CA2343006A1

CA2343006A1 - Compositions and methods for the treatment of immune related diseases

Info

Publication number: CA2343006A1
Application number: CA002343006A
Authority: CA
Inventors: Sherman Fong; Audrey Goddard; Austin L. Gurney; Daniel Tumas; William I. Wood
Original assignee: Individual
Current assignee: Genentech Inc
Priority date: 1998-09-17
Filing date: 1999-09-15
Publication date: 2000-03-23
Also published as: JP2003524600A; WO2000015797A2; WO2000015797A3; AU6498499A; IL141551A0; KR20010085816A

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 within the major histocompatibility 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 G 1 transition) and results in the expression of a high affinity receptor for IL-2 and sometimes IL-4. The activated T cell progresses through the cycle proliferating and differentiating into memory cells or effector cells.
In addition to the signals mediated through the TCR, activation of T cells involves additional costimulation induced by 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-l, CD56, etc.
T-cell proliferation in a mixed lymphocyte culture or mixed lymphocyte reaction (MLR} is an established indication of the ability of a compound to stimulate the immune system. In many immune responses. inflammatory cells infiltrate the site of injury or infection. The migrating cells may be neutrophilic, eosinophilic, monocytic or lymphocytic. Histologic examination of the affected tissues provides evidence of an immune stimulating or inhibiting responseCurrent Protocols in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.
Immune related diseases can be treated by suppressing the immune response.
Using neutralizing antibodies that inhibit molecules having immune stimulatory activity would be beneficial in 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 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.

Accordingly, 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PRO326 polypeptide. In one aspect, the antibody mimics the activity of a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide (an agonist antibody) or conversely the antibody inhibits or neutralizes the activity of a PR0245, PRO217, PR0301, PR0266, PR0335, PRO331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 molecuie, 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 in 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 anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 antibody, and vectors and recombinant host cells comprising 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide that inhibit one or more of the functions or activities of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide.
In a further embodiment, the invention concerns isolated nucleic acid molecules that hybridize to the complement of the nucleic acid molecules encoding the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 ribozyme and/or triple helix sequence which, in turn, may be used in regulation of the amplified genes.
In another embodiment, the invention concerns a method for determining the presence of a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide comprising exposing a cell suspected of containing the polypeptide to an anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR033 I or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 in the mammal from which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method of diagnosing an immune disease in a mammal. comprising (a) contacting an anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 in the test sample indicates the presence of tumor 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, fluorimetry, 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-PR0245, PR02I7, PR0301, PR0266, PR0335, PR033 I or PR0326 antibody and a carrier (e.g. a buffer) in suitable packaging. The kit preferably contains instructions for using the antibody to detect the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide. In a preferred aspect, the active agent is a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 antibody.
A further embodiment is a method for identifying a compound capable of inhibiting the expression and/or activity of a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide by contacting a candidate compound with a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide is immobilized on a solid support. In another aspect, the non-immobilized component carries a detectable label.
Brief Description of the Drawings Figures lA-D and Table 4 show hypothetical exemplifications for using the below described method to determine % amino acid sequence identity (Figures lA-B) and %
nucleic acid sequence identity (Figures 1C-D) using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid sequence of a hypothetical polypeptide of the invention of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "PRO"
polypeptide of interest is being compared, "PRO-DNA" represents a hypothetical "PRO"-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid molecule of interest is being compared, "X", "Y" and "Z" each represent different hypothetical amino acid residues and "N", "L" and "V" each represent different hypothetical nucleotides.
Figures 2A-P and Table S provide the complete source code for the ALIGN-2 sequence comparison computer program. This source code may be routinely compiled for use owa UMX
operating system to provide the ALIGN-2 sequence comparison computer program.
Figure 3 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0245(L1NQ219), wherein the nucleotide sequence (SEQ
ID NO: 1) is a clone designated herein as "DNA35638". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 4 and Table 6 show the amino acid sequence (SEQ ID NO: 2) of a native sequence PR0245 polypeptide as derived from the coding sequence of Figure 3. Also shown are the approximate locations of various other important polypeptide domains if known.
Figure 5 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0217(UNQ 191 ), wherein the nucleotide sequence (SEQ ID NO: 3) is a clone designated herein as "DNA33094". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 6 and Table 7 show the amino acid sequence (SEQ ID NO: 4) of a native sequence PR0217 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 cDNA containing a nucleotide sequence encoding native sequence PR0301(LJNQ264), wherein the nucleotide sequence (SEQ
ID NO: 5) is a clone designated herein as "DNA40628". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 8 and Table 8 show the amino acid sequence (SEQ ID NO: 6) of a native sequence PR0301 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 PR0266 (LINQ233), wherein the nucleotide sequence (SEQ ID NO: 7) is a clone designated herein as "DNA37150". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 10 and Table 9 show the amino acid sequence (SEQ ID NO: 8) of a native sequence PR0266 polypeptide as derived from the coding sequence of Figure 9. Also shown are the approximate locations of various other important polypeptide domains if known.
Figure 11 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0335 (UNQ287V), wherein the nucleotide sequence (SEQ ID NO: 9) is a clone designated herein as "DNA41388". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 12 and Table 10 show the amino acid sequence (SEQ ID NO: 10) of a native sequence PR0335 polypeptide as derived from the coding sequence of Figure I 1. Also shown are the approximate locations of various other important polypeptide domains if known.
Figure 13 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0331 (UNQ292), wherein the nucleotide sequence (SEQ
ID NO: 11) is a clone designated herein as "DNA40981". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 14 and Table 11 show the amino acid sequence (SEQ ID NO: 12) of a native sequence PR0331 polypeptide as derived from the coding sequence of Figure 13. Also shown are the approximate locations of various other important polypeptide domains if known.
Figure 15 shows the nucleotide sequence of a cDNA containing a nucleotide sequence encoding native sequence PR0326 (UNQ287), wherein the nucleotide sequence (SEQ
ID NO: 13) is a clone designated herein as "DNA37140". Also presented in bold font and underlined are the positions of the respective start and stop codons.
Figure 16 and Table 12 show the amino acid sequence (SEQ ID NO: 14) of a native sequence PR0331 polypeptide as derived from the coding sequence of Figure 15. 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 inciuded 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, neoplasia, etc.
The term "T cell mediated" disease means a disease in which T cells directly or indirectly mediate or otherwise contribute to a morbidity in a mammal. The T cell mediated disease may be associated with cell mediated effects, lymphokine mediated effects, etc., and even effects associated with B cells if the B cells are stimulated, for example, by the lymphokines secreted by T cells.
Examples of immune-related and inflammatory diseases. some of which are imune or T cell mediated, which can be treated according to the invention include systemic lupus erythematosis, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjsgren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrornbocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial 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 and fibrotic lung diseases such as 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 eosinophilic pneumonias, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft -versus-host-disease. Infectious diseases include AIDS (HIV
infection), hepatitis A, B, C, D, and E, 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.
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 (neutrophilic, eosinophilic, monocytic, lymphocytic cells), antibody production, auto-antibody production, complement production, 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 inflammatory cells (neutrophilic, eosinophilic, monocytic, lymphocytic) into cellular 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.
"Chronic" administration refers to administration of the agents) 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.

"Carriers" as used herein include pharmaceutically 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 earner is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrzns; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
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. I~3~, I~zs, y9o and Re~gb), 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 adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g.
paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinoreibine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, 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 onapristone.
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 overexpressine 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 G 1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G 1 also spill over into S-phase arrest, for example, DNA
alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, 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 "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide;
inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs} such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-l, IL-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-alpha or TNF-beta; 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 culture and biologically active equivalents of the native sequence cytokines.
As used herein, a "PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide" refers to a native sequence PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 having the same amino acid sequence as a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 derived from nature. Such native sequence PR024~. PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 can be isolated from nature or can be produced by recombinant and/or synthetic means. The term specifically encompasses naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326. In one embodiment of the invention, the native sequence PR0245, PR02I7, PR0301, PR0266, PR0335, PR0331 or PR0326 is a mature or full-length native sequence PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or comprising the amino acid sequences shown in Figures 4, 6, 8, 10, 12, 14 and 16.
The term "polypeptide of the invention" refers to each individual PR0245, PR0217, PR0301. PR0266, PR0335, PR0331 or PR0326 polypeptide. All disclosures in this specification which refer to the "polypeptide of the invention" or to "the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 polypeptide of the invention individually. The term "compound of the invention"
includes the polypeptide of the invention, as well as agonist antibodies for and antagonist antibodies to these polypeptide, peptides or small molecules having agonist or antagonist activity developed from the polypeptide, etc.
The term "polypeptide of the invention" also includes variants of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptides. A "variant" polypeptide means an active polypeptide as defined below having at least about 80% amino acid sequence identity with the amino acid sequence of the PR024~, PR0217, PR0301, PR0266, PR0335, PR0331 or polypeptides . Such variant polypeptides include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and/or C-terminus, as well as within one or more internal domains. Ordinarily, a variant polypeptide will have at least about 80% amino acid sequence identity, more preferably at least about 81% amino acid sequence identity, more preferably at least about 82% amino acid sequence identity, more preferably at least about 83%
amino acid sequence identity, more preferably at least about 84% amino acid sequence identity, more preferably at least about 85% amino acid sequence identity, more preferably at least about 86%
amino acid sequence identity, more preferably at least about 87% amino acid sequence identity, more preferably at least about 88% amino acid sequence identity, more preferably at least about 89%
amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, more preferably at least about 91 % amino acid sequence identity, more preferably at least about 92%
amino acid sequence identity, more preferably at least about 93% amino acid sequence identity, more preferably at least about 94% amino acid sequence identity, more preferably at least about 95%
amino acid sequence identity, more preferably at least about 96% amino acid sequence identity, more preferably at least about 97% amino acid sequence identity, more preferably at least about 98%
amino acid sequence identity and yet more preferably at least about 99% amino acid sequence identity with the amino acid sequence of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptides.
Variants do not encompass the native polypeptide sequence.
Ordinarily, variant polypeptides of the invention are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30 amino acids in length, more often at least about 40 amino acids in length, more often at least about SO
amino acids in length, more often at least about 60 amino acids in length, more often at least about 70 amino acids in length, more often at least about 80 amino acids in length, more often at least about 90 amino acids in length, more often at least about 100 amino acids in length, more often at least about 150 amino acids in length, more often at least about 200 amino acids in length, more often at least about 300 amino acids in length, or more.
"Percent (%) amino acid sequence identity" with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are Il identical with the amino acid residues in a sequence of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptides, 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 amino 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, ALIGN-2 or Megalign (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 obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 2A-P.
The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Figures 2A-P 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.
TXLJ510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Figures 2A-P. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX
V4.OD. AlI sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given amino 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 given amino 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 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 amino 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, Figures 1 A-B demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program.
However, % 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.
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, mufti-pass e-value = 0.01, constant for mufti-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 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 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 given amino 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 amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
Also included within the term "polypeptides of the invention" are polypeptides which in the context of the amino acid sequence identity comparisons performed as described above, include amino acid residues in the sequences compared that are not only identical, but also those that have similar properties. These polypeptides are termed "positives". 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 1 below) of the amino acid residue of interest. For purposes herein, the % value of positives of a given amino 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 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 amino acid sequence B, the %
positives of A to B will not equal the % positives of B to A.

An " invention variant polynucleotide" or "invention variant nucleic acid sequence" means a nucleic acid molecule which encodes an active polypeptide of the invention as defined herein and which has at least about 80% nucleic acid sequence identity with the nucleotide acid sequence of DNA35638, DNA33094, DNA40628, DNA37150, DNA41388, DNA40981, or DNA37140 or a specifically derived fragment thereof. Ordinarily, an invention variant poiynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least about 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity, more preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably at least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence of DNA35638, DNA33094, DNA40628, DNA37150, DNA41388, DNA40981, or DNA37140 or a derived fragment thereof.
Variants do not encompass the native nucleotide sequence. In this regard, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of invention variant polynucleotides having at least about 80% nucleic acid sequence identity to nucleotides DNA35638, DNA33094, DNA40628, DNA37I~0, DNA41388, DNA40981, or DNA37140 will encode a polypeptide having an amino acid sequence which is identical to the amino acid sequence of a PR0245, PR0217, PR0301, PR0266, PR033s, PR0331 or PR0326 or the amino acid sequence encoded by the clones deposited with the ATCC described below.
Ordinarily, invention variant polynucleotides are at least about 30 nucleotides in length, often at least about 60 nucleotides in length, more often at least about 90 nucleotides in length, more often at least about 120 nucleotides in length, more often at least about 150 nucleotides in length, more often at least about 180 nucleotides in length, more often at least about 210 nucleotides in length, more often at least about 240 nucleotides in length, more often at least about 270 nucleotides in length, more often at least about 300 nucleotides in length, more often at least about 450 nucleotides in length, more often at least about 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.

"Percent (%) nucleic acid sequence identity" with respect to the invention polypeptide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in an invention polypeptide-encoding 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 ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (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, %
nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Figures 2A-P.
The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Figures 2A-P 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.
TXLJ510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Figures 2A-P. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX
V4.OD. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, 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, Figures 1 C-D 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".
Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program.
However, % 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.
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, mufti-pass e-value = 0.01, constant for mufti-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSiJM62.
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 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.
In other embodiments, invention variant polynucleotides are nucleic acid molecules that encode an active polypeptide of the invention and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding the full-length invention polypeptide. Invention variant polypeptides include those that are encoded by an invention variant polynucleotide.
An "isolated" nucleic acid molecule encoding a polypeptide of the invention is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the polypeptide-encodin~ nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule encoding a polypeptide of the invention includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express a polypeptide of the invention where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence. and a ribosome 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 ribosome 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.
"Stringency" of hybridization reactions is readily determinable by one of ordinary 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 hybridizable 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 Bio(ouy, Wiley Interscience Publishers, ( 1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: ( I ) employ low ionic strength and high temperature for washing, for example 0.015 M
sodium chioride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at SOC;

(2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1%
bovine serum albumin/0. I % Ficoll/0. I % polyvinylpyrrolidone/SOmM sodium phosphate buffer at pH
6.~ with 750 mM sodium chloride, 7~ mM sodium citrate at 42C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), SO mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ug/ml), 0.1% SDS, and 10% dextran sulfate at 42C, with washes at 42C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at SSC, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at SSC.
"Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: 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 37C in a solution comprising: 20% formamide, 5 x SSC (150 mM
NaCI, 15 mM
trisodium citrate), SO 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-SOC. 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 polypeptide of the invention 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 amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
"Active" or "activity" in the context of variants of the polypeptide of the invention refers to forms) of proteins of the invention which retain the biologic and/or immunologic activities of a native or naturally-occurnng polypeptide of the invention.
"Biological activity" in the context of an antibody or another molecule that can be identified by the screening assays disclosed herein (e.g. an organic or inorganic small molecule, peptide, etc.) is used to refer to the ability of such molecules to induce or inhibit infiltration of inflammatory cells into a tissue, to stimulate or inhibit T-cell proliferation and to stimulate or inhibit lymphokine release by cells. Another preferred activity is increased vascular permeability or the inhibition thereof.
The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide of the invention 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 polypeptide of the invention disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides of the invention , peptides, small organic molecules, etc.
A "small molecule" is defined herein to have a molecular weight below about 600 daltons.
"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the samestructural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity.
Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. The term "antibody" is used in the broadest sense and specifically covers, without limitation, intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 antibody is an antibody which immunologically binds to a PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide. The antibody may bind to any domain of the polypeptide of the invention which may be contacted by the antibody. For example, the antibody may bind to any extracellular domain of the polypeptide and when the entire polypeptide is secreted, to any domain on the polypepetide which is available to the antibody for binding.
"Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light- chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarily-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR
regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., NIHPubl.
No.91-3242, Vol. I, pages 647-669 ( 1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The term "hypervariable region" or "complementarily-determining regions"
(CDRs) as used herein define a subregion within the variable region of extreme sequence variability of the antibody, which form the antigen-binding site and are the main determinants of antigen specificity. According to one definition, they can be, for example, residues (Kabat nomenclature) 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region and residues (Kabat nomenclature) 31-35 (H1), 50-65 (H2), 95-102 (H3) in the heavy chain variable region. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD.
[ 1991 ]. Alternatively, or in combination with the region defined by Kabat, the hypervariable region can be the "hypervariable loop", comprising, for example, residues (Chothia nomenclature) 26-32 (L1), 50-53 (L2), 91-96 (L3) in the light chain variable region and residue (Chothia nomenclature) 26-32 (Hl), 53-SS (L2) and 96-101 (L3); Chothia and Lesk, J. Mol. Biol. 196: 901-917 [1987].
"Framework" or "FR" residues are those variable domain residues of relatively low sequence variability which lie in between the CDR regions.
"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')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Ena.
8(10):1057-1062 [1995]);
single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen- binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc"
fragment, whose PR0245, PR0217, PR0301, Pro266, pro335, pro331 or pro326 reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 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 -binding 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 VH-VL dimer.
Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only 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 (CH 1 ) 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 hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues) 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 (K) and lambda (~,), based on the amino acid sequences of 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 into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called called a, 8, E, 7, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
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. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [ 1975], or may be made by recombinant DNA methods (see, c.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. See also U.S Patent Nos. 5,750,373, 5,571,698, 5,403.484 and 5,223,4()9 which describe the preparation of antibodies using phagemid and phage vectors.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Moirison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 [1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarily-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, 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 sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 ( 1986); Reichmann et al., Nature, 332:323-329 [1988]; and Presta, Curr. O~ Struct. Biol., 2:593-596 (1992). The term "humanized antibody" includes a "primatized" antibody where the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest. Antibodies containing residues from Old World monkeys are also possible within the invention. See, for example, U.S. Patent Nos. 5,658,570; 5,693,780; 5,681,722;
5,750,105; and 5,756,096.
Antibodies and fragments thereof in this invention also include "affinity matured" antibodies in which an antibody 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 in the affinity of the matured antibody for the antigen relative to the starting antibody. Typically, the starting antibody 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 in which many 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 Theranv, 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 antibody 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/Technolo~y 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994);
Fishwild et al., Nature BiotechnoloQV 14, 845-51 (1996); Neuberger, Nature Biotechnoloey 14, 826 ( 1996}; Lonberg and Huszar, Intern. Rev. lmmunol. 13 65-93 ( 1995).
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and V~, domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to forth the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-3 I 5 ( 1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). 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 93/11161; and Hollinger 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 environment 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 compound of the invention will be purified ( 1 ) to greater than 95% by weight of the compound 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-terminal 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 stain. Isolated compound, e.g. antibody or polypeptide, includes the compound in situ within recombinant cells since at least one component of the compound's natural environment will not be present. Ordinarily, however, isolated compound 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 compound, e.g. antibody or polypeptide, so as to generate a "labelled" compound. 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 compound 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), polysaccharides (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, phospholipids and/or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the Iiposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
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.
II. Compositions and Methods of the Invention 1. Preparation of th~o)ypeptides of the invention The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 (UNQ219, UNQ191, UNQ264, UNQ233, UNQ287V, UNQ292 or UNQ287 respectively). In particular, cDNA encoding a PR0245, PR0217, PR0301, PR0266, PR033~, PR03 ~ 1 or PR0326 polypeptide has 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 LJNQ 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 DNA35638, DNA33094. DNA40628, DNA37150, DNA41388, DNA40981 and DNA37140 as well as all further native homologues and variants included in the foregoing definition of PR0245, PR0217. PR0301, PR026G, PR0335, PR0331 or PR0326, will be referred to as PR0245, PR0217, PR0301, PR0266. PR0335, PR0331 or PR0326 or simply as "the polypeptide of the invention", regardless of their origin or mode of preparation.
The description below relates primarily to production of the polypeptide of the invention by culturing cells transformed or transfected with a vector containing nucleic acid which encodes of the 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 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); Mernfield, J. Am. Chem. Soc., 85:2149-2154 ( 1963)]. In vitro 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 polypeptide of the invention may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length polypeptide.
In addition to the full-length native sequence polypeptides described herein, it is contemplated that variants can be prepared. Variants can be prepared by introducing appropriate nucleotide changes into the DNA, and/or by synthesis of the desired polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the polypeptide of the invention, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence or in various domains of the polypeptide of the invention described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S.
Patent No. 5,364,934.
Variations may be a substitution, deletion or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the native sequence polypeptide sequence. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the polypeptide of the invention. Guidance in 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 polypeptide of the invention with that of homologous known protein molecules and minimizing the number of amino 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 leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about I 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.
Polypeptide fragments of the polypeptides of the invention are also within the scope of the invention. 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 invention polypeptide.
Polypeptide fragments may be prepared by any of a number of conventional techniques.
Desired peptide fragments may be chemically synthesized. An alternative approach involves generating fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave 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, polypeptide fragments share at least one biological and/or immunological activity with the native invention polypeptide.
In particular embodiments, conservative substitutions of interest are shown in Table 1 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 1, or as further described below in reference to amino acid classes, are introduced and the products screened.

