies, 1 ml) was added and the mixture was incubated at 37°C for 30 minutes The transformants were then plated onto 20 standard 150 mm LB plates containing ampicilhn and incubated for 16 hours (37°C) Positive colonies were scraped off the plates and the DNA was isolated from the bacterial pellet using standard protocols, e g CsCl gradient The purified DNA was then carried on to the yeast protocols below

The yeast methods were divided into three categories (1 ) Transformation of yeast with the plasmid/cDNA combined vector, (2) Detection and isolation of yeast clones secreting amylase, and (3) PCR amplification of the insert directly from the yeast colony and purification ot the DNA for sequencing and further analysis The yeast strain used was HD56-5A (ATCC 90785) This strain has the following genotype MAT alpha, ura3 52, leu2-3, leu2 1 12, hιs3-l l , hιs3 15, MAL + , SUC + , GAL +

Transformation was performed based on the protocol outlined by Gietz et al , Nucl Acid Res . 20 1425 (1992) Transformed cells were then inoculated from agar mto YEPD complex media broth (100 ml) and grown overnight at 30^CC The YEPD broth was prepared as described in Kaiser et al , Methods in Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p 207 ( 1994) The overnight culture was then diluted to about 2 x IO⁶ cells/ml (approx OD600 = 0 1) into fresh YEPD broth (500 ml) and regrown to 1 x 10⁷ cells/ml (approx OD600 = 0 4-0 5) The cells ere then harvested and prepared for transformation by transfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5 minutes the supernatant discarded and then resuspended mto sterile water, and centrifuged again in 50 ml falcon tubes at 3 500 rpm in a Beckman GS 6KR centrifuge The supernatant was discarded and the cells were subsequently washed with LiAc/TE (10 ml 10 mM Tris HCl 1 mM EDTA pH 7 5, 100 mM Lι,OOCCH,) and resuspended into LiAc/TE (2 5 ml) Transformation took place b\ mixing the prepared cells ( 100 μl) with freshly denatured single stranded salmon testes DNA (Lofstrand Labs, Gaithersburg MD) and transforming DNA (1 μg, vol < 10 μl) in microfuge tubes The mixture was mixed briefly by vortexmg, then 40% PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris HCl, 1 mM EDTA 100 mM Lι,OOCCH„ pH 7 5) was added This mixture was gentl) mixed and incubated at 30°C while agitating for 30 minutes The cells were then heat shocked at 42°C for 15 minutes, and the reaction vessel centrifuged m a microfuge at 12,000 rpm for 5 10 seconds decanted and resuspended into TE (500 μl, 10 mM Tris HCl 1 mM EDTA, pH 7 5) followed by recentπfugation The cells were then diluted into TE (1 ml) and aliquots (200 μl) were spread onto the selective media previously prepared in 150 mm growth plates (VWR)

Alternatively instead of multiple small reactions, the transformation was performed using a single large scale reaction wherein reagent amounts were scaled up accordingly

The selective media used was a synthetic complete dextrose agar lacking uracil (SCD Ura) prepared as described in Kaiser et al , Methods in Yeast Genetics Cold Spring Harbor Press, Cold Spring Harbor, NY, p 208-210 (1994) Transformants were grown at 30°C for 2-3 days

The detection of colonies secreting amylase was performed by including red starch m the selective growth media Starch was coupled to the red dye (Reactive Red 120, Sigma) as per the procedure described by Biely et al , Anal Biochem . 172 176-179 (1988) The coupled starch was incorporated mto the

SCD Ura agar plates at a final concentration of 0 15 % (w/v), and was buffered with potassium phosphate to a pH of 7 0 (50 100 mM final concentration)

The positive colonies were picked and streaked across fresh selective media (onto 150 mm plates) in order to obtain well isolated and identifiable single colonies Well isolated single colonies positive for amylase secretion were detected by direct incorporation of red starch into buffered SCD Ura agar Positive colonies were determined by their ability to break down starch resulting in a clear halo around the positive colony visualized directly

4 Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by a toothpick and diluted into sterile water (30 μl) in a 96 well plate At this time, the positive colonies were either frozen and stored for subsequent analysis or immediately amplified An aliquot of cells (5 μl) was used as a template for the PCR reaction in a 25 μl volume containing 0 5 μl Klentaq (Clontech, Palo Alto, CA), 4 0 μl 10 mM dNTP s (Perkin Elmer-Cetus), 2 5 μl Klentaq buffer (Clontech), 0 25 μl forward oligo 1 , 0 25 μl reverse oligo 2,

12 5 μl distilled water The sequence of the forward oligonucleotide 1 was

5 -TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3 (SEQ ID NO 17) The sequence of reverse oligonucleotide 2 was

5 -CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT 3 ' (SEQ ID NO 18)

PCR was then performed as follows a Denature 92°C 5 minutes

3 cycles of Denature 92°C, 30 seconds

Anneal 59°C 30 seconds

Extend 72°C, 60 seconds

3 cycles of Denature 92°C, 30 seconds

Anneal 57°C, 30 seconds

Extend 72°C, 60 seconds

25 cycles of Denature 92°C, 30 seconds

Anneal 55°C, 30 seconds

Extend 72°C, 60 seconds

Hold 4°C

The underlined regions of the oligonucleotides annealed to the ADH promoter region and the amylase region, respectively, and amplified a 307 bp region from vector pSST-AMY 0 when no insert was present Typically, the first 18 nucleotides of the 5' end of these oligonucleotides contained annealing sites for the sequencing primers, wherein these sequences may differ depending upon the sequencing primers employed Thus, the total product of the PCR reaction from an empty vector was 343 bp However, signal sequence-fused cDNA resulted in considerably longer nucleotide sequences Following the PCR, an aliquot of the reaction (5 μl) was examined by agarose gel electrophoresis in a 1 % agarose gel using a Tπs-Borate-EDTA (TBE) buffering system as described by Sambrook et al , supra Clones resulting in a single strong PCR product larger than 400 bp were further analyzed by DNA sequencing after purification with a 96 Qiaquick PCR clean-up column (Qiagen Ine , Chatsworth, CA)

5 Identification of Full-length Clone

A cDNA sequence isolated in the above screen was then compared to a variety of expressed sequence tag (EST) databases which included public EST databases (e g , GenBank) and a proprietary EST DNA database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA) to identify existing homologies The homology search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods in Enzvmology 266 460-480 (1996)) Those comparisons resulting in a BLAST score of 70 (or in some cases 90) or greater that did not encode known proteins were clustered and assembled mto consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington)

Based on the consensus sequence, oligonucleotide probes were generated and used to screen a human fetal brain (LIB 153) library prepared as described above The cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does not contain the Sfil site, see, Holmes et al , Science, 253 1278-1280 (1991)), and the cDNA size cut was less than 2800 bp PCR primers (forward and reverse) were sy nthesized forward PCR primer 5'-CCAAACTCACCCAGTGAGTGTGAGC 3 (SEQ ID NO 19) reverse PCR primer 5' TGGGAAATCAGGAATGGTGTTCTCC 3' (SEQ ID NO 20) Additionall) a synthetic oligonucleotide hybridization probe was constructed which had the following nucleotide sequence hybridization probe

5'-CTTGTTTTCACCATTGGGCTAACTTTGCTGCTAGGAGTTCAAGCCATGCC 3 ' (SEQ ID NO 21 ; In order to screen several libraries for a source of a full-length clone DNA from the libraries was screened by PCR amplification with the PCR primer pair identified above A positive library was then used to isolate clones encoding the PR0725 gene using the probe oligonucleotide and one of the PCR primers A full length clone was identified that contained a single open reading frame with an apparent translational initiation site at nucleotide positions 161-163 and ending at the stop codon found at nucleotide positions 455-457 (Figure 5, SEQ ID NO 5) The predicted polypeptide precursor is 98 amino acids long has a calculated molecular weight of approximately 1 1 ,081 daltons and an estimated pi of approximatel) 6 68 Analysis of the full-length PR0725 sequence shown m Figure 6 (SEQ ID NO 6) evidences the presence of the following a signal peptide from about amino acid 1 to about amino acid 20, a potential

N-glycosylation site from about amino acid 72 to about amino acid 75 and a tyrosine kinase phosphor) lation site from about amino acid 63 to about ammo acid 70 Clone DNA52758 1399 has been deposited with ATCC on April 14, 1998 and is assigned ATCC deposit no 209773

Analysis of the amino acid sequence of the full-length PR0725 polypeptide suggests that it possesses no significant sequence similarity to any known protein However, an analysis of the Dayhoff database

(version 35 45 SwissProt 35) evidenced some degree of homology between the PR0725 amino acid sequence and the following Dayhoff sequences, POL BLVAU, PSSP RAT, CELC36C5 7, AF019234 , 148862 P_R12498, P_P10125, P_R26861 , A64527 and P_W20495

D Isolation of cDNA clones Encoding Human PRO 1031

The extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins from the Swiss-Prot public protein database were used to search expressed sequence tag (EST) databases The EST databases included public EST databases (e g . GenBank, Merck/Wash U ) and a proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals Palo Alto, CA) The search was performed using the computer program BLAST or BLAST2 (Altshul et al , Methods m Enzvmologv

266 460-480 (1996)) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequence Those comparisons resulting in a BLAST score of 70 (or m some cases 90) or greater that did not encode known proteins were clustered and assembled mto consensus DNA sequences with the program "phrap" (Phil Green, University of Washington, Seattle, Washington) An initial consensus DNA sequence was assembled relative to other EST sequences using phrap

The initial consensus DNA sequence was extended using repeated cycles of BLAST and phrap to extend the consensus sequence as far as possible using the sources of EST sequences discussed above Based on the consensus sequences and other information provided herein Merck W74558 (clone 344649) was further examined DNA sequencing gave the full length DNA sequence for PRO 1031 [herein designated as DNA59294 1381] and the derived protein sequence for PRO1031

The entire nucleotide sequence of DNA59294 1381 is shown in Figure 7 (SEQ ID NO 7) Clone DNA59294 1381 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 42 44 and ending at the stop codon at nucleotide positions 582 584 (Figure 7) The predicted polypeptide precursor is 180 ammo acids long (Figure 8) The full length PRO1031 protein shown m Figure 8, SEQ ID NO 8, has an estimated molecular weight of about 20,437 and a pl of about 9 58 Clone DNA59294-1381 has been deposited with the ATCC Regarding the sequence, it is understood that the deposited clone contains the correct sequence, and the sequences provided herein are based on known sequencing techniques

Analysis of the amino acid sequence of the full length PRO 1031 polypeptide suggests that it is a novel cytokine Still analyzing the amino acid sequence of SEQ ID NO 8, the putative signal peptide is at about ammo acids 1-20 of SEQ ID NO 8 An N glycosylation site is at about amino acids 75-78 of SEQ

ID NO 8 A region having sequence identity with IL-17 is at about amino acids 96 180 The corresponding nucleotides can be routinely determined given the sequences provided herein

E Isolation of cDNA Clones Encoding Human PRO200

Probes based on an expressed sequence tag (EST) identified from the Incyte Pharmaceuticals database due to homology with VEGF were used to screen a cDNA library derived from the human glioma cell line G61 In particular, Incyte Clone "INC 1302516' was used to generate the following four probes

(SEQ ID NO 22) 5 -ACTTCTCAGTGTCCATAAGGG 3 ,

(SEQ ID NO 23) 5 -GAACTAAAGAGAACCGATACCATTTTCTGGCCAGGTTGTC-3' , (SEQ ID NO 24) 5'-CACCACAGCGTTTAACCAGG 3 , and (SEQ ID NO 25) 5 -ACAACAGGCACAGTTCCCAC 3' Nine positives were identified and characterized Three clones contained the full coding region and were identical in sequence Partial clones were also identified from a fetal lung library and were identical with the glioma derived sequence with the exception of one nucleotide change which did not alter the encoded amino acid

EXAMPLE 2 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay 24)

This example shows that certain PRO polypeptides of the invention are active as a stimulator of the proliferation of stimulated T lymphocytes Compounds which stimulate proliferation of lymphocytes are useful therapeutically where enhancement of an immune response is beneficial A therapeutic agent may take the form of antagonists of the PRO polypeptide of the invention, for example murine human chimeric, humanized or human antibodies against the polypeptide

The basic protocol for this assay is described in Current Protocols in Immunology, unit 3 12, edited by JE Cohgan, AM Kruisbeek, DH Marglies, EM Shevach, W Strober National Insitutes of Health, Published by John Wiley & Sons, Ine

More specificall) . in one assay variant, peripheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs) If desired the cells are frozen in fetal bovine serum and DMSO after isolation Frozen cells may be thawed overnight in assay media (37°C, 5 % CO,) and then washed and resuspended to 3xl0⁶ cells/ml of assay media (RPMI, 10% fetal bovme serum

1 % penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non essential ammo acids 1 % pyruvate) The stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads)

The assay is prepared by plating in triplicate wells a mixture of

100 1 of test sample diluted to 1 % or to 0 1 % , 50 1 of irradiated stimulator cells, and

50 1 of responder PBMC cells 100 microhters of cell culture media or 100 microhter of CD4-IgG is used as the control The wells are then incubated at 37°C, 5 % CO, for 4 days On day 5, each well is pulsed with tπtiated thy midine (1 0 mC/well Amersham) After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated In another variant of this assa) , PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice The cells are teased from freshly harvested spleens in assay media (RPMI, 10% fetal bovine serum, 1 % penicillin/streptomycin, 1 % glutamine, 1 % HEPES, 1 % non-essential amino acids, 1 % pyruvate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to lxlO⁷ cells/ml of assay media The assay is then conducted as described above