Table 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;

norleucine leu Leu (L) norleucine; ile; val;

met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; 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) ile; leu; met; phe;

ala; norleucine leu Substantial modifications in function or immunological identity of the invention polypeptide are accomplished by selecting substitutions 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 conforniation, (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, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asp, gln, his, lys, arg;
(S) 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 substitution sites or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as oligonucleotide-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., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:31 S ( 1985)j, restriction selection mutagenesis [Wells et al., Philos. Traps. R. Soc. London SerA, 317:415 ( 1986)] or other known techniques can be performed on the cloned DNA to produce the variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine.
Alanine 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 amino 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 isoteric amino acid can be used.
Covalent modifications of polypeptides of the invention are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an invention polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking the invention polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-polypeptide antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl 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 -amino 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 invention 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 polypeptide (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. 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 polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence polypeptide (for O-linked glycosylation sites). The amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the 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 polypeptide of the invention is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the polypeptide of the invention may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino 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., 118:131 ( 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. Enzvmol., 138:350 (1987).
Another type of covalent modification comprises linking the invention polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835;
4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.

The polypeptide of the present invention may also be modif ed in a way to form a chimeric molecule comprising the invention polypeptide fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a ehimeric molecule comprises a fusion of the invention polypeptide 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 polypeptide of the invention. The presence of such epitope-tagged forms of the polypeptide of the invention can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the polypeptide of the invention to be readily purified by affinity 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-histidine-glyeine (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 9E10 antibodies thereto [Evan et al., Molecular and Cellular BioloQV, 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., BioTechnoloev, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an -tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; 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 ehimeric molecule may comprise a fusion of the polypeptide of the invention 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 Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an invention polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG 1 molecule. For the production of immunoglobulin fusions see also US
Patent No.

5,428,130 issued June 27, 1995.
Isolation of DNA Encodine the Polypeptide of the Invention DNA encoding the polypeptide of the invention may be obtained from a cDNA
library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level.
Accordingly, human DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The gene encoding the polypeptide of the invention may also be obtained from a genomic library or by oligonucleotide synthesis.
Libraries can be screened with probes (such as antibodies to the polypeptide of the invention or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA 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 Laboratory Press, 1989). An alternative means to isolate the gene encoding the polypeptide of the invention 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 in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling.
Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., su ra.
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available 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 through sequence alignment using computer software programs such as ALIGN, DNAstar, and INHERIT
which employ various algorithms to measure homology.
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 conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.
ii. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described herein for production of the polypeptides of the invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture 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., s_~ra.

Methods of transfection are known to the ordinarily skilled artisan, for example, CaP04 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 or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium 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 withaut such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virolo~y, X2:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations 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, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enz~molo~v, 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 prokaryates include but are not limited to eubacteria, 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. coli strain W3110 (ATCC 27,325) and KS 772 (ATCC
53,635).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding the polypeptides of the invention. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
Suitable host cells for the expression of glycosylated polypeptides of the invention are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf~3, 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 CV 1 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 ovary cells/-DHFR -(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
USA, 77:4216 ( 1980));
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 ( 1980)); human lung cells (W 138, 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.

iii. Selection and Use of a Replicable Vector The nucleic acid (e.g., cDNA or genomic DNA) encoding the polypeptides of the invention may be inserted into a replicable 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, phagemid 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 sites) 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 polypeptide of the invention may be produced recombinantly 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 DNA
encoding the polypeptide of the invention 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, peniciilinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces alpha-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. 1990), or the signal described in WO
90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion 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 cloning 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 2u 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., ampicillin, 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 nucleic acid encoding the polypeptide of the invention, 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 hp 1 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 1 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 nucleic acid sequence encoding the polypeptide of the invention 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 beta-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 Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide of the invention.
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 ReQ., 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, triosephosphate 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 described in EP 73,657.
Transcription of the polypeptide of the invention 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 polypeptide of the invention 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, alpha-fetoprotein, and insulin). Typically, 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 coding sequence of the polypeptide of the invention, but is preferably located at a site S' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will 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 the polypeptide of the invention .
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of the polypeptide of the invention in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 ( 1981 ); Mantei et aL, Nature, 281:40-46 ( 1979); EP
117,060; and EP I 17,05 8.
iii. Detecting Gene Expression Gene expression may be measured in a sample directly, for example, 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. 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 immunological methods, such as immunohistochemical staining of cells or 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 the inventive polypeptide or against a synthetic peptide based on the DNA
sequences provided herein or against exogenous sequence fused to DNA encoding the polypeptide of the invention and encoding a specific antibody epitope.
iv. Purification of Polypeptide Forms of the polypeptide of the invention 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 the polypeptide of the invention can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
It may be desired to purify the polypeptide of the invention from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as DEAE;
chromatofocusing; 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 polypeptide of the invention . Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in EnzymoloQV, 182 ( 1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York ( 1982). The purification steps) selected will depend, for example, on the nature of the production process used and the particular polypeptide of the invention produced.
Tissue Distribution The location of tissues expressing the 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 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 polypeptide of the invention or against a synthetic peptide based on the DNA sequences encoding the polypeptide of the invention or against an exogenous sequence fused to a DNA encoding a polypeptide of the invention and encoding a specific antibody epitope.
General techniques for generating antibodies, and special protocols for Northern blotting and in situ hybridization are provided below.
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-PR0245. PR0217, PR0301, PR0266. PR0335, PR0331 or PR0326 antibodies to inhibit the effect of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-I58 (CRC Press, Inc., 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 insolubilized 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 antibody binds to the analyte, thus forming an insoluble three-part complex.
See, e.g., U.S. 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 assay). 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.
4. 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 J. E. Coligan, A. M. Kruisbeek, D. H.
Marglies, E. M. Shevach, W.
Strober, National Insitutes of Health, Published by John Wiley & Sons, Inc. In this assay, the ability of a test compound to stimulate the proliferation of activated T cells is assayed. A suspension of responder T cells is cultured with allogeneic stimulator cells and the proliferation of T cells is measured by uptake of tritiated 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 in responsiveness in this assay in part reflect differences in IL-2 production by the responding cells. The MLR results can be verified by a standard lymphokine (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 crosslinking increases lymphokine 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 glycoproteins in the Ig 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, C.
A. and Allison, J. P., Curr. Opin. Immunol. ( 1997) 9:396. Schwartz, R. I-L, Cell ( 1992) 7 I :1065;
Linsey, P. S. and Ledbetter, J. A., Annu. Rev. Immunol. (1993) 11:191; June, C. H. et al., Immunol.
Today ( 1994) 15:321; Jerkins, M. K., Immunity ( 1994) 1:405. In a costimulation assay, the polypeptides of the invention are assayed for T cell costimulatory or inhibitory activity.
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 in 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 PR024~, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 polypeptide or an agonist antibody therefor.
Direct use of a stimulating compound as in the invention has been validated in experiments with 4-1 BB glycoprotein, a member of the tumor necrosis factor receptor family, which binds to a ligand (4-1BBL) expressed on primed T cells and signals T cell activation and growth. Alderson, M.
E. et al., J. Immunol. ( 1994) 24:2219.
The use of an agonist stimulating compound has also been validated experimentally.
Activation of 4-1BB by treatment with an agonist anti-4-1BB antibody enhances eradication of tumors. Hellstrom, I. and Hellstrom, K. E., Crit. Rev. Immunol. ( 1998) 18:1.
Immunoadjuvant therapy for treatment of tumors, described in 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 anti-CTLA-4 antibodies which enhance T cell proliferation, presumably by removal of the inhibitory signal caused by CTLA-4 binding. Walunas, T. L. et al., Immunity ( 1994) 1:405.
On the other hand, polypeptides of the invention, as well as other compounds of the invention, which are direct inhibitors of T cell proliferation/activation and/or lymphokine 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 in 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 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 andlor lymphokine secretion. Blocking the stimulating effect of the polypeptides suppresses the immune response of the mammal. This use has been validated in experiments using an anti-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.

5. Animal Models The results of the cell based in vitro assays can be further verified using in vivo animal 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 antagonists 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 rejection. 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, H. Jr. and Sachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., Raven Press, NY, 1989, 889-992. A suitable procedure is described in detail in Current Protocols in Immunology, above, unit 4.4. Other transplant rejection models which can be used to test the compounds of the invention are the allogeneic heart transplant models described by Tanabe, M. et al., Transplantation (1994) 58:23 and Tinubu, S. A. 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 vivo 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 in the central nervous system. EAE is generally considered to be a relevant animal model for MS in humans. Bolton, C., Multiple Sclerosis (1995) I :143. Both acute and relapsing-remitting models have been developed. The compounds of the invention can be tested for T cell stimuiatory or inhibitory activity against immune mediated demyelinating 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 oligodendrocytes or Schwann cells are grafted into the central nervous system as described in Duncan, I. D. 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 elicitation phase. The elicitation 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, A. M.
Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc., 1994, unit 4.2.
See also Grabbe, S. and Schwarz, T., Immun. Today 19(1):37-44 (1998) .
An animal model for arthritis is collagen-induced arthritis. This model shares clinical, histological and immunological 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 compounds of the invention can be tested for activity against autoimmune arthritis using the protocols described in Current Protocols in Immunology, above, units I5.5. See also the model using a monoclonal antibody to CD18 and VLA-4 integrins described in Issekutz, A. C. 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 compounds of the invention for activity and effectiveness in the treatment of asthma are described by Wolyniec, W. W. et al., Am. J. Respir. Cell Mol. Biol.
(1998) 18:777 and the references cited therein.
Additionally, the 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, M. P. et al., Nat. Med. ( 1997) 3:183, in which the mice demonstrate histopathologic skin lesions resembling psoriasis. Another suitable model is the human skin/scid mouse chimera prepared as described by Nickoloff, B. J. 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, L1.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., Cell 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 in 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 er al., Proc. Nati. Acad. Sci. USA 89, 6232-636 ( 1992).
The expression of the transgene in 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 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. Typically, 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 into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined 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, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. 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 germ to create a "knock out" animal.
Progeny harboring the homologously recombined 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 recombined 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.

6. ImmunoAdjuvant Therany In one embodiment, the immunostimulating 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, BAGS 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, C. 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 vivo.
Melero. I. et al., Nature Medicine ( 1997) 3:682; Kwon, E. D. et aL, Proc.
Natl. Acad. Sci. USA
( 1997) 94:8099; Lynch, D. H. et al., Nature Medicine ( 1997) 3:625; Finn, O.
J. and Lotze, M. T., J.
Immunol. ( 1998) 21:114. The stimulatory 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/aetivation and an antitumor response to tumor antigens. The growth regulating, cytotoxic, or chemotherapeutic agent may be administered in conventional amounts using known administration regimes. Immunostimulating activity by the compounds of the invention allows reduced amounts of the growth regulating, cytotoxic, or chemotherapeutic agents thereby potentially lowering the toxicity to the patient.
Screenine Assays for Drug_Candidates Screening assays for drug candidates are designed to identify compounds that bind 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 particulariy suitable for identifying small molecule drug candidates.
Small molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptide-immunogiobulin fusions, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic 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 poiypeptide 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 carnes 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-protein 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 arid Nathans [Proc.
Natl. Acad. Sci. USA 89, 5789-5793 (199I)]. 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 in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL 1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for beta-galactosidase.
A complete kit (MATCHMAKERT"') for identifying protein-protein 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 in specif c protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
In order to fmd compounds that interfere with the interaction of a gene identified herein and other infra- or extracellular components can be tested, a reaction mixture is usually prepared containing the product of the gene and the infra- 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 infra- or extracellular component present in the mixture is monitored as described above. The formation of a complex in the control reactions) 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.

8. Compositions and Methods for the Treatment of Immune Related Diseases The compositions useful in the treatment of immune related diseases include, without limitation, antibodies, small organic and inorganic molecules, peptides, phosphopeptides, antisense and ribozyme molecules, triple helix molecules, etc. that inhibit or stimulate immune function, for example, T cell proliferation/activation, lymphokine release, or immune cell infiltration.
For example, antisense RNA and RNA molecule act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. When antisense DNA is used, oligodeoxyribonucleotides 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 ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g. Rossi, Current Biolo~y 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 single-stranded and composed of deoxynucleotides. The base composition of these oiigonucleotides is designed such that it promotes triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of purines or pyrimidines 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.
Antibodies Some of the most promising drug candidates according to the present invention are antibodies and antibody fragments which may inhibit (antagonists) or stimulate (agonists) T cell proliferation, eosinophil infiltration, etc.
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 andlor adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. 'The immunizing agent may include the polypeptide of the invention or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-'CDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
ii. Monoclonal Antibodies Antibodies which recognize and bind to the polypeptides of the invention or which act as agonist therefor may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma -methods, such as those described by Kohler and Milstein, Nature, 256:495 ( 1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of 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 polypeptide of the invention 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 hybridoma cell [coding, 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 hybridoma 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 phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Tvpe Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromveloma 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 Techn9ues and Applications, Marcel Dekker, Inc., New York, ( 1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide of the invention or having similar activity as the polypeptide of the invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro 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 by 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, su ra]. 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 i~r vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example. protein A-Sepharose, hydroxyapatite 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 in place of the homologous murine sequences [U.S.
Patent No. 4,816,567; Morrison et al., su ra] 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 of 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 are preferably 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 heavy 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 amino acid residue or are deleted so as to prevent crosslinking.
In vitro 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.
iii. Human and Humanized Antibodies The 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')2 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 of 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 comprise substantially all of at least one, and typically two, variable domains, 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 of a 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. Humanization can be essentially performed following the method of Winter and coworkers [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 analoeous 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 Th_ eraov, 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 antibody 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/TechnoloQV 10, 779-783 ( 1992); Lonberg et al., Nature 368 856-859 ( 1994); Morrison, Nature 368, 8I2-13 (1994); Fishwild et al.. Nature Biotechnolo~y 14, 845-51 (1996); Neuberger, Nature BiotechnoloQV 14, 826 ( 1996); Lonberg and Huszar, Intern. Rev.
Immunol. 13 65-93 ( 1995).
iv. Bisgecific 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 may be for the polypeptide of the invention, 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 coexpression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 [ 1983]). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (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 in WO 93/08829, published 13 May 1993. and in Traunecker et al., EMBO J., 10:3655-3659 (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 hinge, 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 cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example. Suresh et al., Methods in Enzymolo~y, 121:210 ( 1986).
v. Heteroconiueate Antibodies 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 crosslinking agents.
For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
vi. Effector function en ineerin~
It may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating an immune related disease, for example.
For example cysteine residues) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cvtotoxicity (ADCC). See Caron et al., J. Exp, Med.
176:1191-1195 (1992) and Shopes, B. 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 which 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).
vii. Immunoconjugates 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 thereofl. or a radioactive isotope (i.e.. a WO 00/15?97 PCT/US99/21547 radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 2'ZBi,'3'I,'3'In, 9°Y and'g6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothioeyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tisue 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) which is conjugated to a cvtotoxic agent (e.g. a radionucleotide).
viii. Immunoli~osomes The proteins, antibodies, etc. disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art, such as described in 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 with a lipid composition comprising phosphatidylcholine. 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 interchange reaction. A chemotherapeutic agent (such as doxorubicin) may be optionally St contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81 ( 19) 1484 ( 1989).
10. Pharmaceutical Compositions The active molecules of the invention, 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, in the form of pharmaceutical compositions.
Therapeutic formulations of the active molecule, preferably a polypeptide or antibody of the invention. are prepared for storage by mixing the active molecule having 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 methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); iow molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpymolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, 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 TWEENr"', PLURONICSTM
or polyethylene glycol (PEG).
Compounds identified by the screening assays of the present invention 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 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 interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, 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 hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT T"' (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic 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 moisture at 37C, 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-disulfide 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.
11. Methods of Treatment It is contemplated that the 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 into 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 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 vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocvtopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis. tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Bane 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 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 eosinophilic pneumonias, idiopathic pulmonary 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. Antibodies either directly or indirectly mediate tissue injury.
Though T lymphocytes have not been shown to be directly involved in 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 joint 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 in 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, vasculitis, and cutaneous ulcers. The second form of extra-articular disease is the so called Felty's syndrome which occurs late in the RA disease course, sometimes after joint disease has become quiescent, and involves the presence of neutropenia, thrombocvtopenia and splenomegaly. This can be accompanied by vasculitis 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, pleuritis, coronary arteritis, intestitial pneumonitis with pulmonary fibrosis, keratoconjunetivitis 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 ItA; some patients which are rhematoid factor positive are classified as juvenile rheumatoid arthritis. The disease is sub-classified into three major categories: pauciarticular, polyarticular, 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 sponylitis, 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 (sclerodetma) has an unknown etiology. A hallmark of the disease is induration of the skin; 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 in many patients. ICAM-1 is often upregulated on the cell surface of fibroblasts 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 peristalsisimotility;
kidney: concentric subendothelial intimal proliferation affecting small arcuate and interlobular arteries with resultant reduced renal cortical blood flow, results in proteinuria, azotemia and hypertension;

skeletal muscle: atrophy, interstitial fibrosis: inflammation; lung:
interstitial pneumonitis and interstitial fibrosis; and heart: contraction band necrosis, scarring/fibrosis.
Idiopathic inflammatory myopathies including dermatomyositis, polymyositis and others are disorders of chronic muscle inflammation of unknown etiology resulting in 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 against 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 vasculitis are diseases in which the primary lesion is inflammation and subsequent damage to blood vessels which results in ischemia/necrosis/degeneration to tissues supplied by the affected vessels and eventual end-organ dysfunction in some cases.
Vasculitides 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 vasculitis group include: systemic necrotizing vasculitis: polyarteritis nodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener's granulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.
Miscellaneous vasculitides include: mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease), isolated CNS
vasculitis, Behet's disease, thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizing venulitis. The pathogenic mechanism of most of the types of vasculitis 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 activation, or both.
Sarcoidosis is a condition of unknown etiology which is characterized by the presence of epithelioid 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 hemoglobinuria 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 in other clinical settings, platelet destructioniremoval 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 thyroglobulin, thyroid microsomal antigen (thyroid peroxidase).
Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune destruction of pancreatic islet (3 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 glomerulonephritis and tubulointerstitial 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 producesiinduces 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 demyelination as a result of damage caused to oligodendrocytes or to myelin 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 etiology is unknown; however, viral infections, genetic predisposition, environment, and autoimmunity all contribute. Lesions contain infiltrates of predominantly T lymphocyte mediated, microglial cells and infiltrating macrophages;
CD4+T lymphocytes are the predominant cell type at lesions. The mechanism of oligodendrocyte cell death and subsequent demyelination is not known but is likely T lymphocyte driven.

Inflammatory and Fibroiic Lung Disease. including Eosinophilic Pneumonias;
Idiopathic Pulmonary Fibrosis. and Hypersensitivity Pneumonitis may involve a disregulated immune-inflammatory response. Inhibition of that response would be of therapeutic benefit.
Autoimmune or Immune-mediated Skin Disease including Bullous Skin 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 Grafr rejection 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, E and 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/derivatives/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 iatrogenic (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 in 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 proliferative response in the MLR (or small molecule agonists or antibodies that affected the same receptor in 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 by the neoplasm in other cells.
Antagonism of such inhibitory molecules (either with antibody, small molecule antagonists or other means) enhances immune-mediated tumor rejection.
Additionally, inhibition of molecules with proinflammatary properties may have therapeutic benefit in reperfusion injury; stroke: myocardial infarction; atherosclerosis;
acute lung injury;

hemorrhagic shock; burn; sepsisiseptic shock; acute tubular necrosis;
endometriosis; degenerative joint disease and pancreatic.
The compounds of the present invention, e.g. 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, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, 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 dosing 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 onapristone (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, CD 11 a, CD
18, ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens disclosed herein may be coadministered 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 coadministered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by a 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 polypeptide of the invention.
For the treatment or reduction in the severity of immune related disease, the appropriate dosage of an a compound of the invention will depend on the type of disease to be treated, as defined above, the severity 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 severity of the disease, about I ug/kg to 15 mg/kg (e.g. 0.1-20mgikg) 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 daily dosage might range from about 1 ug/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.
12. 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 in the composition is usually a 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.
13. Diagnosis and Prosnosis 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 in 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 qualitatively 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 cvtometry, fluorimetry, or other techniques known in the art. 'These techniques are particularly suitable, if the overexpressed gene encodes a cell surface protein Such binding assays are performed essentially as decribed above.

In situ detection of antibody binding to the marker gene products can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a histological specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of 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 only, and are not intended to Limit the scope of the present invention in any way.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
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. Unless otherwise noted, the present invention uses standard procedures of recombinant DNA technology, such as those described hereinabove and in 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, inc., N.Y., 1990; Harlow et al., Antibodies: A
Laboraton~ Manual, Cold Spring Harbor Press, Cold Spring Harbor, 1988; Gait, M.J., Oligonucleotide Svnthesis, IRL
Press, Oxford, 1984; R.I. Freshney, Animal Cell Culture, 1987; Coligan et al..
Carrrent Protocols in Immunology, 1991.

Isolation of cDNA clones Encodin~ Human PR0245. PR0217. PR0301, PR0266.
PR0335. PR0331 or PR0326 I Isolation of cDNA Clones Encoding Human PR0245 The extracellular domain (ECD) sequences (including the secretion signal, if any) of from about 950 known secreted proteins li-om 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 (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzy_molo~y 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 encoding PR0245 was assembled relative to the other identified EST sequences, where the consensus sequence was designated herein as DNA30954, and the polypeptide showed some structural homology to transmembrane protein receptor tyrosine kinase proteins.
Based on the DNA30954 consensus sequence, oligonucleotides were synthesized to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for PR024~.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO: 15) reverse PCRprimer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO: 16) Additionally, a synthetic oligonucleotide hybridization probe was constructed from the consensus DNA30954 sequence which had the following nucleotide sequence:
hybridization probe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3' (SEQ ID NO:
17) 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 PR0245 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. 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 NotI site, linked with blunt to SaII
hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD
that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 ( 1991 )) in the unique XhoI and NotI
sites.
DNA sequencing of the clones isolated as described above gave the full-length DNA
sequence for PR0245 [herein designated as UNQ219 (DNA35638)) and the derived protein sequence for PR0245.
The entire nucleotide sequence of UNQ219 (DNA35638) is shown in Figure 3 (SEQ
ID NO:
1 ). Clone UNQ219 (DNA35638) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 89-91 [Kozak et al., supra) and ending at the stop codon at nucleotide positions 1025-1027 (Fig. 3). The predicted polypeptide precursor is 312 amino acids song (Figure. 4; PR0245; SEQ ID NO: 2). Clone UNQ219 (DNA35638) has been deposited with ATCC
on September 17, 1997 and is assigned ATCC Deposit No. 209265.
Analysis of the amino acid sequence of the full-length PR0245 suggests that a portion of it possesses 60% amino acid identity with the human c-myb protein and, therefore, may be a new member of the transmembrane protein receptor tyrosine kinase family.
II. Isolation of cDNA clones Encoding PR0217 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST
databases. The EST databases included public databases (e.g., Dayhof, GenBank), and proprietary databases (e.g. LIFESEQT"', Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altschul, SF and Gish ( 1996), Methods in Enzvmology 266:
460-80 ( 1996); http://blast.wustl/edu/blast/README.html) as a comparison of the ECD protein sequences to a 6 frame translation of the EST sequences. Those comparisons with 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, WA; (http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
Consensus DNA sequences encoding EGF-like homologues were assembled (DNA28726, DNA28730 and DNA28760} using phrap. In some cases, the consensus DNA sequence was extended using repeated cycles of blast and phrap to extend the consensus sequence as far as possible using the three sources of EST sequences listed above.
Based on this 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. The pair of forward and reverse PCR primers (rotated as *.f and *.r, respectively) may range from 20 to 30 nucleotides (typically 24), and are designed to give a PCR
product of 100-1000 by in length. The probe sequences (rotated as *.p) are typically 40-55 by (typically 50) in length. In some cases additional oligonucleotides are synthesized when the consensus sequence is greater than 1-1.5 kbp. In order to screen several libraries for a source of 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 by the in vivo cloning procedure using the probe oligonucleotide and one of the PCR primers.
This library was used to isolate DNA32279, DNA32292 and DNA33094 was fetal kidney, fetal lung and fetal lung, respectively.

RNA for the construction of the cDNA libraries was isolated using standard isolation protocols, e.g., Ausubel et al., Current Protocols in Molecular Biology, from tissue or cell line sources or it was purchased from commercial sources (e.g., Clontech). The cDNA libraries used to isolate the cDNA
clones were constructed by standard methods (e.g., Ausubel et al.) using commercially available reagents (e.g., Invitrogen). The cDNA was primed with oligo dT containing a Noti site, linked with blunt to SaII hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoesis, and cloned in a defined orientation in a suitable cloning vector (pRKSB or pRKSD) in the unique XhoI and NotI sites.
A cDNA clone was sequenced in its entirety. The entire nucleotide sequence of EGF-like homologue PR0217 is shown in Figure 5 (SEQ ID NO: 3). The predicted polypeptide is 379 (PR0217;
Figure 6; SEQ ID NO: 4) amino acids in length with a molecule weight of approximately 41.52 kDa.
The oligonucleotide sequences used in the above procedure were the following:
28726.p (OLI500) (SEQ ID NO: 18) GGGTACACCTGCTCCTGCACCGACGGATATTGGCTTCTGGAAGGCC
28726.f (OLI 502) (SEQ ID NO: 19) ACAGATTCCCACCAGTGCAACC
28726.r (OLI 503) (SEQ ID NO: 20) CACACTCGTTCACATCTTGGC
28730.p (OLI 516) (SEQ ID NO: 21) AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA
28730.f (OLI 517) (SEQ ID NO: 22) AGAGTGTATCTCTGGCTACGC
28730.r (OLI 518) (SEQ ID NO: 23) TAAGTCCGGCACATTACAGGTC
28760.p (OLI 617) (SEQ ID NO: 24) CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCTGCATC
28760.f (OLI 618) (SEQ ID NO: 25) AAAGACGCATCTGCGAGTGTCC
28760.r (OLI 619) (SEQ ID NO: 26) TGCTGATTTCACACTGCTCTCCC
III. Isolation of cDNA clones Encodine Human PR0301 The extracellular domain (ECD) sequences (including the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot public database were used to search EST
databases. The EST databases included public EST databases (e.g., GenBank), a proprietary EST
database (LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzvmology, 266:460-480 ( 1996); http://blast.wustl/edu/blast/README.html] as a comparison of the ECD
protein sequences to a 6 frame translation of the EST sequences. 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; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
A consensus DNA sequence encoding DNA35936 was assembled using phrap. In some cases, the consensus DNA sequence was extended using repeated cycles of blast and phrap to extend the consensus sequence as far as possible using the three sources of EST
sequences listed above.
Based on this consensus sequence, oligoriucleotides 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. Forward and reverse PCR primers (notated as *.f and *.r, respectively) may range from 20 to 30 nucleotides (typically about 24), and are designed to give a PCR product of 100-1000 by in length. The probe sequences (notated as *.p) are typically 40-55 by (typically about 50) in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than 1-1.5 kbp. In order to screen several libraries for a source of 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 by the in vivo cloning procedure suing the probe oligonucleotide and one of the PCR primers.
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 PR0301 gene using the probe oligonucleotide and one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal kidney. The cDNA libraries used to isolated the cDNA clones were constructed by standard methods using commercially available reagents (e.g., Invitrogen, San Diego, CA; Clontech, etc.) The cDNA was primed with oligo dT containing a NotI site, linked with blunt to SaII
hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD
that .does not contain the SfiI site; see, Holmes et al., Science, 253: I 278-1280 1,1991 )) in the unique XhoI and NotI
sites.
A cDNA clone was sequenced in its entirety. The full length nucleotide sequence of native sequence PR0301 is shown in Figure 7 (SEQ ID NO: 5). Clone DNA40628 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 52-54 [Kozak et al., supra] (Fig. 7). The predicted polypeptide (PR0301: Figure 8: SEQ ID NO: 6) is 299 amino acids WO 00!15797 PCT/US99I21547 long with a predicted molecular weight of 32583 daltons and pI of 8.29. Clone DNA40628 has been deposited with ATCC and is assigned ATCC deposit No. 209432.
Based on a BLAST and FastA sequence alignment analysis of the full-length sequence, PR0301 shows amino acid sequence identity to A33 antigen precursor (30%) and coxsackie and adenovirus receptor protein (29%).
The oligonucleotide sequences used in the above procedure were the following:
OLI2162 (35936.f1) (SEQ ID NO: 27) TCGCGGAGCTGTGTTCTGTTTCCC
OLI2163 (35936.p 1 ) (SEQ ID NO: 28) TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT
OLI2164 (35936.f2) (SEQ ID NO: 29) ACACCTGGTTCAAAGATGGG
OLI2165 (35936.r1) (SEQ ID NO: 30) TAGGAAGAGTTGCTGAAGGCACGG
OLI2166 (35936.f3) (SEQ ID NO: 31) TTGCCTTACTCAGGTGCTAC
OLI2167 (35936.r2) (SEQ ID NO: 32) ACTCAGCAGTGGTAGGAAAG
IV Isolation of cDNA Clones Encoding Human PR0266 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 (LIFESEQr"'', Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzymolo~y 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; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html}.
Based on an expression sequence tag 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 PR0266. 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 by in length. The probe sequences are typically 40-55 by in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1-I.Skbp. In order to screen several libraries for a full-length clone. DNA from the libraries was screened by PCR
amplification, as ber Ausubel et al., Current Protocols in Molecular Bioloey;
with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest by the iu vivo clongin procedure using the probe oligonucleotide and one of the primer pairs.
A pair of PCR primers ( forward and reverse) were synthesized:
forward PCR~rimer 5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO: 33) reverse PCR primer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID NO: 34) Additionally, a synthetic oligonucleotide hybridization probe was constructed which had the following nucleotide sequence:
hybridization probe 5'-GGTGGCTATACATGGATAGCAATTACCTGGACACGCTGTCCCGGG-3' (SEQ ID NO: 35) 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 PR0266 gene using the probe oligonucleotide and one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal brain tissue. 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 NotI site, linked with blunt to SaII
hemikinased adaptors, cleaved with NotI, sized appropriately by gel electrophoresis. and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRKSB is a precursor of pRKSD
that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 ( 1991 )) in the unique XhoI and NotI
sites.
DNA sequencing of the clones isolated as described above ;aye the full-length DNA
sequence for PR0266 [herein designated as UNQ233 (DNA37150)] and the derived protein sequence for PR0266.
The entire nucleotide sequence of LTNQ233 (DNA37150) is shown in Figure 9 (SEQ
ID NO:
7). Clone UNQ233 (DNA37150) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1-3 [Kozak et al., su ra] and ending at the stop codon after nucleotide position 2088. The predicted polypeptide precursor is 696 amino acids long (Figure 10;
PR0266: SEQ ID NO: 8). Clone UNQ233 (DNA37150) has been deposited with ATCC
and is assigned ATCC deposit no. 209401.
Analysis of the amino acid sequence of the full-length PR0266 polypeptide suggests that portions of it possess significant homology to a SLIT protein. thereby indicating that PR0266 may be a novel leucine rich repeat protein.

V. Isolation of cDNA Clones Encodins Human PR0335, PR0331 or PR0326 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~, Incvte Pharmaceuticals, Palo Alto, CA). The search was performed using the computer program BLAST or BLAST2 (Altshul et al., Methods in Enzymoloev 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; http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
A consensus DNA sequence was assembled relative to other EST sequences using phrap.
Based on the 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 PR0335, PR0331 or PR0326. 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 by in length. The probe sequences are typically 40-55 by in length. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about I-I .Skbp. 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 BioloQV, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest by the in vivo clongin procedure using the probe oligonucleotide and one of the primer pairs.
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 PR0335, PR0331 or PR0326 gene using the probe oligonucleotide and one of the PCR primers.
RNA for construction of the cDNA libraries was isolated from human fetal kidney tissue (PR0335 and PR0326) and human fetal brain (PR0331 ). 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 NotI site, linked with blunt to SaII 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; pRKSB is a precursor of pRKSD that does not contain the SfiI site; see, Holmes et al., Science, 253:1278-1280 ( 1991)) in the unique XhoI and NotI sites.
DNA sequencing of the clones isolated as described above gave the full-length DNA
sequence for PR0335 (Figure 1 I: SEQ ID NO: 9), PR0331 (Figure 13; SEQ ID NO:
11 ) or PR0326 (Figure 15: SEQ ID NO: 13) and the derived protein sequence for PR0335 (Figure 12: SEQ ID NO:
10), PR0331 (Figure 14; SEQ ID NO: 12) or PR0326 (Figure 16; SEQ ID NO: 14).
The nucleic acid encoding PR0335 was deposited with the ATCC on 2 June 1998 and is assigned ATCC Accession No. 209927; the nucleic acid encoding PR0331 was deposited with the ATCC on 7 November 1997 and is assigned ATCC Accession No. 209439; and the nucleic acid encoding PR0326 was deposited with the ATCC on 21 November 1997 and is assigned ATCC Accession No. 209489.
Anaiysis of the amino acid sequence of the full-length PR0335, PR0331 or polypeptide suggests that portions of it possess significant homology to the LIG-1 protein, thereby indicating that PR0335, PR0331 and PR0326 may be a novel LIG-1-related protein.

Stimulatory Activity in Mixed Lymphocvte Reaction (MLR) Assay (No. 24) This example shows that the 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. Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an immune response is beneficial. A therapeutic agent may take the form of antagonists of the 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 J. E. Coligan, A. M. Kruisbeek, D. H. Marglies, E. M. Shevach, W.
Strober, National Insitutes of Health, Published by John Wiley & Sons, Inc.
More specifically, 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, S% C02 ) and then washed and resuspended to 3 x 106 cellslml of assay media (RPMI; 10% fetal bovine serum, 1°io penicillin/streptomycin, 1%
glutamine, 1% HEPES, 1% non-essential amino 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 pl of irradiated stimulator cells and 50 p,l of responder PBMC cells.