Positive increases over control are considered positive with increases of greater than or equal to 180% being preferred However, any value greater than control indicates a stimulatory effect for the test protein The results are shown in Table 7 below

Table 7

PRO PRO Concentration Percent Increase Over Control

PR0526 15 6 nM 302

PR0526 1 56 nM 272

PR0719 1 07 nM 262

PR0719 0 107 nM 130

PR0725 25 60 nM 264

PR0725 2 56 nM 189

PRO 1031 5 60 nM 285

PRO 1031 0 56 nM 147 EXAMPLE 3 Skin Vascular Permeability Assay (Assay 64)

This assay sho s that certain PRO polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosmophil and PMN infiltration at the site of injection of the animal Compounds which stimulate an immune response are useful therapeutically where stimulation of an immune response is beneficial This skm vascular permeability assay is conducted as follows Hairless guinea pigs weighing 350 grams or more are anesthetized with ketamme (75-80 mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM) A sample of purified PRO polypeptide of the invention or a conditioned media test sample is injected lntradermally onto the backs of the test animals with 100 μl per injection site It is possible to have about 10-30, preferably about 16-24, inaction sites per animal One μl of Evans blue dye (1 % in physiologic buffered saline) is injected intracardially Blemishes at the injection sites are then measured (mm diameter) at 1 hr and 6 hr post injection Animals were sacrificed at 6 hrs after injection

Each skin injection site is biopsied and fixed in formalin The skins are then prepared for histopathologic evaluation Each site is evaluated for inflammatory cell infiltration into the skin Sites with visible inflammatory cell inflammation are scored as positive Inflammatory cells may be neutrophihc, eosinophihc , monocytic or lymphocytic At least a minimal peπvascular infiltrate at the injection site is scored as positve, no infiltrate at the site of injection is scored as negative. The following polypeptides tested positive in this assay: PRO200.

EXAMPLE 4 Human Co-Stimulation Assay

In addition to the activation signal mediated by the T cell receptor, T cell activation requires a costimulatory signal. One costimulatory signal is generated by the interaction of B7 (CD3) with CD28. In this assay, 96 well plates are coated with CD3 with or without CD28 and then human peripheral blood lymphocytes followed by a test protein, are added Proliferation of the lymphocytes is determined by tπtiated thymidine uptake A positive assay indicates that the test protein provided a stimulatory signal for lymphocyte proliferation Material

1) Hyclone D-PBS without Calcium, Magnesium

2) Nunc 96 well certified plates #4-39454

3) Anti-human CD3 Amac 0178 200 μg/ml stock

4) Anti-human CD28 Biodesign P42235M 5) Media: Gibco RPMI 1640 + 10% Intergen #1020-90 FBS, 1 % Glu, 1 % P/S, 50 μg/ml Gentamycm, 10 mM Hepes Fresh for each assay

6) Tπtiated Thymidine

7) Frozen human peripheral blood lymphocytes (PBL) collected via a leukophoresis procedure

Plates are prepared by coating 96 well flat bottom plates with antι-CD3 antibody (Amac) or antι-CD28 antibody (Biodesign) or both Hyclone D-PBS without calcium and magnesium Antι-CD3 antibody is used at 10 ng/well (50.1 of 200 ng/ml) and antι-CD28 antibody at 25 ng/well (50:1 of 0.5 μg/ml) in 100.1 total volume PBLs are isolated from human donors using standard leukophoresis methods The cell preparations are frozen in 50% fetal bovine serum and 50% DMSO until the assay is conducted Cells are prepared by thawing and washing PBLs in media resuspending PBLs m 25 mis of media and incubating at 37°C, 5 % CO, o\ ernight

In the assay procedure, the coated plate is washed twice with PBS and the PBLs are washed and resuspended to lxlO⁶ cells/ml using lOOμl/well 100 μl of a test protein or control media are added to the plate making a total volume per well of 200 μl The plate is incubated for 72 hours The plate is then pulsed for 6 hours with tritiated thymidine (1 mC/well, Amersham) and the PBLs are harvested from the plates and evaluated for uptake of the tritiated thymidine

The results of this assay for compounds of the invention are shown in Table 8 below Positive increases over control are considered positive with increases of greater than or equal to 180% being preferred However, any value greater than control indicates a stimulatory effect for the test protein PBMC means peripheral blood mononuclear cells

Table 8

PRO PRO Concentration Percent Increase Over Control

PR0526 6 24 nM (CD4) 420

PR0526 6 24 nM (PBMC) 234

PR0719 0 428 nM (CD4) 197

PR0719 0 428 nM (PBMC) 245

PR0725 10 24 nM (CD4) 265

PR0725 10 24 nM (PBMC) 147

PRO 1031 2 24 nM (CD4) 784

PRO 1031 2 24 nM (PBMC) 398

EXAMPLE 5 Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay 67)

This example shows that certain polypeptides of the invention are active as inhibitors of the proliferation of stimulated T- lymphocytes Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an immune response is beneficial The procedure was the same as in Example 2 Any decreases below control is considered to be a positive result for an inhibitory compound, with decreases of less than or equal to 80% being preferred However, any value less than control indicates an inhibitory effect for the test protein

Table 9

PRO PRO Concentration Percent Decrease Below Control

PRO200 4 7 nM 82 7 PRO200 47 nM 63 7 EXAMPLE 6 / ; situ Hybridization

In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping In situ hybridization was performed following an optimized version of the protocol by Lu and

Gillett, Cell Vision 1 : 169-176 (1994). using PCR-generated "P-labeled πboprobes. Briefly, formalm-fixed, paraffin-embedded human tissues were sectioned, deparaffimzed, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37°C, and further processed for in situ hybridization as described by Lu and Gillett, supra A P^-P] UTP-labeled antisense πboprobe was generated from a PCR product and hybridized at 55 °C overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.³P-Riboprobe synthesis

6.0 μl (125 mCi) of "P-UTP (Amersham BF 1002, SA < 2000 Ci/mmol) were speed vac dried. To each mbe containing dried^,3P-UTP, the following ingredients were added 2.0 μl 5x transcription buffer 1 O μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM : 10 μ; each of 10 mM GTP, CTP & ATP + 10 μl H₂0) 1.0 μl UTP (50 μM) 1.0 μl Rnasin

1.0 μl DNA template (lμg) 1.0 μl H,O

1.0 μl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)

The tubes were incubated at 37°C for one hour. 1.0 μl RQ1 DNase were added, followed by incubation at 37°C for 15 minutes. 90 μl TE (10 mM Tris pH 7.6/lmM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Mιcrocon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE were added. 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III were added to

3 μl of loading buffer. After heating on a 95^CC heat block for three minutes, the gel was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70 °C freezer one hour to overnight

³³P-Hvbridization A Pretreatment of frozen sections

The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes The trays were placed in 55 °C incubator tor five minutes to reduce condensation The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H,0) After deprotemation in 0 5 μg/ml proteinase K for 10 minutes at 37°C (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5 x SSC for 10 minutes at room temperature The sections were dehydrated in 70% , 95 % , 100% ethanol, 2 minutes each B Pretreatment of paraffin-embedded sections

The slides were deparaffimzed placed in SQ H,0. and rmsed twice in 2 x SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in

250 ml RNase-free RNase buffer, 37 C, 15 minutes) - human embryo, or 8 x proteinase K (100 μl in 250 ml Rnase buffer, 37°C, 30 minutes) - lormahn tissues Subsequent rinsing m 0.5 x SSC and dehydration were performed as described above.

C Prehybndization

The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper The tissue was covered w ith 50 μl of hybridization buffer (3 75g Dextran Sulfate + 6 ml SQ

H,0), vortexed and heated in the microwave for 2 minutes with the cap loosened After cooling on ice, 18.75 ml formamide, 3.75 ml 20 x SSC and 9 ml SQ H,0 were added, the tissue was vortexed well, and incubated at 42 °C for 1-4 hours.

D. Hybridization

1.0 x IO⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95 °C for 3 minutes The slides were cooled on ice, and 48 μl hybridization buffer were added per slide After vortexing, 50 μl^""P mix were added to 50 μl prehybndization on slide. The slides were incubated overnight at 55 °C.

E Washes

Washing was done 2 x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC +

16 ml 0.25M EDTA, V_f=4L), followed by RNaseA treatment at 37°C for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer = 20 μg/ml), The slides were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows. 2 hours at 55 °C, 0.1 x SSC, EDTA (20 ml

20 x SSC + 16 ml EDTA, V_f=4L).

F Results

DNA59294-1381

Expression was observed m muscle, certain types of smooth muscle in the adult and in skeletal and smooth muscle in the human fetus. Expression in adult human was in smooth muscle of tubular organs including pancreas, small intestine, stomach, colon and gall bladder. In fetal tissues there was moderate to high diffuse expression in skeletal muscle in the axial skeleton and limbs. There was weak expression in the smooth muscle of the intestinal wall and testis The IL17 homologue gene was localized to human chromosome 5q32-34 by radiation hybrid analysis using the Stanford Radiation Hybrid Panel G3 of human/CHO clones EXAMPLE 7 In situ Hybridization in Cells and Diseased Tissues

The in situ hybridization method of Example 6 is used to determine gene expression analyze the tissue distribution of transcription, and follow changes in specific mRNA synthesis for the genes/DNAs and the proteins of the invention in diseased tissues isolated from human individuals suffering from a specific disease These results show more specifically where in diseased tissues the genes of the invention are expressed and are more predictive of the particular localization of the therapeutic effect of the inhibitory or stimulatory compounds of the invention (and agonists or antagonists thereof) m a disease Hybridization is performed according to the method of Example 6 using one or more of the following tissue and cell samples

(a) lymphocytes and antigen presenting cells (dendritic cells, langherhans cells, macrophages and monocytes, NK cells), (b) lymphoid tissues normal and reactive lymph node, thymus, Bronchial Associated Lymphoid

Tissues, (BALT), Mucosal Associated Lymphoid Tissues (MALT),

(c) human disease tissues Synovium and joint of patients with arthritis and degenerative joint disease, colon from patients with inflammatory bowel disease including ulcerative colitis and Crohns' disease, skm lesions from psoriasis and other forms of dermatitis, lung tissue including BALT and tissue lymph nodes from chronic and acute bronchitis, pneumonia, pneumonitis, pleuπtis, lung tissue including BALT and tissue lymph nodes from asthma, nasal and sinus tissue from patients with rhinitis or sinusitis, bram and spinal cord from multiple sclerosis, alzheimer's disease and stroke, kidney from nephritis, glomerulonephπtis and systemic lupus erythematosis, liver from infectious and non infectious hepatitis and tissues from neoplasms/cancer Expression is observed in one or more cell or tissue samples indicating localization of the therapeutic effect of the compounds of the invention (and agonists or antagonists thereof) in the disease associated with the cell or tissue sample DNA59294-1381

There was low diffuse expression in the smooth muscle (tunica musculaπs) in 5 specimens with chronic inflammatory bowel disease

EXAMPLE 8 Use of PRO as a hybridization probe

The following method describes use of a nucleotide sequence encoding PRO as a hybridization probe DNA comprising the coding sequence of full-length or mature PRO as disclosed herein is employed as a probe to screen for homologous DNAs (such as those encoding naturally occurring variants of PRO) in human tissue cDNA libraries or human tissue genomic libraries

Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions Hybridization of radiolabeled PRO derived probe to the filters is performed m a solution of 50% formamide, 5x SSC, 0 1 % SDS, 0 1 % sodium pyrophosphate, 50 mM sodium phosphate, pH 6 8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20 hours Washing of the filters is performed in an aqueous solution of 0 lx SSC and 0 1 % SDS at 42°C DNAs having a desired sequence identity with the DNA encoding full-length native sequence PRO can then be identified using standard techniques known in the art

EXAMPLE 9 Expression of PRO in E coli

This example illustrates preparation of an unglycosylated form of PRO by recombinant expression

The DNA sequence encoding PRO is initially amplified using selected PCR primers The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector A variety of expression vectors may be employed An example of a suitable vector is pBR322 (derived from E coli, see Bolivar et al , Gene. 2 95 ( 1977)) which contains genes for ampicillin and tetracychne resistance The vector is digested with restriction enzyme and dephosphorylated The PCR amplified sequences are then ligated mto the vector The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokmase cleavage site), the PRO coding region, lambda transcriptional terminator, and an argU gene The ligation mixture is then used to transform a selected E coli strain using the methods described in Sambrook et al , supra Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing

Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics The overnight culture may subsequently be used to inoculate a larger scale culture The cells are then grown to a desired optical density, during which the expression promoter is turned on

After culturing the cells for several more hours, the cells can be harvested by centrifugation The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubihzed PRO protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein

PRO may be expressed m E coli in a poly-His tagged form, using the following procedure The DNA encoding PRO is initially amplified using selected PCR primers The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokmase The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E coli host based on strain 52 (W31 10 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(ladq) Transformants are first grown in LB containing 50 mg/ml carbenicilhn at 30°C with shaking until an O D 600 of 3 5 is reached Cultures are then diluted 50 100 fold into CRAP media (prepared by mixing 3 57 g (NH₄),S0₄, 0 71 g sodium cιtrate^»2H,0, 1 07 g KC1, 5 36 g Difco yeast extract, 5 36 g Sheffield hycase SF in 500 mL water, as well as 1 10 mM MPOS, pH 7 3, 0 55 % (w/v) glucose and 7 mM MgS0₄) and grown for approximately 20-30 hours at 30 °C with shaking Samples are removed to verify expression by SDS PAGE analysis, and the bulk culture is centrifuged to pellet the cells Cell pellets are frozen until purification and refolding