WO OO/i5797 PCT/US99/21547 100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control. The wells are then incubated at 37°C, 5% C02 for 4 days. On day 5 and each well is pulsed with tritiated thymidine (i.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 assay, 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 lx 107 cells/ml of assay media.
The assay is then conducted as described above using a sample having a PRO concentration obtained by diluting a stock solution. The results of this assay for compounds of the invention are shown 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.
Table 2 PRO PRO Concentration Percent Increase Over Control PR0245 0.1 % I 89.7 0.1% 193.7 " 1.0% 212.5 " I .0% 300.5 PR0217 0.1 % 74.5 " 1.0% 89.5 " 0.99 nM 97.0 " 9.9 nM 122.3 " 0.25 nM 144.8 " 2.5 nM 126.9 PR0301 50.0% 109.4 " 70.0 nM 133.7 " 700.0 nM 83.6 0.1% 58.7 PR0301 I .0% 127.7 " 0.1% 181.7 " I .0% 187.3 " 0.1 % 127.5 " 1.0% 108.3 PR0266 0.1 % 136.4 " 0.1 % 139.2 " 1.0% 189.8 " 1.0% 245.1 PR0335 50.0% 91.0 " 50.0% 103.8 0.1 % 130.0 " 1.0% 180.2 PR0331 50.0% 155.5 " 0.1 % 169.3 " 1.0% 128.1 " 0.1 % 129.3 " 1.0% 162.5 PR0326 50.0% 91.0 " 50.0% 103.8 " 0.1% 130.0 " 1.0% I 80.2 Skin Vascular Permeability Assay (No. 64 This assay shows that certain polypeptides of the invention stimulate an immune response and induce inflammation by inducing mononuclear cell, eosinophil and PMN
infiltration at the site of injection of the animal. This skin vascular permeability assay is conducted as follows. Hairless guinea pigs weighing 350 grams or more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg xylazine intramuscularly (IM). A sample of purified polypeptide of the invention or a conditioned media test sample is injected intraderrnally onto the backs of the test animals with 10011L per injection site. It is possible to have about 10-30, preferably about. 16-24, injection sites per animal.
One mL 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 was biopsied and fixed in formalin. The skins were then prepared for histopathalogic evaluation. Each site was evaluated for inflammatory cell infiltration into the skin. Sites with visible inflammatory cell inflammation were scored as positive. Inflammatory cells can be neutrophilic, eosinophilic, monocytic or lymphocytic. The results of this test for compounds of the invention is shown below.
In the Table below, at least a minimal perivascular infiltrate at the injection site is scored as positve, no inf ltrate at the site of injection is scored as negative.
Table 3 PRO Hours Post In~,ection Infiltrate Desi nt~ a PR0245 24 hr positive PR0217 24 hr positive PR0301 24 hr positive PR0266 24 hr positive PR0335 24 hr positive PR0331 24 hr positive PR0326 24 hr positive Human Co-Stimulation Assav 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 tritiated 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 ~tg/ml stock 4) Anti-human CD28 Biodesign P42235M
5) Media: Gibco RPMI 1640 + 10 % Intergen #1020-90 FBS, 1% Glu, 1% P/S, 50 ~g/ml Gentamycin, 10 mM Hepes. Fresh for each assay.
6) Tritiated Thymidine 7) Frozen human peripheral blood lymphocytes (PBL) collected via a leukophoresis procedure Plates are prepared by coating 96 well flat bottom plates with anti-CD3 antibody {Amac) or anti-CD28 antibody (Biodesign) or both in Hyclone D-PBS without calcium and magnesium. Anti CD3 antibody is used at 10 ng/well (SO~tI of 200 ng/ml) and anti -CD28 antibody at 25 ng/well (SO p.l of 0.5 p,g/ml) in 100 p,l 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 in 25 mls of media and incubating at 37°C, 5% C02 overnight.
In the assay procedure, the coated plate is washed twice with PBS and the PBLs are washed and resuspended to 1 x 106 cells/ml using 100 ~,L /well. 100 p,l of a test protein or control media are added to the plate making a total volume per well of 200 p.L. The plate is incubated for 72 hours.
The plate is then pulsed for 6 hours with tritiated thyrnidine ( 1 mC/well;
Amersham) and the PBLs are harvested from the plates and evaluated for uptake of the tritiated thymidine.

In 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 33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for I S minutes at 37°C, and fizrther processed for in situ hybridization as described by Lu and Gillett, supra. A [33P] UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at SSC overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
33p_RiboQrobe synthesis 6.0 p,l ( 125 mCi) of 33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried 33P-UTP, the following ingredients were added:
2.0 p,l Sx transcription buffer 1.0 p.l DTT ( 100 mM ) 2.0 wl NTP mix (2.5 mM : I Ow l; each of 10 mM GTP, CTP ~ ATP + 10~ I H20) 1.0 wl UTP (50 p,M) 1.0 ~l Rnasin 1.0 ~1 DNA template ( 1 ~.g) 1.0 ~1 HBO
The tubes were incubated at 37°C for one hour. 1.0 ~L RQ 1 DNase were added, followed by incubation at 37°C for 1 S minutes. 90 ~,L TE ( 10 mM Tris pH 7.6/ 1 mM
EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-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 ~L TE were added. 1 ~,L 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 p.L of the probe or 5 ~.L of RNA Mrk III were added to 3 ~.L of loading buffer. After heating on a 95C 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 -70C freezer one hour to overnight.
33p-Hybridization Pretreatment offrozen 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 SSC
incubator for 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 H20). After deproteination in 0.5 p,g/ml proteinase K for minutes at 37°C ( 12.5 ~,L 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.
Pretreatment ofparaffin-embedded sections The slides were deparaffinized, placed in SQ
H20, and rinsed twice in 2 x SSC at room temperature, for 5 minutes each time.
The sections were deproteinated in 20 ~g/ml proteinase K (500 ~.L of 10 mg/ml in 250 ml RNase-free RNase buffer;
37C, 15 minutes ) - human embryo, or 8 x proteinase K ( 100 1xL in 250 ml Rnase buffer, 37C, 30 minutes) - formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
Prehybridization The slides were laid out in plastic box lined with Box buffer (4 x SSC, 50% formamide) - saturated filter paper. The tissue was covered with 50 ~L of hybridization buffer (3.75g Dextran Sulfate + 6 ml SQ H20), 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
H20 were added, the tissue was vortexed well, and incubated at 42C for 1-4 hours.

Hvbridization 1.0 x 106 cpm probe and 1.0 ~,L tRNA (50 mg/ml stock) per slide were heated at 95C for 3 minutes. The slides were cooled on ice, and 48 1tL
hybridization buffer were added per slide. After vortexing, 50 ~L 33P mix were added to 50 ~L
prehybridization on slide. The slides were incubated overnight at SSC.
Washes Washing was done 2x 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 37C for 30 minutes (500 p,L of 10 mg/ml in 250 ml Rnase buffer= 20 ug/ml), The slides were washed 2x10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows:
2 hours at SSC, 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, V~4L).
DNA 35638 ll TM receptor) Expression was observed in the endothelium lining of a subset of fetal and placental vessels.
Endothelial expression was confined to these tissue blocks. Expression was also observed over intermediate trophoblast cells of placenta.
Oligo C-120N: (SEQ ID NO: 36) GGA TTC TAA TAC GAC TCA CTA TAG GGC TGC GGC G(iC TCA GGT CTT CAG TT
Oligo c-120P (SEQ ID NO: 37) CTA TGA AAT TAA CCC TCA CTA AAG GGA GCA TGG GAT GGG GAG GGA TAC GG
DNA 33094 (EGF Homolos) A highly distinctive expression pattern was observed. In the human embryo expression was obseerved in outer smooth muscle layer of the GI tract, respiratiry cartilage, branching respiratory epithelium, osteoblasts, tendons, gonad, in the optic nerve head and developing dermis. In the adult, expression was observed in the epidermal pegs of the chimp tongue, the basal epithelial myoepithelial cells of the prostate and urinary bladder. Expression was also found in the alveolar lining cells of the adult lung, mesenchymal cells juxtaposed to erectile tissue in the penis and the cerebral cortex (probably glial cells). In the kidney, expression was only seen in disease, in cells surrounding thyroidized renal tubules.
Oligo D-200V (SEQ ID NO: 38) CTA TGA AAT TAA CCC TCA CTA AAG GGA ATA GCA GGC CAT CCC AGG ACA
Oligo D-2002 (SEQ ID NO: 39) CTA TGA AAT TAA CCC TCA CTA AAG GGA TGT CTT CCA TGC CAA CCT TC

In situ Hybridization in Cells and Diseased Tissues The in situ hybridization method of Example S 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) in a disease. Hybridization is performed according to the method of Example 5 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 ~ Skin lesions from Psoriasis and other forms of dermatitis ~ Lung tissue including BALT and tissue lymph nodes from Chronic and acute bronchitis, pneumonia, pneumonitis, pleuritis ~ Lung tissue including BALT and tissue lymph nodes from Asthma ~ nasal and sinus tissue from patients with rhinitis or sinusitis ~ Brain and Spinal cord from Multiple Sclerosis. Alzheimer's Disease and Stroke ~ Kidney from Nephritis, Glomerulonephritis and Systemic Lupus Erythematosis ~ Liver from Infectious and non-infectious Hepatitis ~ Tissues from NeoplasmslCancer.
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 thereofl in the disease associated with the cell or tissue sample.
DNA 35638 (PR0245) was found to be expressed in inflamed human tissues (psoriasis, inflammatory bowel disease (IBD), inflamed kidney, inflamed lung, hepatitis (liver block), normal tonsil, adult and chimp (multiblocks). Expression was present in the endothelium/intima of large vessels in the lung afflicted with chronic inflammation, in the superficial dermal vessels of the psoriatic skin, in arterioles in a specimen of chronic sclerosing nephritis, and in capillaries including the perifollucular sinuses of the tonsil. These results indicate that PR0245 is immunostimulatory (enhances T lymphocyte proliferation in the MLR and costimulation) and has proinflammatory properties (induces a neutrophjil infiltrate in vivo).

Use of PR0245 PR0217 PR0301 PR0266 PR0335 PR0331 or PR0326 as a hybridization probe The following method describes use of a nucleotide sequence encoding PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 as a hybridization probe.
DNA comprising the coding sequence of full-length or mature PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 (as shown in Figures 4, 6, 8, 10, 12, 14 and 16) is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326) 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326-derived probe to the filters is performed in a solution of 50% formamide, Sx 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 O.lx SSC and 0.1% SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length native sequence PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 can then be identified using standard techniques known in the art.

Expression of PR0245 PRO 17 PR0301 PR0266, PR0335, PR0331 or PR0326 in E. coli This example illustrates preparation of an unglycosylated form of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 by recombinant expression in E. coli.

The DNA sequence encoding PR0245, PRO217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into 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 enterokinase cleavage site), the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 solubilized PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.
PR0245, PR0217, PR0301 and PR0266 were expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding PR0245, PR0217, PR0301 and FR0266 was initially amplified using selected PCR primers. The primers contained 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 enterokinase. The PCR-amplified, poly-His tagged sequences were then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W31 LO fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).
Transformants were first grown in LB containing 50 mg/ml carbenicillin at 30C with shaking until an O.D.600 of 3-5 was reached. Cultures were then diluted SO-100 fold into CRAP media (prepared by mixing 3.57 g (~4)2504~ 0.71 g sodium citrate.2H20, 1.07 g KCI, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgS04) and grown for approximately 20-30 hours at 30C with shaking. Samples were removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets were frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of O. LM and 0.02 M, respectively, and the solution was stirred overnight at 4C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant was 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.
Depending the clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column was washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein were pooled and stored at 4C. Protein concentration was estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
The proteins were refolded by diluting 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 were chosen so that the final protein concentration was between 50 to 100 micrograms/ml. The refolding solution was stirred gently at 4C for 12-36 hours.
The refolding reaction was 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 was filtered through a 0.22 micron filter and acetonitrile was added to 2-10% final concentration. The refolded protein was chromatographed on a Poros RI/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 were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were 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 resin. 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 endotoxin from the samples.
Fractions containing the desired folded PR0245, PR0217, PR0301 and PR0266 proteins, respectively, were pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins were 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.

Expression of PR024~ PR0217 PR0301 PR0266, PR0335. PR0331 or PR0326 in mammalian cells This example illustrates preparation of a potentially glycosylated form of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 by recombinant expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 DNA is ligated into pRKS with selected restriction enzymes to allow insertion of the PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 DNA using ligation methods such as described in Sambrook et al., su ra. The resulting vector is called pRKS-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326.
In one embodiment, the selected host cells may 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 ug pRKS-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 DNA is mixed with about 1 ug DNA
encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 uL of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 uL 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 culture 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 culture medium containing 200 uCi/ml 3sS-cysteine and 200 uCi/ml 3sS_methionine. 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 rnay be dried and exposed to film for a selected period of time to reveal the presence of PR0245, PR0217, PR0301, PR0266. PR0335, PR0331 or PR0326 polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PR0245, PR0217, PR0301, PR0266, PR0335. PR0331 or PR0326 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 ug pRKS-PR0245. PR0217, PR0301. PR0266, PR0335, PR0331 or PR0326 DNA is added. The cells are first concentrated from the spinner t7ask 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 for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium. ~ ug/ml bovine insulin and 0.1 ug/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed PR0245, PR0217.
PRO301, PR0266, PR0335, PR0331 or PR0326 can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 can be expressed in CHO cells. The pRKS-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 can be transfected into CHO cells using known reagents such as CaP04 or DEAF-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3'S-methionine. After determining the presence of PR0245. PR0217. PR0301, PRO266, PR0335, PR0331 or PR0326 polypeptide, the culture 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PRO326 can then be concentrated and purified by any selected method.
Epitope-tagged PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 may also be expressed in host CHO cells. The PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 may be subcloned out of the pRKS vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged PR0245. PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 insert 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 culture medium containing the expressed poly-His tagged PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 can then be concentrated and purified by any selected method. such as by Ni2+-chelate affinity chromatography.
PR0245, PR0217 and PR0301 were expressed in CHO cells by both a transient and a stable expression procedure.
Stable expression in CHO cells was performed using the following procedure.
The proteins were expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins were fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs were subcloned in a CHO
expression 8l 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 cDNAs. The vector used expression in 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 were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect (Quiagen), Dosper or Fugene (Boehringer Mannheim). The cells were grown and described in Lucas et al., supra.
Approximately 3 x 10-' cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA were thawed by placement into water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 ~m filtered PS20 with 5% 0.2 ~m diafiltered fetal bovine serum). The cells were then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells were transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37C. After another 2-3 days, a 250 mL, 500 mL
and 2000 mL
spinners were seeded with 3 x 105 cells/mL. The cell media was 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 was actually used. 3L production spinner is seeded at 1.2 x 106 cells/mL. On day 0, the cell number pH
were determined. On day 1, the spinner was sampled and sparging with filtered air was commenced.
On day 2, the spinner was sampled, the temperature shifted to 33C, 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). Throughout the production, pH was adjusted as necessary to keep at around 7.2. After 10 days, or until viability dropped below 70%, the cell culture was harvested by centrifugtion and filtering through a 0.22 ~.m filter. The filtrate was either stored at 4C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins were purified using a Ni-NTA
column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was 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/min. at 4C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was 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 -80C.
Immunoadhesin (Fc containing) constructs of were purified from the conditioned media as follows. The conditioned medium was 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 was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 ~tL of 1 M
Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity was assessed by SDS
polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.
PR0326 was also produced by transient expression in COS cells.

Expression of PR0245 PR02I7 PR030I PR0266. PRO335. PR0331 or PR0326 in Yeast The following method describes recombinant expression of PR0245, PRO217;
PR0301, PR0266, PR0335, PR0331 or PR0326 in yeast.
First, yeast expression vectors are constructed for intracellular production or secretion of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 from the ADH2/GAPDH
promoter. DNA encoding PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326. For secretion, DNA encoding PR0245, PR0217, PR0301, PR026G, PR0335. PR033I or PR0326 can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH
promoter, a native PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326.
Yeast cells, such as yeast strain AB 110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.
Recombinant PR0245, PR02I7, PR0301, PR0266, PRO335, PR0331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 may further be purified using selected column chromatography resins.

Exuression of PR0245, PR0217, PR0301, PR0266, PR0335, PR0~3lorPR0326 in Baculovirus-Infected Insect Cells The following method describes recombinant expression of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 in Baculovirus-infected insect cells.
The sequence coding for PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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 PR0245, PR0217, PR0301, PR0266, PR0335. PR0331 or PR0326 or the desired portion of the coding sequence of PR.0245. PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 [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 Spodoptera frugiperda ("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 PR0245, PR02I7, PR0301, PR0266, PR0335, PR0331 or PR0326 can then be purified, for example, by Ni2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf~ cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, S~ cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9: 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted SO-fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol, pH 7.8) and filtered through a 0.45 um filter. A Niz+-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 AZBO 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 AZgo 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 Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen}. Fractions containing the eluted His,o-tagged PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0245, PR0217, PR0301, PR0266; PR0335, PR0331 or PR0326 can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.
PR0245, PR0301 and PR0266 were expressed in baculovirus infected Sf~ insect cells.
While the expression was actually performed in a 0.5-2 L scale, it can be readily scaled up for larger (e.g. 8 L) preparations. The proteins were expressed as an IgG construct (immunoadhesin), in which the protein extracellular region was fused to an IgG 1 constant region sequence containing the hinge, CH2 and CH3 domains and/or in poly-His tagged forms.
Following PCR amplification, the respective coding sequences were subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the vector and Baculogold baculovirus DNA (Pharmingen) were co-transfected into 105 Spodoptera fi~crgiperda ("Sf~") cells (ATCC CRL 1711), using Lipofectin (Gibco BRL).
pb.PH.IgG and pb.PH.His are modifications of the commercially available baculovirus expression vector pVL1393 (Pharmingen), with modified polylinker regions to include the His or Fc tag sequences. The cells were grown in Hink's TNM-FH medium supplemented with 10%
FBS
(Hyclone). Cells were incubated for 5 days at 28C. The supernatant was harvested and subsequently used for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH
medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. Cells were incubated for 3 days at 28C. The supernatant was harvested and the expression of the constructs in the baculovirus expression vector was determined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B beads (Pharmacia) for IgG
tagged proteins followed by SDS-PAGE analysis comparing to a known concentration of protein standard by Coomassie blue staining.
The first viral amplification supernatant was used to infect a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells were incubated for 3 days at 28C. The supernatant was harvested and filtered.
Batch binding and SDS-PAGE analysis was repeated, as necessary, until expression of the spinner culture was confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) was harvested by centrifugation to remove the cells and filtered through 0.22 micron filters.
For the poly-His tagged constructs, the protein construct were purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media were pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and S mM imidazole at a flow rate of 4-S ml/min. at 4C. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 2S ml G2S
Superfine (Pharnlacia) column and stored at -80C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the conditioned media as follows. The conditioned media were 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 was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH
3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity of the proteins was verified by SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid sequencing by Edman degradation.
PR024S, PR0217, PR0301, PR0266, PR0331 and PR0326 were also expressed in baculovirus infected High-S cells using an analogous procedure.