E coli paste from 0 5 to 1 L fermentations (6 10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tπs, pH 8 buffer Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0 l M and 0 02 M respectively , and the solution is stirred overnight at 4 °C This step results in a denatured protein with all cysteine residues blocked by sulfitohzation The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 m The supernatant is diluted with 3 5 volumes of metal chelate column buffer (6 M guanidine 20 mM Tris, pH 7 4) and filtered through 0 22 micron filters to clarify The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7 4 The protein is eluted with buffer contaimng 250 mM imidazole Fractions containing the desired protein are pooled and stored at 4°C Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence

The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of 20 mM Tris, pH 8 6, 0 3 M NaCI, 2 5 M urea 5 mM cysteine 20 mM glycine and 1 mM

EDTA Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml The refolding solution is stirred gently at 4 °C for 12 36 hours The refolding reaction is quenched by the addition of TFA to a final concentration of 0 4% (pH of approximately 3) Before further purification of the protein, the solution is filtered through a 0 22 micron filter and acetonitrile is added to

2-10% final concentration The refolded protein is chromatographed on a Poros Rl/H reversed phase column using a mobile buffer of 0 1 % TFA with elution with a gradient of acetonitrile from 10 to 80% Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions contaming homogeneous refolded protein are pooled Generally the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resm Aggregated species are usually eluted at higher acetonitrile concentrations In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxm from the samples

Fractions containing the desired folded PRO polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution Proteins are formulated into 20 mM Hepes, pH 6 8 with 0 14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered Many of the PRO polypeptides disclosed herein were successfully expressed as described above

EXAMPLE 10 Expression of PRO in mammalian cells

This example illustrates preparation of a potentially glycosylated form of PRO by recombinant expression in mammalian cells The vector, pRK5 (see EP 307,247, published March 15 1989) is employed as the expression vector Optionally the PRO DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the PRO DNA using ligation methods such as described in Sambrook et al , supra The resulting vector is called pRK5-PRO

In one embodiment, the selected host cells ma) be 293 cells Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics About 10 μg pRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al , Cell, 31 543 (1982)] and dissolved in 500 μl of 1 mM Tns-HCl, 0 1 mM EDTA, 0 227 M CaCl, To this mixture is added, dropwise, 500 μl of

50 mM HEPES (pH 7 35). 280 mM NaCI 1 5 mM NaP0₄, and a precipitate is allowed to form for 10 minutes at 25°C The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37°C The culmre medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds The 293 cells are then washed with serum 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 replaced with culture medium (alone) or culmre medium containing 200 μCi/ml^,3S-cysteιne and 200 μCi/ml^1SS methionme After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15 % SDS gel The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of PRO polypeptide The culmres containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested m selected bioassays

In an alternative technique, PRO may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al , Proc Natl Acad Sci . 12 7575 (1981) 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-PRO DNA is added The cells are first concentrated from the spmner flask by centrifugation and washed with PBS The DNA-dextran precipitate is incubated on the cell pellet for four hours The cells are treated with 20% glycerol tor 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0 1 μg/ml bovine transferrin After about four days, the conditioned media is centrifuged and filtered to remove cells and debris The sample containing expressed PRO can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography

In another embodiment, PRO can be expressed m CHO cells The pRK5-PRO can be transfected into CHO cells using known reagents such as CaP0₄ or DEAE-dextran As described above, the cell cultures can be incubated, and the medium replaced with culmre medium (alone) or medium containing a radiolabel such as³⁵S-methιonme After determining the presence of PRO polypeptide, the culmre medium may be replaced with serum free medium Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested The medium containing the expressed PRO can then be concentrated and purified by any selected method

Epitope tagged PRO may also be expressed in host CHO cells The PRO may be subcloned out of the pRK5 vector The subclone insert can undergo PCR to fuse m frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector The poly-his tagged PRO inseπ can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones

Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector Labeling may be performed as described above to verify expression The culmre medium containing the expressed pol) His tagged PRO can then be concentrated and purified by any selected method, such as by Nf⁺-chelate affinity chromatography

PRO may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure Stable expression in CHO cells is performed using the following procedure The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e g extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly His tagged form

Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al , Current Protocols of Molecular Biology, Unit 3 16, John

Wiley and Sons (1997) CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA s The vector used expression m CHO cells is as described in Lucas et al , Nucl Acids Res 24 9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR) DHFR expression permits selection for stable maintenance of the plasmid following transfection

Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect" (Quiagen), Dosper^* or Fugene" (Boehringer Mannheim) The cells are grown as descπbed in Lucas et al , supra Approximately 3 x 10⁷ cells are frozen in an ampule for further growth and production as described below The ampules containing the plasmid DNA are thawed by placement mto water bath and mixed by vortexing The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0 2 μm filtered PS20 with 5 % 0 2 μm diafiltered fetal bovine serum) The cells are then ahquoted into a 100 mL spinner containing 90 mL of selective media After 1 2 days the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37°C After another 2 3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3 x 10' cells/mL The cell media is exchanged with fresh media by centrifugation and resuspension in production medium Although any suitable CHO media may be employed, a production medium described in U S Patent No 5, 122,469, issued June 16, 1992 may actually be used A 3L production spinner is seeded at 1 2 x 10⁶ cells/mL On day 0, the cell number pH ie determined On day 1 , the spinner is sampled and sparging with filtered air is commenced

On day 2, the spinner is sampled, the temperature shifted to 33°C, and 30 mL of 500 g/L glucose and 0 6 mL of 10% antifoam (e g , 35 % polydimethylsiloxane emulsion Dow Corning 365 Medical Grade Emulsion) taken Throughout the production, the pH is adjusted as necessary to keep it at around 7 2 After 10 days, or until the viability dropped below 70% , the cell culmre is harvested by centrifugation and filtering through a 0 22 μm filter The filtrate was either stored at 4°C or immediately loaded onto columns for purification For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen) Before purification, imidazole is added to the conditioned media to a concentration of 5 mM The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7 4, buffer containing 0 3 M NaCI and 5 mM imidazole at a flow rate of 4-5 ml/mm at 4°C After loading the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0 25 M imidazole The highly purified protein is subsequently desalted into a storage buffer containing 10 mM

Hepes, 0 14 M NaCI and 4 % mannitol, pH 6 8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80°C

Immunoadhesin (Fc containing) constructs are purified from the conditioned media as follow s The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6 8 After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3 5 The eluted protein is immediately neutralized by collecting 1 ml fractions mto tubes containing 275 μL of 1 M Tris buffer, pH 9 The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins The homogeneity is assessed by SDS polyacry lamide gels and by N terminal ammo acid sequencing by Edman degradation

Many of the PRO polypeptides disclosed herein were successfull) expressed as described above