Preparation of Antibodies that Bind PR024S PR0217. PR0301, PR0266,PR0335.
PR0331 or This example illustrates preparation of monoclonal antibodies which can specifically bind PR024S, PR0217, PR0301, PR0266, PR033S, PR0331 or PR0326.
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified PR024S, PR0217, PR030I, PR0266, PR033S, PR0331 or PR0326, fusion proteins containing PR024S, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326, and cells expressing recombinant PR024S, PR0217, PR0301, PR0266, PR033S, PR0331 or PR0326 on the cell surface.
Selection of the immunogen can be made by the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PR024S. PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally 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 in 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-PR0245. PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326. 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 in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, 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 PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326. Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against PR0245, PR0217, PR0301, PR0266.
PR0335, PR0331 or PR0326 is within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-PR0245, PR0217, PR0301, PR0266, PR0335, PR0331 or PR0326 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.
Deposit of Material The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC):
Material ATCC Deb. No. Deposit Date DNA40981 209439 7 November 1997 DNA37140 209489 21 November 1997 2 June 1.998 DNA35638 209265 17 September 1997 DNA37150 209401 17 October 1997 DNA33094 209256 16 September 1997 DNA32292 209258 16 September 1997 DNA32279 209259 16 September 1997 DNA40628 209432 7 November 1997 This deposit 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 culture 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 I.
I4 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.

PRO XX~CXX3tXXXX~C,~CXXX {Length = 1 ~ amino acids) Comparison Protein X3~XXYYYYYYY (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
polvpeptide) divided by I S = 33.3%
PRO XXXXXXXXXX (Length = 10 amino acids) Comparison Protein XX3{XXYYYYYYZZYZ (Length == I S 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 10 = 50%
PRO-DNA 1'IhFNNNNI'flJ1'f2JNNNN (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%
PRO-DNA (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%

/*
* C-C increased from t 2 to I S
* Z is average of EQ
* B is average of ND
* match with stop is M; stop-stop = 0; J (joker) match = 0 *~
#define M -8 /* value of a match with a stop */
int _day(26][26J = {
/* 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, I,-1; 1, 0,-t,-2; l, 0, M, I, 0; 2, 1, 1. 0, 0; 6, 0,-3, 0}, /* B */ { 0, 3,-4, 3, 2,-S, 0, 1: 2, 0, 0,-3,-2, 2, M; I , 1, 0, 0, 0, 0,-2,-S, 0,-3, 1 }, /* C */ {-2,-4,15; S; 5,-4: 3; 3, 2, 0,-5,-b,-5,-4, M,-3; 5.-4. 0,-2, 0,-2,-8, 0, 0; 5}, /* D *l { 0, 3; 5, 4, 3.-b, 1, I: 2, 0, 0,-4; 3, 2. M; 1, 2,-l, 0, 0, 0; 2; 7, 0.-4, 2}, /* E */ { 0, 2; 5, 3, 4: S, 0, I; 2, 0, 0,-3: 2, 1, M; l, 2,-l, 0, 0, 0; 2; 7, 0,X1, 3}, /* F */ {-4; S,-4: 6; 5. 9,-S; 2, l, 0,-5, 2, 0,-4, M,-S; S,-4; 3; 3, 0; l, 0, 0, 7; S), /* G */ { 1, 0; 3. 1, 0; S, 5; 2; 3, 0,-2,-4,-3, 0 -M,-1; I; 3. I, 0, 0; I; 7, 0; 5, 0}, /* H */ {-1, 1; 3, I, I: 2: 2, 6; 2, 0, 0; 2,-2, 2, M, 0, 3, 2,-I; l, 0; 2; 3, 0, 0, 2}.
/* i *, {-1; 2; 2.-2,-2, 1,-3.-2, 5, 0,-2, 2, 2,-2, M,-2; 2; 2; I, 0, 0, 4,-5, 0; I,-2}, !* J *,' { 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}, /* K */ {-1. 0; 5, 0, 0.-S: 2, 0; 2. 0, S; 3, 0, I . M; I, 1, 3, 0, 0, 0, 2;
3, 0,-4. 0), /* L */ {-2; 3.-b.-4,-3, 2.-4,-2, 2. 0; 3, b. 4; 3 _M; 3,-2; 3,-3; 1. 0, 2,-2, 0,-I,-2}.
/* M *l {-1: 2,-S,-3; 2, 0; 3; 2, 2, 0, 0, 4, b,-2, M; 2; I . 0; 2: 1, 0, 2, 4, 0; 2,-I }, /* N */ { 0. 2.-4, 2, 1.-4. 0. 2; 2. 0, 1: 3: 2, 2._M,-t, 1, 0, 1, 0. 0.-2,-4, 0; 2, l }.
/* O */ { M, M, M, M, M, M, M, M, M, M, M, M. M. M. O. M,-M._M,-M -M -M _M _M -M _M -M}, /* P */ { l,-1; 3: L-l: S: 1, 0; 2, 0; 1,-3; 2.-l,_M, 6, 0, 0, 1, 0, 0,-1,-b, 0.-S, 0}, /* Q */ { 0, l; 5. 2. 2; S; I. 3: 2Ø 1,-2,-1, 1 _M, 0, 4. 1,-l,-1, 0.-2.-5.
0.-4. 3), /* R */ {-2, 0,-4; I; 1,-4, 3, 2; 2, 0, 3,-3, 0, 0, M, 0, I, 6, 0,-I, 0,-2, 2, 0,~, 0), /* S */ { l, 0, 0, 0, 0; 3, 1; 1,-1, 0, 0,-3,-2, l,_M, l; I, 0, 2, l, 0; l; 2, 0,-3, 0}, /* T */ { l, 0; 2, 0, 0; 3, 0; 1, 0, 0, 0; 1; I, O, M, 0; 1,-1, l, 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; l,-I,-2, 4, 0,-2, 2, 2; 2, M: 1; 2; 2: I, 0, 0, 4; 6.
0, 2,-2}, J* W */ {-b; 5; 8; 7; 7, 0; 7; 3; 5, 0,-3; 2,-4,-4,_M, 6,-S, 2,-2; S, 0; 6,17, 0, 0; 6}, /* X */ { 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; S, 0,-1. 0.-4.-I, 2: 2._A'I: S.-4,-4; 3,-3, 0; 2.
0. 0,10.}.
/* Z */ { 0, l; 5, 2, 3; S, 0, 2.-2, 0, 0; 2,-l, I _M, 0, 3, 0, 0, 0, 0; 2,-b, 0,~, 4}
);
Page 1 of day.h TABLE 5 (cont.) /*

*/

#include<stdio.h>

#include<ctype.h>

#defineMAXJMP /* max jumps in a diag */
l6 #defineMAXGAP 1* don't continue to penalize 24 gaps larger than this */

#defineIMPS 1024 /* max jmps in an path */

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

#defineDMA? 3 /* value of matching bases */

#defineDMIS 0 /* penalty for mismatched bases */

#defineDINSO 8 /* penalty for a gap */

#defineDINS I /* penalty per base */

#definePINSO 8 /* penalty for a gap */

#de5nePINS 4 /* penalty per residue !/

strvct jmp {

short n[MAXJMP]; % * size of jmp (neg for defy) */

unsignedshortx[MAXJMPJ; !* base no. of jmp in seq x */

}; /* limiu seq to 2~16 -1 */

struct diag {

int score; /* score at last jmp */

long offset; /* offset of prev block */

short ijmp; I* current jmp index *I

struct /* list of jmps */
}; jmp jp;

struct path {

int spc; /* number of leading spaces */

shortn[JMPS];/* size of jmp (gap) */

int x[JMPS];/* loc of jmp (last elem before }; gap) */

char *ofile;/* output file name'/

char *namex[2]; /* seq names: getseqs() */

char *prog;/* prog name for em msgs */

char *seqx[2];/* seqs: getseqsQ */

int dmax;/* best diag: nw() */

int dmax0;/* final diag */

int dna; l* set ifdna: main() */

int endgaps;/* set if penalizing end gaps */

int gapx, gapy;
/*
total gaps in seqs */

int len0, lent;
l*
seq lens */

int ngapx, ngapy;
/*
total size of gaps */

int smax;/* max score: nw()'/

int *xbm;/* bitmap for matching *!

long offset;/* current offset in jmp tile */

structdiag *dx; /* holds diagonals */

structpath pp[2];l* holds path forseqs */

char *callocQ,*malloc(), *index(), *strcpy();

char *getseq(),*g_calloc();

Page 1 of nw.h TABLE 5 (cont.l /* Needleman-Wunsch alignment program * usage: props file! filet * where file! and tile2 are two dna or two protein sequences.
* The sequences can be in upper- 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 in the tile "align.out"
* The program may create a unp tile in itmp 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 _pbvai[26] =
I , 2~( 1 «('D _'A'))~( 1 «('N .'A')). 4. 8. 16. 32. 64.
128, 256. OxFFFFFFF. I«10. I«I1, 1«12. t«13. I«14.
1«I5. 1«16. I«17, 1«18. 1«19, 1«20, I«21, 1«22, 1 «23, 1 «24, 1 «25 ~( 1 «('E -'A'))~( 1 «('Q -'A')) main(ac, av) main int ac;
char *av[];
prop = av[0];
if(ac!=3) ( fprintf(stdetr,"usage: %s file! file2\n", prop);
fprintf(stderr,"where filet and filet are two dna or two protein sequences.kt");
fprintf(stdert,"The sequences can be in upper- or lower-case\n");
fprinttrstderr,"Any lines beginning with ;' or'<' are ignored\n");
fprintt(stderr,"Output is in the file \"align.out\"\n");
exit( I );
]
namex[0] = av[ 1 ];
namex[ 1 ] = av[2];
seqx[O] = getseq(namex[0]. &IenO);
seqx[ I ] = getseq(namex[ 1 ], &len I );
xbm = (dna)? dbval : _pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "atign.out"; /* output tile */
nwQ; /* fill in the matrix. get the possible jmps */
readjmps(): /* get the actual jmps */
printQ; /* print slats. alignment */
cleanup(0); I* unlink any tmp files *l Page I of nw.c TABLE 5 (cont~
/* do the alignment, return best score: main( ) * dna: values in Fitch and Smith, PNAS, 80. 1382-1386, 1983 * pro: PAM 250 values * When scores are equal, we prefer mismatches to any gap, prefer * a new gap to extending an ongoing gap. and prefer a gap in seqx * to a gap in seq y.
*~
nw( ) nw {
char *px, *py; !* seqs and ptrs *1 int *ndely, *dely; * keep track ofdely */
int ndelx, delx; . * keep track of delx */
int *tmp; /* for swapping row(1, rowl */
int mis; /* score for each type */
int ins0, insl; /* insertion penalties */
register id; /* diagonalindex */
register ij; 1* jmp index */
register *col0, *coll; l* score for curr.last row */
register xx, yy; /* index into seqs */
dx = (struct diag *)g calloc("to get diags", IenO+Ienl+I, sizeof(struct diag));
ndely = (int *)g calloc("to get ndely". lenl+I. sizeof(int));
dely-(int *)g calfoc("to get dely". lenl+I, sizeof(int));
col0 = (int *)g calloc("to get col0", lenl+I, sizeof(int));
toll = (int *)g calloc("to get toll".lent+l,sizeof(int));
ins0 = (dna)? DINSO : PINSO;
ins l = (dna)? DINS 1 : PINS 1;
smax = -10000;
if (endgaps) {
for (col0[0] = defy[O] _ -ins0, yy = l; yy <= len I; yy++) {
colo[yy] = dely[yyJ = solo[yy-I ] - insl;
ndely[yyJ = yy;
col0[OJ=0; /* Waterman Bull Math Biol 84 */
else for (yY = 1, YY <= ten 1; yy++) dely[yy] _ -ins0:
* fill in match matrix *~
for (px = seqx[0], xx = l; xx <= len0; px++_ xx++) {
/* initialize first entry in col */
if (endgaps) {
if (xx = t ) cot I [0] = delx = -1 ins0+ins I );
else ndelx = xx;
f else {
colt[0] = delx = col0[OJ - insl:
coil[0] = 0:
delx = -ins0:
ndelx = 0;
Paee 2 of nw.c TABLE 5 (cont. ) ...nw for (pY = seqx[ 1 ], YY = 1; YY <= len I : pYi'i', YY++) {
mis = col0[yy-I ];
if (dna) mis+=(xbm(*px-'A']&xbm[*py-A'])? DMAT: DMIS;
else mis+= day[*px-A'](*PY-'A']:
/* update penalty for del in x seq;
* favor new del over ongong dei * ignore MAXGAP if weighting endgaps */
if (endgaps ~~ ndely[yy] < MAXGAP) {
if (col0(yy] - ins0 >= dely[yy]) {
dely[yy) = col0[yy]-(ins0+insl);
ndely[yy] = I ;
} else {
dely[yy] = ins l ;
ndely(yy1++;
}
} else {
if (col0[yy] - (ins0+ins l ) >= dely[yy]) {
dely[yy] = col0[yy] - (ins0+insl );
ndely[yy] = I
} else ndely[yy]++;
/* update penalty for del in y seq;
* favor new del over ongong del */
if (endgaps /~ ndelx < MAXGAP) {
if (col l [yy-I ] - ins0 >= delx) {
deli = col I [yy- l ] - (ins0+ins I ):
ndelx = l ;
} else {
delx = insl;
ndelx++;
} else {
if (col I [yy-1 ] - (ins0+ins t ) >= delx) {
delx = col I [yy-1 ] - (ins0+ins t );
ndelx = I ;
} else ndelx++;
}
/* pick the maximum score: we're favoring * mis over any del and deix over dely */
Page 3 of nw.c WO 00/15797 _ PCT/US99/21547 TABLE 5 (cont.l id=xx-yy+lenl-I;
if (mis >= delx && mis >= dely[yy]) col l [yyJ = mis;
else if (delx >= dely[yyJ) {
coil{yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~~ (ndelx >= MAXJMP
&& xx > dx[id] jp.x[ij]+MX) ~~ mis > dx[idJ.score+DINSO)) {
dx[id].ijmp++;
if (++ij >= MAXJMP) {
writcjmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struet jmp) + sizeof(offset);
]
dx[id] jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx:
dx(id].score = delx;
else {
toll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx(idJ.jp.n[0] && (!dna p (ndely[yyJ >= MAXJMP
&& xx > dx[idJ.jp.x[ij]+MX) ~~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij >= MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
j dx[id];jp.n[ij] _ -ndely[yy];
dx[id] jp.x[ijJ = xx;
dx[idj.score = dely(yy]:
if (xx = len0 && yy < len l ) {
/* last col if(endgaps) toll[yyl-= ins0+insl*(lenl-yy);
if (col l [yy] > smax) {
smax = col l [yy];
dmax = id;

if (endgaps && xx < IenO) coll(yy-Ij = ins0+insl*(len0-xx);
if (col I [yy-I ] > smax) {
smax = col 1 [yy-1 ];
dmax = id;
tmp = col0; col0 = col l : col I = tmp;
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)col I ); ] Page 4 of nw.c ...nw TABLE 5 (cont.) ~*
* print() -- only routine visible outside this module *
* static:
* getmat() -- trace back best path, count matches: print() * pr align() -- print alignment of described in array p[]: print() * dumpblock() -- dump a block of lines with numbers, stars: pr align() * nums( ) -- put out a number line: dumpblock( ) * putline( ) -- put out a line (name, [num], seq, [num]): dumpblock( ) * stars() - -put a line of stars: dumpblock() * stripname( ) -- strip any path and prefix from a seqname */
#include "nw.h°
#define SPC 3 #define P_LiNE 256 /* maximum output line *1 #def;ne P SPC 3 /* space between name or num and seq */
extern _day(26][26];
int olen; /* set output line length */
FILE *fx; /* output file *.' print() print {
int Ix, ly, firstgap, lastgap; /* overlap *l if ((fx = fopen(ofile, "w")) ~ 0) {
fprintt(stderr,"%s: can't write %s~n", prog, otile);
cleanup(1);
]
fprintf(fx. "<first sequence: %s (length = %d)~n", namex[0], IenO);
fptintf(fx, "<second sequence: °/*s (length = °/*d)~rt", namex( 1 ], len I );
olen = 60;
lx = IenO;
ly= lenl;
firstgap = lastgap = 0;
if (dmax < len 1 - l ) { /* leading gap in x */
pp[0].spc = firstgap = lenl - dmax - 1;
IY = PP[0]~sPc;

else if (dmax > len I - I ) { ; * leading gap in y *!
pp( I ].spc = tirstgap = dmax - (len 1 - I );
Ix = pP[ I ]~sPc;
]
if (dmax0 < IenO - I ) { /* trailing gap in x *!
lastgap = len0 - dmax0 - I :
lx = lastgap;
]
else if (dmax0 > IenO - l ) { l* trailing gap in y */
lastgap = dmax0 - (IenO - I );
ly = lastgap;
getmat(Ix. ly, firstgap, lastgap);
pr_align();
Page t of nwprint.c TABLE 5 (cont. ) l*
* trace back the best path, count matches */
static getmat(lx, ly, firstgap, lastgap) getmat int Ix, ly; /' "core" (minus endgaps) */
int firstgap, lastgap; J* leading trailing overlap */
int nm, i0, il, siz0, sizl;
char outx[32];
double pct;
register n0.nl;
register char *p0,*pl;
/* get total matches. score '1 i0=il=siz0=sizl=0;
p0 = seqx[0] + pp[ 1 ].spc;
P1 ° se9x[1] + PP[0]~sPc:
n0=PP[I]~sPc+ 1:
nl = pp[0].spc + 1;
nm = 0;
while ( *p0 && *p l ) {
if(siz0) {
pl++;
nl++:
siz0--;
else if (sizl ) {
p0++;
n0++;
sizl--;
else {
if (xbm[*p0= A']&xbm[ *pl = A' nm++;

if = PP[0].x[i0]) (n0++

siz0 =
pp[0].n[i0++];

if = pp[ 1 (nl++].x[i I ]) sizl =pp[1].n[il++J;