EXAMPLE 1 1 Expression of PRO m Yeast

The following method describes recombinant expression of PRO in yeast First, yeast expression vectors are constructed for intracellular production or secretion of PRO from the ADH2/GAPDH promoter DNA encoding PRO and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PRO For secretion, DNA encoding PRO can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native PRO signal peptide or other mammalian signal peptide, or, for example, a yeast alpha factor or mvertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PRO

Yeast cells, such as yeast strain AB1 10, can then be transformed with the expression plasmids described above and culmred in selected fermentation media The transformed yeast supernatants can be analyzed by precipitation with 10% tπchloroacetic acid and separation by SDS PAGE, followed by staining of the gels with Coomassie Blue stam Recombinant PRO can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters The concentrate containing PRO may further be purified using selected column chromatography resins Many of the PRO polypeptides disclosed herein were successfull) expressed as described above EXAMPLE 12 Expression of PRO in Baculovirus Infected Insect Cells

The following method describes recombinant expression of PRO in Baculov lrus infected insect cells

The sequence coding for PRO is fused upstream of an epitope tag contained within a baculovirus expression vector Such epitope tags include poly his tags and immunoglobulin tags (like Fc regions of IgG)

A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) Briefly the sequence encoding PRO or the desired portion of the coding sequence of PRO such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5 and 3 regions The 5 primer may incorporate flanking (selected) restriction enzyme sites The product is then digested with those selected restriction enzymes and subcloned into the expression vector

Recombinant baculovirus is generated by co transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopteia frugψerda ("Sf9 ) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO BRL) After 4 5 days of incubation at 28°C the released viruses are harvested and used for further amplifications Viral infection and protein expression are performed as described by O Reilley et al , Baculovirus expression vectors A Laboratory Manual, Oxford Oxford

University Press (1994)

Expressed poly his tagged PRO can then be purified, tor example, by N⁺ -chelate affinity chromatography as follows Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al , Nature. 362 175-179 (1993) Briefly, Sf9 cells are washed, resuspended in somcation buffer (25 mL Hepes, pH 7 9, 12 5 mM MgCl,, 0 1 mM EDTA, 10% glycerol, 0 1 % NP-40, 0 4 M KC1), and sonicated twice for 20 seconds on ice The sonicates are cleared by centrifugation, and the supernatant is diluted 50 fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol, pH 7 8) and filtered through a 0 45 μm filter A Nf⁺-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer The filtered cell extract is loaded onto the column at 0 5 mL per minute The column is washed to baseline

A₂₈₀ with loading buffer, at which point fraction collection is started Next, the column is washed with a secondary wash buffer (50 mM phosphate, 300 mM NaCI 10% glycerol, pH 6 0), which elutes nonspecifically bound protein After reaching A,₈₀ baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer One mL fractions are collected and analyzed by SDS PAGE and silver staining or Western blot with Nf⁺-NTA conjugated to alkaline phosphatase (Qiagen)

Fractions containing the eluted Hιs_l0-tagged PRO are pooled and dialyzed against loading buffer

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography

Many of the PRO polypeptides disclosed herein were successfully expressed as described above EXAMPLE 13 Preparation of Antibodies that Bind PRO

This example illustrates preparation of monoclonal antibodies which can specifically bind PRO Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Godmg, supra Immunogens that may be employed include purified PRO, fusion proteins containing PRO, and cells expressing recombinant PRO on the cell surface Selection of the lmmunogen can be made by the skilled artisan without undue experimentation

Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or lntrapeπtoneally in an amount from 1 -100 micrograms Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot pads The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified m the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional immunization injections Serum samples may be periodically obtained from the mice by retro orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies After a suitable antibody titer has been detected, the animals "positive" for antibodies can be lmected with a final intravenous injection of PRO Three to four days later, the mice are sacrificed and the spleen cells are harvested The spleen cells are then fused (using 35 % polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. l , available from ATCC, No CRL 1597 The fusions generate hybridoma cells which can then be plated m 96 well tissue culmre plates containing HAT (hypoxanthine, aminopteπn, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids The hybridoma cells will be screened in an ELISA for reactivity against PRO Determination of

"positive" hybridoma cells secreting the desired monoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected lntrapeπtoneally into syngeneic Balb/c mice to produce ascites containing the anti-PRO monoclonal antibodies Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed

EXAMPLE 14 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety of standard techniques in the art of protein purification For example, pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide is purified by immunoaffimty chromatography using antibodies specific for the PRO polypeptide of interest In general, an immunoaffimty column is constructed by covalently coupling the anti-PRO polypeptide antibody to an activated chromatographic resin

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N J ) Likewise monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A Partially purified immunoglobulin is covalently attached to a chromatographic resm such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology ) The antibody is coupled to the resm, the resm is blocked, and the derivative resin is washed according to the manufacturer's instructions Such an lmmunoaffinity column is utilized in the purification of PRO polypeptide by preparing a fraction from cells containing PRO polypeptide in a soluble form This preparation is derived by solubihzation of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art Alternatively, soluble PRO polypeptide containing a signal sequence may be secreted in useful quantity into the medium m which the cells are grown A soluble PRO polypeptide-contammg preparation is passed over the immunoaffimty column, and the column is washed under conditions that allow the preferential absorbance of PRO polypeptide (e g , high ionic strength buffers in the presence of detergent) Then, the column is eluted under conditions that disrupt antibody /PRO polypeptide binding (e g , a low pH buffer such as approximately pH 2 3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is collected

EXAMPLE 15 Drug Screening

This invention is particularly useful for screening compounds by using PRO polypeptides 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 be free solution, affixed to a solid support, borne on a cell surface, or located mtracellularly One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the PRO polypeptide or fragment Drugs are screened against such transformed cells in competitive binding assays Such cells, either in viable or fixed form, can be used for standard binding assays One may measure, for example, the formation of complexes between PRO polypeptide or a fragment and the agent being tested Alternatively, one can examine the diminution m complex formation between the PRO polypeptide and its target cell or target receptors caused by the agent being tested

Thus, the present invention provides methods of screening for drugs or any other agents which can affect a PRO polypeptide associated disease or disorder These methods comprise contacting such an agent with an PRO polypeptide or fragment thereof and assaying (I) for the presence of a complex between the agent and the PRO polypeptide or fragment, or (n) for the presence of a complex between the PRO polypeptide or fragment and the cell, by methods well known in the art In such competitive binding assays, the PRO polypeptide or fragment is typically labeled After suitable incubation, free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to PRO polypeptide or to interfere with the PRO polypeptide/cell complex

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to a polypeptide and is described in detail WO 84/03564, published on September 13, 1984 Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate such as plastic pms or some other surface As applied to a PRO polypeptide, the peptide test compounds are reacted with PRO polypeptide and washed Bound PRO poly peptide is detected by methods well known in the art Purified PRO polypeptide can also be coated directly onto plates for use m the aforementioned drug screening techniques In addition non neutralizing antibodies can be used to capmre the peptide and immobilize it on the solid support

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptide specifically compete with a test compound for binding to PRO polypeptide or fragments thereof In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigemc determinants with PRO polypeptide

EXAMPLE 16 Rational Drug Design

The goal of rational drug design is to produce structural analogs of biologically active polypeptide of interest (i e a PRO polypeptide) or of small molecules with which they interact, e g agonists, antagonists, or inhibitors Any of these examples can be used to fashion drugs which are more active or stable forms of the PRO polypeptide or which enhance or interfere with the function of the PRO polypeptide in vivo (c f , Hodgson, Bio/Technology. 9 19 21 (1991))

In one approach, the three-dimensional structure of the PRO polypeptide, or of an PRO polypeptide-inhibitor complex, is determined by x ray crystallography, by computer modeling or, most typically, by a combination of the two approaches Both the shape and charges of the PRO polypeptide must be ascertained to elucidate the structure and to determine active sιte(s) of the molecule Less often, useful information regarding the structure of the PRO polypeptide may be gained by modeling based on the structure of homologous proteins In both cases, relevant structural information is used to design analogous PRO polypeptide-hke molecules or to identify efficient inhibitors Useful examples of rational drug design may include molecules which have improved activity or stability as shown by Braxton and Wells, Biochemistry 31 7796-7801 (1992) or which act as inhibitors, agonists, or antagonists of native peptides as shown by

Athauda et al , J Biochem . 1 13 742-746 (1993)

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

By virtue of the present invention, sufficient amounts of the PRO polypeptide may be made available to perform such analytical studies as X-ray crystallography In addition, knowledge of the PRO polypeptide amino acid sequence provided herein will provide guidance to those employ mg computer modeling techniques in place of or in addition to x-ray crystallography EXAMPLE 17 Biological activities of PRO1031

For IL-6 release, human foreskin fibroblast cells (ATCC) were cultured in MEM media (10% FBS) with the test protein After incubation for 18 hr at 37°C and 5 % CO,, conditioned media were assayed for IL-6 using an ELISA kit (R&D systems) For TNF-α secretion, human leukemia monocytic THP-1 cells were cultured in RPMI media (10% FBS) with the test protein After incubation for 18hr at 37°C and 5 % CO,, conditioned media were quantitated for TNF using an ELISA assay kit (R&D systems)

One of the biological activities of IL-17 is the induction of IL 6 release in fibroblast cells ( Yao et al , 1995, J Immunol 155 5483) To assess the biological functions of PRO1031 , human foreskin fibroblasts were treated with the protein and assa) ed for IL 6 production In contrast to the high level of IL-6 induced by IL-17, PRO1031 failed to stimulate IL-6 secretion in fibroblast cells A human leukemic monocytic cell lme, THP 1 , reported to express IL-17 receptor (Yao et al, 1997, Cytokine 9 794), was then used to assay for the stimulation of TNF a release by IL 17 and PRO1031 While IL 17 induced only a low level of TNF in THP-1 cells, PRO 1031 (as an Fc fusion protein) stimulated TNF production in THP 1 cells A control Fc fusion protein had no effect Similar results were obtained with his tagged protein

To further characterize the stimulation of TNF x release by PRO 1031 , the time course and concentration dependence of the response were assayed in THP 1 cells The results mdicate that PRO1031 stimulates the release of TNF-α m a time- and concentration-dependent manner The EC₅₀ for PRO1031 stimulation is 2 4 nM

These results indicate that PRO 1031 promotes the release of proinflammatory cytokines Such stimulator proteins are useful in treating immune related diseases characterized by poor or inadequate immune function For example , PRO 1031 may be used in immunoadjuvant therapy An antagonist antibody binding to PRO 1031 will inhibit the proinflammatory action of the protein and is useful in inhibiting the immune resonse Such an antibody is useful to treat, for example, immune mediated diseases such as rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, etc using the compositions and methods of administration described above

Deposit of Material

The following materials have been deposited with the American Type Culture Collection, 10801 University

Blvd , Manassas, VA 201 10-2209, USA (ATCC)

Table 10

Material ATCC # ATCC Deposit Date

DNA44184-1319 209704 March 26, 1998

DNA52758-1399 209773 April 14, 1998

DNA59294-1381 209866 May 14, 1998

DNA40646-1327 209705 March 26, 1998

DNA29101-1272 209653 March 5, 1998 These deposits was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culmre of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC. which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC 122 and the Commissioner's rules pursuant thereto (including 37 CFR 1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

What is claimed

1 A composition comprising a PR0526, PR0719, PR0725 PRO1031 or PRO200 polypeptide, agonist or fragment thereof and a carrier or excipient, useful for

(a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof,

(b) stimulating or enhancing an immune response in a mammal in need thereof, or (c) increasing the proliferation of T lymphocytes in a mammal in need thereof in response to an antigen

2 Use of a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide, agonist or a fragment thereof to prepare a composition useful for (a) increasing infiltration of inflammatory cells into a tissue of a mammal in need thereof,

(b) stimulating or enhancing an immune response in a mammal in need thereof, or

(c) increasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen

3 A composition comprising a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide, antagonist or a fragment thereof and a carrier or excipient, useful for

(a) decreasing infiltration of inflammatory cells into a tissue of a mammal m need thereof,

(b) inhibiting or reducing an immune response in a mammal in need thereof, or

(c) decreasing the proliferation of T-lymphocytes in a mammal in need thereof in response to an antigen

4 Use of a PR0526, PR0719, PR0725, PRO 1031 or PRO200 polypeptide, antagonist or a fragment thereof to prepare a composition useful for

(a) decreasing infiltration of inflammatory cells into a tissue of a mammal in need thereof, (b) inhibiting or reducing an immune response in a mammal in need thereof, or

(c) decreasing the proliferation of T-lymphocytes in a mammal m need thereof in response to an antigen

5 A method of treating an immune related disorder, such as a T cell mediated disorder, in a mammal m need thereof, comprising administering to the mammal an effective amount of a PR0526,

PR0719, PR0725, PRO 1031 or PRO200 polypeptide, an agonist antibody thereof, an antagonist antibody thereto, or a fragment thereof

6 The method of Claim 5, wherein the disorder is selected from systemic lupus erythematosis. rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vascuhtis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuπa), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune -mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mel tus, immune-mediated renal disease (glomerulonephπtis, tubulomterstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillam-Barre syndrome, and chronic inflammatory demyelinatmg polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis. Crohn's disease), gluten-sensitive enteropathy, and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunologic diseases of the lung such as eosinophihc pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft -versus-host-disease.

7. The composition or use of any of the preceding claims, wherein the agonist or antagonist is a monoclonal antibody.

8 The composition or use of any of the preceding claims, wherein the agonist or antagomst is an antibody fragment or a single-chain antibody.

9 The composition or use of Claims 7 or 8. wherein the antibody has non-human complementarity determining region (CDR) residues and human framework region (FR) residues

10 A method for determining the presence of a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide, comprising exposing a cell suspected of containing the polypeptide to an antι-PR0526, PR0719, PR0725, PRO1031 or PRO200 antibody and determining binding of the antibody to the cell

11 A method of diagnosing an immune related disease in a mammal, comprising detectmg the level of expression of a gene encoding a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher expression level m the test sample as compared to the control sample indicates the presence of immune related disease in the mammal from which the test tissue cells were obtained. 12 A method of diagnosing an immune related disease in a mammal comprising (a) contacting an antι-PR0526, PR0719 PR0725, PRO1031 or PRO200 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the antibody and the polypeptide in the test sample wherein formation of said complex is indicative of the presence of said immune related disease in said mammal

13 An immune related disease diagnostic kit comprising an anti PR0526, PR0719, PR 0725, PRO 1031 or PRO200 antibody or fragment thereof and a carrier in suitable packaging

14 The kit of Claim 13 further comprising instructions for using the antibody to detect a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide

15 An article of manufacture, comprising a container, a label on the container, and a composition comprising an active agent contained withm the container wherein the composition is effective for inhibiting or reducing an immune response in a mammal, the label on the container indicates that the composition can be used for treating an immune related disease, and the active agent in the composition is an agent inhibiting the expression and/or activity ofa PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide

16 The article of manufacture of Claim 15 wherein said active agent is an antι-PR0526, PR0719, PR0725, PRO 1031 or PRO200 antibody

17 A method for identifying a compound capable of inhibiting the expression or activity of a PR0526, PR0719, PR0725, PRO1031 or PRO200 polypeptide, comprising contacting a candidate compound with the polypeptide under conditions and for a time sufficient to allow these two components to interact

18 The method of Claim 17, wherein the candidate compound or the PR0245, PR0217, polypeptide is immobilized on a solid support

19 The method of Claim 18, wherein the non-immobilized component carries a detectable label 20 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of the ammo acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10)

21 Isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequence shown in Figure 1 (SEQ ID NO 1), Figure 3 (SEQ ID NO 3), Figure 5 (SEQ ID NO 5), Figure 7 (SEQ ID NO 7) or Figure 9 ( SEQ ID NO 9)

22 Isolated nucleic acid having at least 80% nucleic acid sequence identit) to a nucleotide sequence selected from the group consisting of the full-length coding sequence ot the nucleotide sequence shown in Figure 1 (SEQ ID NO 1), Figure 3 (SEQ ID NO 3), Figure 5 (SEQ ID NO 5), Figure 7 (SEQ ID NO 7) or Figure 9 (SEQ ID NO 9)

23 Isolated nucleic acid having at least 80% nucleic acid sequence identity to the full length coding sequence of the DNA deposited under any of the ATCC accession numbers shown in Table 10

24 A vector comprising the nucleic acid of any one of Claims 20 to 23

25 The vector of Claim 24 operably linked to control sequences recognized by a host cell transformed with the vector

26 A host cell compπsing the vector of Claim 24

27 The host cell of Claim 26, wherein said cell is a CHO cell

28 The host cell of Claim 26, wherein said cell is an E coli

29 The host cell of Claim 26, wherein said cell is a yeast cell

30 A process for producing a PRO polypeptides comprising culturing the host cell of Claim

26 under conditions suitable for expression of said PRO polypeptide and recovering said PRO polypeptide from the cell culmre

31 An isolated polypeptide having at least 80% ammo acid sequence identity to an ammo acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO 2),

Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10) 32 An isolated polypeptide scoring at least 80% positives when compared to an ammo acid sequence selected from the group consisting of the amino acid sequence shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10)

33 An isolated polypeptide having at least 80% ammo acid sequence identity to an amino acid sequence encoded by the full length coding sequence of the DNA deposited under any of the ATCC accession numbers shown in Table 8

34 A chimeric molecule comprising a polypeptide according to any one of Claims 31 to 33 fused to a heterologous ammo acid sequence

35 The chimeric molecule of Claim 34, wherein said heterologous amino acid sequence is an epitope tag sequence

36 The chimeric molecule of Claim 34, wherein said heterologous ammo acid sequence is a

Fc region of an immunoglobulin

37 An antibody which specifically binds to a polypeptide according to any one of Claims 31 to 33

38 The antibody of Claim 37, wherein said antibody is a monoclonal antibody, a humanized antibody or a single-chain antibody

39 Isolated nucleic acid having at least 80% nucleic acid sequence identity to (a) a nucleotide sequence encoding the polypeptide shown m Figure 2 (SEQ ID NO 2), Figure

4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10), lacking its associated signal peptide,

(b) a nucleotide sequence encoding an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10), with its associated signal peptide, or

(c) a nucleotide sequence encoding an extracellular domain of the polypeptide shown m Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10), lacking its associated signal peptide 40 An isolated polypeptide having at least 80% ammo acid sequence identity to

(a) the polypeptide shown m Figure 2 (SEQ ID NO 2) Figure 4 (SEQ ID NO 4) Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10), lacking its associated signal peptide,

(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10) with its associated signal peptide, or

(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID NO 2), Figure 4 (SEQ ID NO 4), Figure 6 (SEQ ID NO 6), Figure 8 (SEQ ID NO 8) or Figure 10 (SEQ ID NO 10) lacking its associated signal peptide

41 A method of stimulating the proliferation of T lymphocytes, said method comprising contactmg T lymphocytes with a PR0526, PR0719, PR0725 or PRO1031 polypeptide in an amount sufficient to stimulate the proliferation of said T lymphocytes

42 The method according to Claim 41 wherein said PR0526 polypeptide has at least about

85 % amino acid sequence identity with the amino acid sequence shown m Figure 2 (SEQ ID NO 2)

43 The method accordmg to Claim 41 , wherein said PR0719 polypeptide has at least about 85 % amino acid sequence identity with the amino acid sequence shown m Figure 4 (SEQ ID NO 4)

44 The method according to Claim 41 , wherein said PR0725 polypeptide has at least about 85 % ammo acid sequence identity with the amino acid sequence shown in Figure 6 (SEQ ID NO 6)

45 The method according to Claim 41 , wherein said PRO 1031 polypeptide has at least about 85 % amino acid sequence identity with the amino acid sequence shown m Figure 8 (SEQ ID NO 8)

46 A method of inhibiting the proliferation of T lymphocytes, said method comprising contacting T lymphocytes with a PRO200 polypeptide in an amount sufficient to inhibit the proliferation of said T lymphocytes

47 The method according to Claim 46, wherein said PRO200 polypeptide has at least about 85 % ammo acid sequence identity with the ammo acid sequence shown m Figure 10 (SEQ ID NO 10)

48 A method of inducing the release of TNF α from cells capable of releasing TNF-α said method comprising contacting said cells with a PRO 1031 polypeptide in an amount sufficient to induce the release of TNF-α from said cells 49 The method according to Claim 48, wherein said PRO 1031 polypeptide has at least about % amino acid sequence identity with the amino acid sequence shown in Figure 8 (SEQ ID NO 8)