Pte;

pl++;

l* pct homology:
* if penalizing endgaps, base is the shorter seq * else, knock off overhangs and take shorter core */
if(endgaps) lx = (IenO < len I )? len0 : len 1;
else lx = (Ix < ly)? (x : ly;
pct= 100.*(double)nm/(double)Ix;
fprintf(fx, "fin");
fprintf(&, "<%d match%s in an overlap of %d: %.2f percent similarity~n", nm, (nm = 1 )? "" : "es", Ix, pct);
Page 2 of nwprinLc TABLE 5 (cont. ) fprintf(fx. "<gaps in tirst sequence: %d", gapx); ...getmat if (gapx ) ;
(void) sprintt(outx, " (°,%d %s%s)", ngapx, (dna)? "base":"residue", (ngapx == I f' "":"s");
tprintf(fx,"%s", outx);
fprintt(fx. ", gaps in second sequence: %d", gapy);
if (gapY) [
(void) sprinit(outx, " (%d %s%s)", ngapy,(dna)? "base":"residue",(ngapy = I)? "":"s");
fprintf(fx,"%s", outx);
if (dna) fprintt(fx.
"\n<score: %d (match = °/xi, mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT, DMIS, DINSO, DINS 1 );
else fprintt(fx, "\n<score: %d (Dayhoti' PAM 250 mania, gap penalty = %d + %d per residue)\n", smax, PINSO, PINS I );
if(endgaps) fprintf(fx, "<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n", tirstgap, (dna)? "base" : "residue". (firsigap == 1 )° "" : "s".
lastgap, (dna)? "base" : "residue", (lastgap = I )'. "" : "s");
else fprintf(fx, "<endgaps not penalized~n");
static nm; /* matches in core -- for checking */

static hnax; /* lengths of stripped file names */

static ij[2]; /* jmp index for a path */

static nc[2]; /* number at start of current line */

static ni[2]; /* current elem number -- for gapping */

static siz[2];

static *ps[2]; /* ptr to current char element */

static *po[2]; /* ptr to next output char char slot */

static out[2][P /* output line */
char LINE];

static star[P-LINE];/* set by stars() char */

/*
* print alignment of described in struct path pp[]
*/
static pyalign() pr align int nn; /* char count *.~
int more;
register i;
for (i = 0. Imax = 0; i < 2; i++) nn = stripname(namex[i]);
if (nn > Imax) Imax = nn;
nc[i]= I;
ni[i]= t:
siz[i] = ij[i] = 0:
ps[i] = seqx[i];
po[i] = out[i];
] Page 3 of nwprint.c TABLE 5 (coot. ) for (nn = nm = 0, more = I : more; ) { ...pr align for (i = more = 0: i < 2; i++) {
/*
* do we have more of this sequence?
*/
if(!*ps[;]) continue;
more++;
if (pp[i].spc) { /* leading space */
*po[;j++=' .
PP[i]~sPc__;
) else if (siz[ij) { /* in a gap */
*po[ij++=~ , siz[ij__;
I
else { /* we're putting a seq element */
*Po[il = *Ps[il:
if (islower(*ps(i])) *ps[i] = touppeK*ps[ij);
po[i]++;
ps[i]++:
/*
* are we at next gap for this seq?
*/
if(ni[i]=pp[i].x[ij[i))) /*
* we need to merge all gaps * at this location s/
siz[i) = pP[iJ.nfil[il+~'l:
while (ni[iJ = pp[i].x[ij[i]]) siz[i] += PP[i].n(ij(i]++):
ni[i]++;
) if (++nn = oleo p ! more && nn) {
dumpblock();
for(i=0: i<2:i++) po[ij = out[i];
nn = 0;
]
J
/*
* dump a block of lines, including numbers, stars: pr align() *%
static dumpblock() dumpblock {
register i;
for (i = 0; i < 2; i++) *po[ij__ _ ~0';
Page 4 of nwprint.c TABLE 5 (cont. ) (void) putc('~n', fx);
for (i = 0; i < 2; i++) if (*out[i] && (*out[i] !_ " p *(po[i]) !_ ")) ( it(i = 0) nums(i);
if(i =0&&*out[1]) stars( );
putline(i):
if (i = 0 && *out( 1 ]) fprintf(fx, star);
if(i= I) nums(i);
/*
* put out a number line: dumpblock() *i static nutns(ix) nums int ix; /* index in out[] holding seq line */
char nline[P LINEJ;
register i,j;
register char *pn, *px, *py;
for (pn = nline, i = 0; i < Imax+P SPC; i++, pn++) *Pn=, , for (i = nc[ix], py ° out[ix]; *pY: PY++. Pn~) ( if(*py="~~ *PY= =7 *Pn=' , else {
if (i%10 = 0 ~~ (i = I && nc[ix] != 1 )) j = (i < 0)~ _i : i;
for (px = pn; j; j /= 10, px--) *px = j% I 0 + '0';
if (i < 0) *Px=, , else *Pn=, , i++;

) *pn = ~0':
nc[ix] = i;
for (pn = nline; *pn; pn++) (void) putc(*pn, fx);
(void) putc('~tt', &);
) l*
* put out a fine (name, [num], seq, (num]): dumpblock() */
static putline(ix) putline int ix;
...dumpblock Page 5 of nwprint.c TABLE 5 (cont. ) int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px !_ ': ; px++, i++) (void) putc(*px, fx);
for (; i < Imax+p SPC; i++) (void) putc(",fx);
/* these count from 1:
* ni[] is current element (from I ) * nc[J is number at start of current line *~
for (px = out[ix]; *px; px++) (void) putc(*px&Ox7F, fx):
(void) putc('~n', fx);
/*
* put a line of stars (seqs always in out[0], out[ 1 ]): dumpblock() *~
static stars() stars [
int i;
register char *p0, *pl, cx, *px;
if (!*out[oJ p (*out[o] _ " &8r. *(po[oJ) __ ") p !*out[ I ] II (*out[ I ] --- " && *(Po( ! l) __ ")) return;
px = star, for (i = Imax+P SPC; i; i--) *pX~=' , for (p0=out(O], pl =out[1]; *p0 && *pl; p0++, pl++) {
if (isalpha(*p0) && isalpha(*p1 )) {
if(xbm[*pU-A']&xbm[*pl-A']) {
CX = '*';
nm++:

else if (!dna ~X.R day[*p0-A'J[*pl-'A'] > 0) cx ='. .
else cx=' , else cx =' , *px++ = cx;

*Px++ ='~n~:
*px =10';
...putline Page 6 of nwprint.c TABLE 5 lcont.) ~*
* strip path or prefix from pn, return len: pr align() */
static stripname(pn) stripname char *pn; I* file name (may be path) *I
register char *px, *py;
PY=0:
for (px = pn; *px; px++) if (*px ='/') PY=Px+ 1;
if (py) (void) strcpy(pn, py);
return(strlen(pn));
Page 7 of nwprint.c TABLE 5 (cont.) /*
* cleanup( ) -- cleanup any tmp tile * getseq() -- read in seq, set dna, len, maxten * g calloc() -- cailoc() with error checkin * readjmps() -- get the good jmps, from tmp tile if necessary * writejmps() -- write a filled array of jmps to a tmp tile: nw( ) *i #include "nw.h"
#include <sysifile.h>
char *jname = "!tmpihomgXXXXXX"; I* tmp file for jmps *I
FILE *fj:
int cleanup(); /* cleanup tmp file */
long lseek();
/*
* remove any tmp file if we blow */
cleanup(i) cleanup int i:
if (fj) exit(i);
(void) unlink(jnamel:
/*
* 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 *file; /* file name */
int *len; /* seq len *l i char line( 1024], *pseq;
register char *px, *py;
int natgc,tlen;
FILE *fp;
if ((fp = fopen(file."r")) = 0) {
tprintf(stderr,"°,'os: can't read %s~n", prop, tile);
exit( I );
) tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =_ ';' II *line ='<' II *line =_ '>') continue;
for (px = tine; *px !='fin'; px++) if(isupper(*px) p islower(*px)) tlen++:
if ((pseq = malloc((unsigned)(tlen+6))) - 0) {
fprintf(stderr."%s: rnalioc( ) failed to get %d bytes for %s~n", prog, tlen+6, file):
exit( l ):

pseq(0] = pseq[ i ] = pseq[2] = pseq[3] _ ",0';
Page 1 ofnwsubr.c WO 00/15797 . PCT/US99/21547 TABLE 5 (cont. ) PY = Pse4 + 4;

*len =
tlen;

rewind(tp);

while (fgets(line.
1024, fp)) {

if('line= ;'~~'line='<'~~'line ='>') continue:

for (px = line;
*px !='Ut';
px++) {

if (isupper(*px)) *py++= *px;

else if (islower(*px)) *Py++ = toupper(*px ):

if (index("ATGCU"'(py-I
))) natgc++;

]
*py++=
~0':

*py = ~0 ;

(void) fclose(fp);

dna = natgc > (tlen/3);

return(pseq+4);

char g cailoc(msg, nx, sz) g_calloc char *msg; /* program. calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *callocQ;
if((px=calloc((unsigned)<tx, (unsigned)sz))=0) {
if (*msg) {
fprintf(stderr, "%s: g_cailoc() failed %s (n=%d, sz=%d)1n", prog, msg. nx, sz);
exit( 1 );
]
J
return(px);
]
/
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main() *~
readjmps( ) readjmps ( int fd = -1;
int siz,i0,il;
register i, j, xx;
if(fj) {
(void) fclose(fj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintt(stde~r, "%s: can't open() %sw", prog, jname);
cleanup( I );
for (i = i0 = i 1 = 0, dmax0 = dmax, xx = IenO; ; i++) {
while (I) {
for (j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j--) ...getseq Page 2 of nwsubr.c ]04 WO 00/15797 _ PCT/US99/21547 TABLE 5 (cont. ) if (j < 0 && dx[dmax].otl'set && fj) {
...readjmps (void) Iseek(fd, dx[dmax].offset, 0):
(void) read(Cd. (char *)&dx[dmaxj.jp, sizeof(struct jmp));
(void) read(fd, (char *)B.dx[dmax].offset, sizeof(dx[dmaxj.offset));
dx[dmax].ijmp= MAXJMP-I;
) else break;
) if (i >= JMPS) {
fprintt(stderr, "%s: too many gaps in alignmentw", prog);
cleanup(1);
) if (j >= 0) siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x[jJ;
dmax += siz;
if (siz < 0) { % * gap in second seq */
PP[I]~n[il] _ _siz;
xx += siz;
/* id=xx-yy+lenl - 1 */
pp[ 1 ].x[i 1 ] = xx - dmax + len 1 - 1;
BaPY~;
ngapy = siz;
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~~ endgaps)? -siz : MAXGAP;
il++;
) else if (siz > 0) { /* gap in first seq */
pp[0].n[i0] = siz;
pp[0].x[i0] = xx;
gaPx+-t':
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~~ endgaps)? siz : MAXGAP;
i0++;
else break;
) /* reverse the order of jmps *i for (j = 0, i0--: j < i0: j++, i0--) i = PP[Ol.nC1); PP[0).n[j] = PP[0).n[i0]; pp[0].n[i0] = i;
i = PPfOI.xG); PPfO).xG) = PP[0).x[i0]; PP[0]~x[i0] = i:
) for (j = 0, i 1--: J < i 1; j++, i 1--) {
i ° PP[ ll.n~]: PP[ 1 ).n~ ) = PP[ I )~n[i I ): PP[ 1 ).n[i 1 ] = i:
i = PP[ I ).x[!]; PP[ 1 ).x~ ) = PP[ 1 ).x[i 1 ]; PP[ 1 ).x[i 1 ] = i;
) if (fd >= 0) (void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
otf'set = 0;
Page 3 of nwsubr.c 1~$

TABLE 5 (cont.) i*
* write a filled jmp struct otfset of the prev one (if any): nw() *i writejmps(ix) writejmps int ix;
char *mktemp();
if(!fj) {
if (mktempQname) < 0) fprintf(stderr, "%s: can't mktemp() %s~n", prog, jname);
cleanup(t);
}
if ((fj = fopen(jname, "w")) = 0) {
fprintf(stderr, "%s: can't write %s~n", prog, jname);
exit( 1 );
}
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx(ix].offset), 1, fj);

MARRSRHRLLLLLLRYLWALGYHKAYGFSAPKDQQWTAVEYQEAILACKTPKKTVSS
RLEWKKLGRSVSFVWQQTLQGDFKNRAEMIDFNIR.IKNVTRSDAGKYRCEVSAPSEQG
QNLEEDTVTLEVLVAPAVPSCEVPSSALSGT'WELRCQDKEGNPAPEYTWFKDGIRLLE
NPRLGSQSTNSSYTMNTKTGTLQFNTVSKLDTGEYSCEARNSVGYRRCPGKRMQVDDLN
ISGIIAAWWALVISVCGLGVCYAQRKGYFSKETSFQKSNSSSKATTMSENVQWLTPV
IPALWKAAAGGSRGQEF
N-glycosylation siteat residues:

Casein kinase II phosphorylation siteat residues:

N-myristoylation siteat residues:
lee-ls8 Amidation site at residues:

MARRSAFPAAALWLWSILLCLLALRAEAGPPQEESLYLWIDAHQARVLIGFEEDILIVS
EGKMAPFTHDFRKAQQRMPAIPVNIHSMNFTWQAAGQAEYFYEFLSLRSLDKGIMADPT
VNVPLLGTVPHKASWQVGFPCLGKQDGVAAFEVDVIVMNSEGNTILQTPQNAIFFKTC
QQAECPGGCRNGGFCNERRICECPDGFHGPHCEKALCTPRCMNGGLCVTPGFCICPPGF
YGVNCDKANCSTTCFNGGTCFYPGKCICPPGLEGEQCEISKCPQPCRNGGKCIGKSKCK
CSKGYQGDLCSKPVCEPGCGAHGTCHEPNKCQCQEGWHGRHCNKRYEASLIHALRPAGA
QLRQHTPSLKKAEERRDPPESNYIW
N-glycosylation site at residues:

Casein kinase II phosphorylation site at residues:

Tyrosine kinase phosphorylation site at residues:

N-myristoylation site at residues:

ATP/GTP-binding site motif A (P-loop) at residues:

EGF-like domain cysteine pattern signature at residues:

MGTKAQVERKLLCLFILAILLCSLALGSVTVHSSEPEVRIPENNPVKLSCAYSGFSSPR
VEWKFDQGDTTRLVCYNNKITASYEDRVTFLPTGITFKSVTREDTGTYTCMVSEEGGNS
YGEVKVKLIVLVPPSKPTVNIPSSATIGNRAVLTCSEQDGSPPSEYTWFKDGIVMPTNP
KSTRAFSNSSYVLNPTTGELVFDPLSASDTGEYSCEARNGYGTPMTSNAVRMEAVERNV
GVIVAAVLVTLILLGILVFGIWFAYSRGHFDRTKKGTSSKKVIYSQPSARSEGEFKQTS
SFLV
N-glycosylation site at residues:

cAMP- and cGMP-dependent protein kinase phosphorylation site at residues:

Casein kinase II phosphorylation site at residues:

N-myristoylation site at residues:

l09 MLLWILLLETSLCFAAGNVTGDVCKEKICSCNEIEGDLHVDCEKKGFTSLQRFTAPTSQ
FYHLFLHGNSLTRLFPNEFANFYNAVSLHMENNGLHEIVPGAFLGLQLVKRLHINNNKI
KSFRKQTFLGLDDLEYLQADFNLLRDIDPGAFQDLNKLEVLILNDNLISTLPANVFQW
PITHLDLRGNRLKTLPYEEVLEQIPGIAEILLEDNPWDCTCDLLSLKEWLENIPKNALI
GRWCEAPTRLQGKDLNETTEQDLCPLKNRVDSSLPAPPAQEETFAPGPLPTPFKTNGQ
EDHATPGSAPNGGTKIPGNWQIKIRPTAAIATGSSRNKPLANSLPCPGGCSCDHIPGSG
LKMNCNNRNVSSLADLKPKLSNVQELFLRDNKIHSIRKSHFVDYKNLILLDLGNNNIAT
VENNTFKNLLDLRWLYMDSNYLDTLSREKFAGLQNLEYLNVEYNAIQLILPGTFNAMPK
LRILILNNNLLRSLPVDVFAGVSLSKLSLHNNYFMYLPVAGVLDQLTSIIQIDLHGNPW
ECSCTIVPFKQWAERLGSEVLMSDLKCETPVNFFRKDFMLLSNDEICPQLYARISPTLT
SHSKNSTGLAETGTHSNSYLDTSRVSISVLVPGLLLVFVTSAFTWGMLVFILRNRKRS
KRRDANSSASEINSLQTVCDSSYWHNGPYNADGAHRWDCGSHSLSD
IS
N-glycosylation site at residues:

CAMP- and cGMP-dependent protein kinase phosphorylation site at residues:

Casein kinase II phosphorylation site at residues:

N-myristoylation site at residues:

Prokaryotic membrane lipoprotein lipid attachment site at residues:

MVDVLLLFSLCLLFHISRPDLSHNRLSFIKASSMSHLQSLREVKLNNNELETIPNLGPV
SANITLLSLAGNRIVEILPEHLKEFQSLETLDLSSNNISELQTAFPALQLKYLYLNSNR
VTSMEPGYFDNLANTLLVLKLNRNRISAIPPKMFKLPQLQHLELNRNKIKNVDGLTFQG
LGALKSLKMQRNGVTKLMDGAFWGLSNMEILQLDHNNLTEITKGWLYGLLMLQELHLSQ
NAINRISPDAWEFCQKI~SELDLTFNHLSRLDDSSFLGLSLLNTLHIGNNRVSYIADCAF
RGLSSLKTLDLKNNEISWTIEDMNGAFSGLDKLRRLILQGNRIRSITKKAFTGLDALEH
LDLSDNAIMSLQGNAFSQMKKLQQLHLNTSSLLCDCQLKWLPQWVAENNFQSFVNASCA
HPQLLKGRSIFAVSPDGFVCDDFPKPQITVQPETQSAIKGSNLSFICSAASSSDSPMTF
AWKKDNELLHDAEMENYAHLRAQGGEVMEYTTILRLREVEFASEGKYQCVISNHFGSSY
SVKAKLTVNMLPSFTKTPMDLTIRAGAMARLECAAVGHPAPQIAWQKDGGTDFPAARER
RMHVMPEDDVFFIVDVKIEDIGVYSCTAQNSAGSISANATLTVLETPSFLRPLLDRTVT
KGETAVLQCIAGGSPPPKLNWTKDDSPLWTERHFFAAGNQLLIIVDSDVSDAGKYTCE
MSNTLGTERGNVRLSVIPTPTCDSPQMTAPSLDDDGWATVGWIIAWCCWGTSLVWV
VIIYHTRRRNEDCSITNTDETNLPADIPSYLSSQGTLADRQDGWSSESGSHHQFVTSS
GAGFFLPQHDSSGTCHIDNSSEADVEAATDLFLCPFLGSTGPMYLKGNVYGSDPFETYH
TGCSPDPRTVLMDHYEPSYIKKKECYPCSHPSEESCERSFSNISWPSHVRKLLNTSYSH
NEGPGMKNLCLNKSSLDFSANPEPASVASSNSFMGTFGKALRRPHLDAYSSFGQPSDCQ
PRAFYLKAHSSPDLDSGSEEDGKERTDFQEENHICTFKQTLENYRTPNFQSYDLDT
N-glycosylation site at residues:

Glycosaminoglycan attachment site at residues:

Casein kinase II phosphorylation site at residues:

Tyrosine kinase phosphorylation site at residues:

N-myristoylation site at residues:

MLNKMTLHPQQIMIGPRFNRALFDPLLVVLLALQLLWAGLVRAQTCPSVCSCSNQFSK
VICVRKNLREVPDGISTNTRLLNLHENQIQIIKVNSFKHLRHLEILQLSRNHIRTIEIG
AFNGLANLNTLELFDNRLTTIPNGAFWLSKLKELWLRNNPIESIPSYAFNRIPSLRRL
DLGELKRLSYISEGAFEGLSNLRYLNLAMCNLREIPNLTPLIKLDELDLSGNHLSAIRP
GSFQGLMHLQKLWMIQSQIQVIERNAFDNLQSLVEINLAHNNLTLLPHDLFTPLHHLER
IHLHHNPWNCNCDILWLSWWIKDMAPSNTACCARCNTPPNLKGRYIGELDQNYFTCYAP
VIVEPPADLNVTEGMAAELKCRASTSLTSVSWITPNGTVMTHGAYKVRIAVLSDGTLNF
TNVTVQDTGMYTCMVSNSVGNTTASATLNVTAATTTPFSYFSTVTVETMEPSQDEARTT
DNNVGPTPVVDWETTNVTTSLTPQSTRSTEKTFTIPVTDINSGIPGIDEVMKTTKIIIG
CFVAITLMAAVMLVIFYKMRKQHHRQNHHAPTRTVEIINVDDEITGDTPMESHLPMPAI
EHEHLNHYNSYKSPFNHTTTVNTINSIHSSVHEPLLIRMNSKDNVQETQI
N-glycosylation site at residues:

cAMP- and cGMP-dependent protein kinase phosphorylation site at residues:

Casein kinase II phosphorylation site at residues:

N-myristoylation site at residues:

MSAPSLRARAAGLGLLLCAVLGRAGRSDSGGRGELGQPSGVAAERPCPTTCRCLGDLLD
CSRKRLARLPEPLPSWVARLDLSHNRLSFIKASSMSHLQSLREVKLNNNELETIPNLGP
VSANITLLSLAGNRIVEILPEHLKEFQSLETLDLSSNNISELQTAFPALQLKYLYLNSN
RVTSMEPGYFDNLANTLLVLKLNRNRISAIPPKMFKLPQLQHLELNRNKIKNVDGLTFQ
GLGALKSLKMQRNGVTKLMDGAFWGLSNMEILQLDHNNLTEITKGWLYGLLMLQELHLS
QNAINRISPDAWEFCQKLSELDLTFNHLSRLDDSSFLGLSLLNTLHIGNNRVSYIADCA
FRGLSSLKTLDLKNNEISWTIEDMNGAFSGLDKLRRLILQGNRIRSITKKAFTGLDALE
HLDLSDNAIMSLQGNAFSQMKKLQQLHLNTSSLLCDCQLKWLPQWVAENNFQSFVNASC
AHPQLLKGRSIFAVSPDGFVCDDFPKPQITVQPETQSAIKGSNLSFICSAASSSDSPMT
FAWKKDNELLHDAEMENYAHLRAQGGEVMEYTTILRLREVEFASEGKYQCVISNHFGSS
YSVKAKLTVNMLPSFTKTPMDLTIRAGAMARLECAAVGHPAPQIAWQKDGGTDFPAARE
RRMHVMPEDDVFFIVDVKIEDIGWSCTAQNSAGS:LSANATLTVLETPSFLRPLLDRTV
TKGETAVLQCIAGGSPPPKLNWTKDDSPLWTERHFFAAGNQLLIIVDSDVSDAGKYTC
EMSNTLGTERGNVRLSVIPTPTCDSPQMTAPSLDDDGWATVGWIIAWCCWGTSLVW
WIIYHTRRRNEDCSITNTDETNLPADIPSYLSSQGTLADRQDGWSSESGSHHQFVTS
SGAGFFLPQHDSSGTCHIDNSSEADVEAATDLFLCPFLGSTGPMYLKGNVYGSDPFETY
HTGCSPDPRTVLMDHYEPSYIKKKECYPCSHPSEESCERSFSNISWPSHVRKLLNTSYS
HNEGPGMKNLCLNKSSLDFSANPEPASVASSNSFMGTFGKALRRPHLDAYSSFGQPSDC
QPRAFYLKAHSSPDLDSGSEEDGKERTDFQEENHICTFKQTLENYRTPNFQSYDLDT
N-glycosylation site at residues:

Glycosaminoglycan attachment site at residues:

Casein kinase II phosphorylation site at residues:

Tyrosine kinase phosphorylation site at residue:

N-myristoylation site at resudues:

Leucine zipper pattern at residues:

ll8 Sequence Listing <110> Genentech, Inc.
Fong, Sherman Audrey Goddard Gurney, Austin L.
Tumas, Daniel Wood, William I.
<120> COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE
RELATED DISEASES
<130> P1624R2 <141> 1999-09-14 <150> US 60/100,858 <151> 1998-09-17 <160> 39 <210> 1 <211> 1295 <212> DNA
<213> artificial sequence <400> 1 cccagaagtt caagggcccc cggcctcctg cgctcctgcc gccgggaccc 50 tcgacctcct cagagcagcc ggctgccgcc ccgggaagat ggcgaggagg 100 agccgccacc gcctcctcct gctgctgctg cgctacctgg tggtcgccct 150 gggctatcat aaggcctatg ggttttctgc cccaaaagac caacaagtag 200 tcacagcagt agagtaccaa gaggctattt tagcctgcaa aaccccaaag 250 aagactgttt cctccagatt agagtggaag aaactgggtc ggagtgtctc 300 ctttgtctac tatcaacaga ctcttcaagg tgattttaaa aatcgagctg 350 agatgataga tttcaatatc cggatcaaaa atgtgacaag aagtgatgcg 400 gggaaatatc gttgtgaagt tagtgcccca tctgagcaag gccaaaacct 450 ggaagaggat acagtcactc tggaagtatt agtggctcca gcagttccat 500 catgtgaagt accctcttct gctctgagtg gaactgtggt agagctacga 550 tgtcaagaca aagaagggaa tccagctcct gaatacacat ggtttaagga 600 tggcatccgt ttgctagaaa atcccagact tggctcccaa agcaccaaca 650 gctcatacac aatgaataca aaaactggaa ctctgcaatt taatactgtt 700 tccaaactgg acactggaga atattcctgt gaagcccgca attctgttgg 750 atatcgcagg tgtcctggga aacgaatgca agtagatgat ctcaacataa 800 gtggcatcat agcagccgta gtagttgtgg ccttagtgat ctccgtttgt 850 ggccttggtg tatgctatgc tcagaggaaa ggctactttt caaaagaaac 900 ctccttccag aagagtaatt cttcatctaa agccacgaca atgagtgaaa 950 atgtgcagtg gctcacgcct gtaatcccag cactttggaa ggccgcggcg 1000 ggcggatcac gaggtcagga gttctagacc agtctggcca atatggtgaa 1050 accccatctc tactaaaata caaaaattag ctgggcatgg tggcatgtgc 1100 ctgcagttcc agctgcttgg gagacaggag aatcacttga acccgggagg 1150 cggaggttgc agtgagctga gatcacgcca ctgcagtcca gcctgggtaa 1200 cagagcaaga ttccatctca aaaaataaaa taaataaata aataaatact 1250 ggtttttacc tgtagaattc ttacaataaa tatagcttga tattc 1295 <210> 2 <211> 312 <212> PRT
<213> artificial sequence <400> 2 Met Ala Arg Arg Ser Arg His Arg Leu Leu Leu Leu Leu Leu Arg Tyr Leu Val Val Ala Leu Gly Tyr His Lys Ala Tyr Gly Phe Ser Ala Pro Lys Asp Gln Gln Val Val Thr Ala Val Glu Tyr Gln Glu Ala Ile Leu Ala Cys Lys Thr Pro Lys Lys Thr Val Ser Ser Arg Leu Glu Trp Lys Lys Leu Gly Arg Ser Val Ser Phe Val Tyr Tyr Gln Gln Thr Leu Gln Gly Asp Phe Lys Asn Arg Ala Glu Met Ile Asp Phe Asn Ile Arg Ile Lys Asn Val Thr Arg Ser Asp Ala Gly Lys Tyr Arg Cys Glu Val Ser Ala Pro Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp Thr Val Thr Leu Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys Glu Val Pro Ser Ser Ala Leu Ser Gly Thr Val Val Glu Leu Arg Cys Gln Asp Lys Glu Gly Asn Pro Ala Pro Glu Tyr Thr Trp Phe Lys Asp Gly Ile Arg Leu Leu Glu Asn Pro Arg Leu Gly Ser Gln Ser Thr Asn Ser Ser Tyr Thr Met Asn Thr Lys Thr Gly Thr Leu Gln Phe Asn Thr Val Ser Lys Leu Asp Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg Arg Cys Pro Gly Lys Arg Met Gln Val Asp Asp Leu Asn Ile Ser Gly Ile Ile Ala Ala Val Val Val Val Ala Leu Val Ile Ser Val Cys Gly Leu Gly Val Cys Tyr Ala Gln Arg Lys Gly Tyr Phe Ser Lys Glu Thr Ser Phe Gln Lys Ser Asn Ser Ser Ser Lys Ala Thr Thr Met Ser Glu Asn Val Gln Trp Leu Thr Pro Val Ile Pro Ala Leu Trp Lys Ala Ala Ala Gly Gly Ser Arg Gly Gln Glu Phe <210> 3 <211> 2033 <212> DNA
<213> artificial sequence <400> 3 ccaggccggg aggcgacgcg cccagccgtc taaacgggaa cagccctggc 50 tgagggagct gcagcgcagc agagtatctg acggcgccag gttgcgtagg 100 tgcggcacga ggagttttcc cggcagcgag gaggtcctga gcagcatggc 150 ccggaggagc gccttccctg ccgccgcgct ctggctctgg agcatcctcc 200 tgtgcctgct ggcactgcgg gcggaggccg ggccgccgca ggaggagagc 250 ctgtacctat ggatcgatgc tcaccaggca agagtactr_a taggatttga 300 agaagatatc ctgattgttt cagaggggaa aatggcacct tttacacatg 350 atttcagaaa agcgcaacag agaatgccag ctattcctgt caatatccat 400 tccatgaatt ttacctggca agctgcaggg caggcagaat acttctatga 450 attcctgtcc ttgcgctccc tggataaagg catcatggca gatccaaccg 500 tcaatgtccc tctgctggga acagtgcctc acaaggcatc agttgttcaa 550 gttggtttcc catgtcttgg aaaacaggat ggggtggcag catttgaagt 600 ggatgtgatt gttatgaatt ctgaaggcaa caccattctc caaacacctc 650 aaaatgctat cttctttaaa acatgtcaac aagctgagtg cccaggcggg 700 tgccgaaatg gaggcttttg taatgaaaga cgcatctgcg agtgtcctga 750 tgggttccac ggacctcact gtgagaaagc cctttgtacc ccacgatgta 800 tgaatggtgg actttgtgtg actcctggtt tctgcatctg cccacctgga 850 ttctatggag tgaactgtga caaagcaaac tgctcaacca cctgctttaa 900 tggagggacc tgtttctacc ctggaaaatg tatttgccct ccaggactag 950 agggagagca gtgtgaaatc agcaaatgcc cacaaccctg tcgaaatgga 1000 ggtaaatgca ttggtaaaag caaatgtaag tgttccaaag gttaccaggg 1050 agacctctgt tcaaagcctg tctgcgagcc tggctgtggt gcacatggaa 1100 cctgccatga acccaacaaa tgccaatgtc aagaaggttg gcatggaaga 1150 cactgcaata aaaggtacga agccagcctc atacatgccc tgaggccagc 1200 aggcgcccag ctcaggcagc acacgccttc acttaaaaag gccgaggagc 1250 ggcgggatcc acctgaatcc aattacatct ggtgaactcc gacatctgaa 1300 acgttttaag ttacaccaag ttcatagcct ttgttaacct ttcatgtgtt 1350 gaatgttcaa ataatgttca ttacacttaa gaatactggc ctgaatttta 1400 ttagcttcat tataaatcac tgagctgata tttactcttc cttttaagtt 1450 ttctaagtac gtctgtagca tgatggtata gattttcttg tttcagtgct 1500 ttgggacaga ttttatatta tgtcaattga tcaggttaaa attttcagtg 1550 tgtagttggc agatattttc aaaattacaa tgcatttatg gtgtctgggg 1600 gcaggggaac atcagaaagg ttaaattggg caaaaatgcg taagtcacaa 1650 gaatttggat ggtgcagtta atgttgaagt tacagcattt cagattttat 1700 tgtcagatat ttagatgttt gttacatttt taaaaattgc tcttaatttt 1750 taaactctca atacaatata ttttgacctt accattattc cagagattca 1800 gtattaaaaa aaaaaaaatt acactgtggt agtggcattt aaacaatata 1850 atatattcta aacacaatga aatagggaat ataatgtatg aactttttgc 1900 attggcttga agcaatataa tatattgtaa acaaaacaca gctcttacct 1950 aataaacatt ttatactgtt tgtatgtata aaataaaggt gctgctttag 2000 ttttttggaa aaaaaaaaaa aaaaaaaaaa aaa 2033 <210> 4 <211> 379 <212> PRT
<213> artificial sequence <400> 4 Met AIa Arg Arg Ser Ala Phe Pro Ala Ala Ala Leu Trp Leu Trp Ser Ile Leu Leu Cys Leu Leu Ala Leu Arg Ala Glu Ala Gly Pro Pro Gln Glu Glu Ser Leu Tyr Leu Trp Ile Asp Ala His Gln Ala Arg Val Leu Ile Gly Phe Glu Glu Asp Ile Leu Ile Val Ser Glu Gly Lys Met Ala Pro Phe Thr His Asp Phe Arg Lys Ala Gln Gln Arg Met Pro Ala Ile Pro Val Asn Ile His Ser Met Asn Phe Thr Trp Gln Ala Ala Gly Gln Ala Glu Tyr Phe Tyr Glu Phe Leu Ser Leu Arg Ser Leu Asp Lys Gly Ile Met Ala Asp Pro Thr Val Asn Val Pro Leu Leu Gly Thr Val Pro His Lys Al.a Ser Val Val Gln Val Gly Phe Pro Cys Leu Gly Lys Gln Asp Gly Val Ala Ala Phe Glu Val Asp Val Ile Val Met Asn Ser Glu Gly Asn Thr Ile Leu Gln Thr Pro Gln Asn Ala Ile Phe Phe Lys Thr Cys Gln Gln Ala Glu Cys Pro Gly Gly Cys Arg Asn Gly Gly Phe Cys Asn Glu Arg Arg Ile Cys Glu Cys Pro Asp Gly Phe His Gly Pro His Cys Glu Lys Ala Leu Cys Thr Pro Arg Cys Met Asn Gly Gly Leu Cys Val Thr Pro Gly Phe Cys Ile Cys Pro Pro Gly Phe Tyr Gly Val Asn Cys Asp Lys Ala Asn Cys Ser Thr Thr Cys Phe Asn Gly Gly Thr Cys Phe Tyr Pro Gly Lys Cys Ile Cys Pro Pro Gly Leu Glu Gly Glu Gln Cys Glu Ile Ser Lys Cys Pro Gln Pro Cys Arg Asn Gly Gly Lys Cys Ile Gly Lys Ser Lys Cys Lys Cys Ser Lys Gly Tyr Gln Gly Asp Leu Cys Ser Lys Pro Val Cys Glu Pro Gly Cys Gly Ala His Gly Thr Cys His Glu Pro Asn Lys Cys Gln Cys Gln Glu Gly Trp His Gly Arg His Cys Asn Lys Arg Tyr Glu Ala Ser Leu Ile His Ala Leu Arg Pro Ala Gly Ala Gln Leu Arg Gln His Thr Pro Ser Leu Lys Lys Ala Glu Glu Arg Arg Asp Pro Pro Glu Ser Asn Tyr Ile Trp <210> 5 c211> 1857 <212> DNA
<213> artificial sequence <400> 5 gtctgttccc aggagtcctt cggcggctgt tgtgtcagtg gcctgatcgc 50 gatggggaca aaggcgcaag tcgagaggaa actgttgtgc ctcttcatat 100 tggcgatcct gttgtgctcc ctggcattgg gcagtgttac agtgcactct 150 tctgaacctg aagtcagaat tcctgagaat aatcctgtga agttgtcctg 200 tgcctactcg ggcttttctt ctccccgtgt ggagtggaag tttgaccaag 250 gagacaccac cagactcgtt tgctataata acaagatcac agcttcctat 300 gaggaccggg tgaccttctt gccaactggt atcaccttca agtccgtgac 350 acgggaagac actgggacat acacttgtat ggtctctgag gaaggcggca 400 acagctatgg ggaggtcaag gtcaagctca tcgtgcttgt gcctccatcc 450 aagcctacag ttaacatccc ctcctctgcc accattggga accgggcagt 500 gctgacatgc tcagaacaag atggttcccc accttctgaa tacacctggt 550 tcaaagatgg gatagtgatg cctacgaatc ccaaaagcac ccgtgccttc 600 agcaactctt cctatgtcct gaatcccaca acaggagagc tggtctttga 650 tcccctgtca gcctctgata ctggagaata cagctgtgag gcacggaatg 700 ggtatgggac acccatgact tcaaatgctg tgcgcatgga agctgtggag 750 cggaatgtgg gggtcatcgt ggcagccgtc cttgtaaccc tgattctcct 800 gggaatcttg gtttttggca tctggtttgc ctatagccga ggccactttg 850 acagaacaaa gaaagggact tcgagtaaga aggtgattta cagccagcct 900 agtgcccgaa gtgaaggaga attcaaacag acctcgtcat tcctggtgtg 950 agcctggtcg gctcaccgcc tatcatctgc attt~cctta ctcaggtgct 1000 accggactct ggcccctgat gtctgtagtt tcacaggatg ccttatttgt 1050 cttctacacc ccacagggcc ccctacttct tcggatgtgt ttttaataat 1100 gtcagctatg tgccccatcc tccttcatgc cctccctccc tttcctacca 1150 ctgctgagtg gcctggaact tgtttaaagt gtttattccc catttctttg 1200 agggatcagg aaggaatcct gggtatgcca ttgacttccc ttctaagtag 1250 acagcaaaaa tggcgggggt cgcaggaatc tgcactcaac tgcccacctg 1300 gctggcaggg atctttgaat aggtatcttg agcttggttc tgggctcttt 1350 ccttgtgtac tgacgaccag ggccagctgt tctagagcgg gaattagagg 1400 ctagagcggc tgaaatggtt gtttggtgat gacactgggg tccttccatc 1450 tctggggccc actctcttct gtcttcccat gggaagtgcc actgggatcc 1500 ctctgccctg tcctcctgaa tacaagctga ctgacattga ctgtgtctgt 1550 ggaaaatggg agctcttgtt gtggagagca tagtaaattt tcagagaact 1600 tgaagccaaa aggatttaaa accgctgctc taaagaaaag aaaactggag 1650 gctgggcgca gtggctcacg cctgtaatcc cagaggctga ggcaggcgga 1700 tcacctgagg tcgggagttc gggatcagcc tgaccaacat ggagaaaccc 1750 tactggaaat acaaagttag ccaggcatgg tggtgcatgc ctgtagtccc 1800 agctgctcag gagcctggca acaagagcaa aactccagct caaaaaaaaa 1850 aaaaaaa 1857 <210> 6 <211> 299 <212> PRT
<213> artificial sequence <400> 6 Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe Ile Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr Val His Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro Val Lys Leu Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Tzp Lys Phe Asp GIn Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gl.y Ile Leu Val Phe Gly Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser Ala Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val <210> 7 <211> 2755 <212> DNA
<213> artificial sequence <400> 7 gggggttagg gaggaaggaa tccaccccca cccccccaaa cccttttctt 50 ctcctttcct ggcttcggac attggagcac taaatgaact tgaattgtgt 100 ctgtggcgag caggatggtc gctgttactt tgtgatgaga tcggggatga 150 attgctcgct ttaaaaatgc tgctttggat tctgttgctg gagacgtctc 200 tttgttttgc cgctggaaac gttacagggg acgtttgcaa agagaagatc 250 tgttcctgca atgagataga aggggaccta cacgtagact gtgaaaaaaa 300 gggcttcaca agtctgcagc gtttcactgc cccgacttcc cagttttacc 350 atttatttct gcatggcaat tccctcactc gacttttccc taatgagttc 400 gctaactttt ataatgcggt tagtttgcac atggaaaaca atggcttgca 450 tgaaatcgtt ccgggggctt ttctggggct gcagctggtg aaaaggctgc 500 acatcaacaa caacaagatc aagtcttttc gaaagcagac ttttctgggg 550 ctggacgatc tggaatatct ccaggctgat tttaatttat tacgagatat 600 agacccgggg gccttccagg acttgaacaa gctggaggtg ctcattttaa 650 atgacaatct catcagcacc ctacctgcca acgtgttcca gtatgtgccc 700 atcacccacc tcgacctccg gggtaacagg ctgaaaacgc tgccctatga 750 ggaggtcttg gagcaaatcc ctggtattgc ggagatcctg ctagaggata 800 acccttggga ctgcacctgt gatctgctct ccctgaaaga atggctggaa 850 aacattccca agaatgccct gatcggccga gtggtctgcg aagcccccac 900 cagactgcag ggtaaagacc tcaatgaaac caccgaacag gacttgtgtc 950 ctttgaaaaa ccgagtggat tctagtctcc cggcgccccc tgcccaagaa 1000 gagacctttg ctcctggacc cctgccaact cctttcaaga caaatgggca 1050 agaggatcat gccacaccag ggtctgctcc aaacggaggt acaaagatcc 1100 caggcaactg gcagatcaaa atcagaccca cagcagcgat agcgacgggt 1150 agctccagga acaaaccctt agctaacagt ttaccctgcc ctgggggctg 1200 cagctgcgac cacatcccag ggtcgggttt aaagatgaac tgcaacaaca 1250 ggaacgtgag cagcttggct gatttgaagc ccaagctctc taacgtgcag 1300 gagcttttcc tacgagataa caagatccac agcatccgaa aatcgcactt 1350 tgtggattac aagaacctca ttctgttgga tctgggcaac aataacatcg 1400 ctactgtaga gaacaacact ttcaagaacc ttttggacct caggtggcta 1450 tacatggata gcaattacct ggacacgctg tcccgggaga aattcgcggg 1500 gctgcaaaac ctagagtacc tgaacgtgga gtacaacgct atccagctca 1550 tcctcccggg cactttcaat gccatgccca aactgaggat cctcattctc 7.600 aacaacaacc tgctgaggtc cctgcctgtg gacgtgttcg ctggggtctc 1650 gctctctaaa ctcagcctgc acaacaatta cttcatgtac ctcccggtgg 1700 caggggtgct ggaccagtta acctccatca tccagataga cctccacgga 1750 aacccctggg agtgctcctg cacaattgtg cctttcaagc agtgggcaga 1800 acgcttgggt tccgaagtgc tgatgagcga cctcaagtgt gagacgccgg 1850 tgaacttctt tagaaaggat ttcatgctcc tctccaatga cgagatctgc 1900 cctcagctgt acgctaggat ctcgcccacg ttaacttcgc acagtaaaaa 1950 cagcactggg ttggcggaga ccgggacgca ctccaactcc tacctagaca 2000 ccagcagggt gtccatctcg gtgttggtcc cgggactgct gctggtgttt 2050 gtcacctccg ccttcaccgt ggtgggcatg ctcgtgttta tcctgaggaa 2100 ccgaaagcgg tccaagagac gagatgccaa ctcctccgcg tccgagatta 2150 attccctaca gacagtctgt gactcttcct actggcacaa tgggccttac 2200 aacgcagatg gggcccacag agtgtatgac tgtggctctc actcgctctc 2250 agactaagac cccaacccca ataggggagg gcagagggaa ggcgatacat 2300 ccttccccac cgcaggcacc ccgggggctg gaggggcgtg tacccaaatc 2350 cccgcgccat cagcctggat gggcataagt agataaataa ctgtgagctc 2400 gcacaaccga aagggcctga ccccttactt agctccctcc ttgaaacaaa 2450 gagcagactg tggagagctg ggagagcgca gccagctcgc tctttgctga 2500 gagccccttt tgacagaaag cccagcacga ccctgctgga agaactgaca 2550 gtgccctcgc cctcggcccc ggggcctgtg gggttggatg ccgcggttct 2600 atacatatat acatatatcc acatctatat agagagatag atatctattt 2650 ttcccctgtg gattagcccc gtgatggctc cctgttggct acgcagggat 2700 gggcagttgc acgaaggcat gaatgtattg taaataagta actttgactt 2750 ctgac 2755 <210> 8 <211> 696 <212> PRT
<213> artificial sequence <400> 8 Met Leu Leu Trp Ile Leu Leu Leu Glu Thr Ser Leu Cys Phe Ala Ala Gly Asn Val Thr Gly Asp Val Cys Lys Glu Lys Ile Cys Ser Cys Asn Glu Ile Glu Gly Asp Leu His Val Asp Cys Glu Lys Lys Gly Phe Thr Ser Leu Gln Arg Phe Thr Ala Pro Thr Ser Gln Phe Tyr His Leu Phe Leu His Gly Asn Ser Leu Thr Arg Leu Phe Pro Asn Glu Phe Ala Asn Phe Tyr Asn Ala Val Ser Leu His Met Glu Asn Asn Gly Leu His Glu Ile Val Pro Gly Ala Phe Leu Gly Leu Gln Leu Val Lys Arg Leu His Ile Asn Asn Asn Lys Ile Lys Ser Phe Arg Lys Gln Thr Phe Leu Gly Leu Asp Asp Leu Glu Tyr Leu Gln Ala Asp Phe Asn Leu Leu Arg Asp Ile Asp Pro Gly Ala Phe Gln Asp Leu Asn Lys Leu Glu Val Leu Ile Leu Asn Asp Asn Leu Ile Ser Thr Leu Pro Ala Asn Val Phe Gln Tyr Val Pro Ile Thr His Leu Asp Leu Arg Gly Asn Arg Leu Lys Thr Leu Pro Tyr Glu Glu Val Leu Glu Gln Ile Pro Gly Ile Ala Glu Ile Leu Leu Glu Asp Asn Pro Trp Asp Cys Thr Cys Asp Leu Leu Ser Leu Lys Glu Trp Leu Glu Asn Ile Pro Lys Asn Ala Leu Ile Gly Arg Val Val Cys Glu Ala Pro Thr Arg Leu Gln Gly Lys Asp Leu Asn Glu Thr Thr Glu Gln Asp Leu Cys Pro Leu Lys Asn Arg Val Asp Ser Ser Leu Pro Ala Pro Pro Ala Gln Glu Glu Thr Phe Ala Pro Gly Pro Leu Pro Thr Pro Phe Lys Thr Asn Gly Gln Glu Asp His Ala Thr Pro Gly Ser Ala Pro Asn Gly Gly Thr Lys Ile Pro Gly Asn Trp Gln Ile Lys Ile Arg Pro Thr Ala Ala Ile Ala Thr Gly Ser Ser Arg Asn Lys Pro Leu Ala Asn Ser Leu Pro Cys Pro Gly Gly Cys Ser Cys Asp His Ile Pro Gly Ser Gly Leu Lys Met Asn Cys Asn Asn Arg Asn Val Ser Ser Leu Ala Asp Leu Lys Pro Lys Leu Ser Asn Val Gln Glu Leu Phe Leu Arg Asp Asn Lys Ile His Ser Ile Arg Lys Ser His Phe Val Asp Tyr Lys Asn Leu Ile Leu Leu Asp Leu Gly Asn Asn Asn Ile Ala Thr Val Glu Asn Asn Thr Phe Lys Asn Leu Leu Asp Leu Arg Trp Leu Tyr Met Asp Ser Asn Tyr Leu Asp Thr Leu Ser Arg Glu Lys Phe Ala Gly Leu Gln Asn Leu Glu Tyr Leu Asn Val Glu Tyr Asn Ala Ile Gln Leu Ile Leu Pro Gly Thr Phe Asn Ala Met Pro Lys Leu Arg Ile Leu Ile Leu Asn Asn Asn Leu Leu Arg Ser Leu Pro Val Asp Val Phe Ala Gly Val Ser Leu Ser Lys Leu Ser Leu His Asn Asn Tyr Phe Met Tyr Leu Pro Val Ala Gly Val Leu Asp Gln Leu Thr Ser Ile Ile Gln Ile Asp Leu His Gly Asn Pro Trp Glu Cys Ser Cys Thr Ile Val Pro Phe Lys Gln Trp Ala Glu Arg Leu Gly Ser Glu Val Leu Met Ser Asp Leu Lys Cys Glu Thr Pro Val Asn Phe Phe Arg Lys Asp Phe Met Leu Leu Ser Asn Asp Glu Ile Cys Pro Gln Leu Tyr Ala Arg Ile Ser Pro Thr Leu Thr Ser His Ser Lys Asn Ser Thr Gly Leu Ala Glu Thr Gly Thr His Ser Asn Ser Tyr Leu Asp Thr Ser Arg Val Ser Ile Ser Val Leu Val Pro Gly Leu Leu Leu Val Phe Val Thr Ser Ala Phe Thr Val Val Gly Met Leu Val Phe Ile Leu Arg Asn Arg Lys Arg Ser Lys Arg Arg Asp Ala Asn Ser Ser Ala Ser Glu Ile Asn Ser Leu Gln Thr Val Cys Asp Ser Ser Tyr Trp His Asn Gly Pro Tyr Asn Ala Asp Gly Ala His Arg Val Tyr Asp Cys Gly Ser His Ser Leu Ser Asp <210> 9 <211> 3659 <212> DNA
<213> artificial sequence <400> 9 gtaactgaag tcaggctttt catttgggaa gccccctcaa cagaattcgg 50 tcattctcca agttatggtg gacgtacttc tgttgttctc cctctgcttg 100 ctttttcaca ttagcagacc ggacttaagt cacaacagat tatctttcat 150 caaggcaagt tccatgagcc accttcaaag ccttcgagaa gtgaaactga 200 acaacaatga attggagacc attccaaatc tgggaccagt ctcggcaaat 250 attacacttc tctccttggc tggaaacagg attgttgaaa tactccctga 300 acatctgaaa gagtttcagt cccttgaaac tttggacctt agcagcaaca 350 atatttcaga gctccaaact gcatttccag ccctacagct caaatatctg 400 tatctcaaca gcaaccgagt cacatcaatg gaacctgggt attttgacaa 450 tttggccaac acactccttg tgttaaagct gaacaggaac cgaatctcag 500 ctatcccacc caagatgttt aaactgcccc aactgcaaca tctcgaattg 550 aaccgaaaca agattaaaaa tgtagatgga ctgacattcc aaggccttgg 600 tgctctgaag tctctgaaaa tgcaaagaaa tggagtaacg aaacttatgg 650 atggagcttt ttgggggctg agcaacatgg aaattttgca gctggaccat 700 aacaacctaa cagagattac caaaggctgg ctttacggct tgctgatgct 750 gcaggaactt catctcagcc aaaatgccat caacaggatc agccctgatg 800 cctgggagtt ctgccagaag ctcagtgagc tggacctaac tttcaatcac 850 ttatcaaggt tagatgattc aagcttcctt ggcctaagct tactaaatac 900 ctgcacattg ggaacaacag agtcagctac attgctgatt gtgccttccg 950 ggggctttcc agtttaaaga ctttggatct gaagaacaat gaaatttcct 1000 ggactattga agacatgaat ggtgctttct ctgggcttga caaactgagg 1050 cgactgatac tccaaggaaa tcggatccgt tctattacta aaaaagcctt 1100 cactggtttg gatgcattgg agcatctaga cctgagtgac aacgcaatca 1150 tgtctttaca aggcaatgca ttttcacaaa tgaagaaact gcaacaattg 1200 catttaaata catcaagcct tttgtgcgat tgccagctaa aatggctccc 1250 acagtgggtg gcggaaaaca actttcagag ctttgtaaat gccagttgtg 1300 cccatcctca gctgctaaaa ggaagaagca tttttgctgt tagcccagat 1350 ggctttgtgt gtgatgattt tcccaaaccc cagatcacgg ttcagccaga 1400 aacacagtcg gcaataaaag gttccaattt gagtttcatc tgctcagctg 1450 ccagcagcag tgattcccca atgacttttg cttggaaaaa agacaatgaa 1500 ctactgcatg atgctgaaat ggaaaattat gcacacctcc gggcccaagg 1550 tggcgaggtg atggagtata ccaccatcct tcggctgcgc gaggtggaat 1600 ttgccagtga ggggaaatat cagtgtgtca tctccaatca ctttggttca 1650 tcctactctg tcaaagccaa gcttacagta aatatgcttc cctcattcac 1700 caagaccccc atggatctca ccatccgagc tggggccatg gcacgcttgg 1750 agtgtgctgc tgtggggcac ccagcccccc agatagcctg gcagaaggat 1800 gggggcacag acttcccagc tgcacgggag agacgcatgc atgtgatgcc 1850 cgaggatgac gtgttcttta tcgtggatgt gaagatagag gacattgggg 1900 tatacagctg cacagctcag aacagtgcag gaagtatttc agcaaatgca 1950 actctgactg tcctagaaac accatcattt ttgcggccac tgttggaccg 2000 aactgtaacc aagggagaaa cagccgtcct acagtgcatt gctggaggaa 2050 gccctccccc taaactgaac tggaccaaag atgatagccc attggtggta 2100 accgagaggc acttttttgc agcaggcaat cagcttctga ttattgtgga 2150 ctcagatgtc agtgatgctg ggaaatacac atgtgagatg tctaacaccc 2200 ttggcactga gagaggaaac gtgcgcctca gtgtgatccc cactccaacc 2250 tgcgactccc ctcagatgac agccccatcg ttagacgatg acggatgggc 2300 cactgtgggt gtcgtgatca tagccgtggt ttgctgtgtg gtgggcacgt 2350 cactcgtgtg ggtggtcatc atataccaca caaggcggag gaatgaagat 2400 tgcagcatta ccaacacaga tgagaccaac ttgccagcag atattcctag 2450 ttatttgtca tctcagggaa cgttagctga caggcaggat gggtacgtgt 2500 cttcagaaag tggaagccac caccagtttg tcacatcttc aggtgctgga 2550 tttttcttac cacaacatga cagtagtggg acctgccata ttgacaatag 2600 cagtgaagct gatgtggaag ctgccacaga tctgttcctt tgtccgtttt 2650 tgggatccac aggccctatg tatttgaagg gaaatgtgta tggctcagat 2700 ccttttgaaa catatcatac aggttgcagt cctgacccaa gaacagtttt 2750 aatggaccac atgagcccag ttacataaag aaaaaggagt gctacccatg 2800 ttctcatcct tcagaagaat cctgcgaacg gagcttcagt aatatatcgt 2850 ggccttcaca tgtgaggaag ctacttaaca ctagttactc tcacaatgaa 2900 ggacctggaa tgaaaaatct gtgtctaaac aagtcctctt tagattttag 2950 tgcaaatcca gagccagcgt cggttgcctc gagtaattct ttcatgggta 3000 cctttggaaa agctctcagg agacctcacc tagatgccta ttcaagcttt 3050 ggacagccat cagattgtca gccaagagcc ttttatttga aagctcattc 3100 ttccccagac ttggactctg ggtcagagga agatgggaaa gaaaggacag 3150 attttcagga agaaaatcac atttgtacct taaacagact ttagaaaact 3200 acaggactcc aaattttcag tcttatgact tggacacata gactgaatga 3250 gaccaaagga aaagcttaac atactacctc aagtgaactt ttatttaaaa 3300 gagagagaat cttatgtttt ttaaatggag ttatgaattt taaaaggata 3350 aaaatgcttt atttatacag atgaaccaaa attacaaaaa gttatgaaaa 3400 tttttatact gggaatgatg ctcatataag aatacctttt taaactattt 3450 tttaactttg ttttatgcaa aaaagtatct tacgtaaatt aatgatataa 3500 atcatgatta ttttatgtat ttttataatg ccagatttct ttttatggaa 3550 aatgagttac taaagcattt taaataatac ctgccttgta ccatttttta 3600 aatagaagtt acttcattat attttgcaca ttatatttaa taaaatgtgt 3650 caatttgaa 3659 <210> 10 <211> 1059 <212> PRT
<213> artificial sequence <400> 10 Met Val Asp Val Leu Leu Leu Phe Ser Leu Cys Leu Leu Phe His Ile Ser Arg Pro Asp Leu Ser His Asn Arg Leu Ser Phe Ile Lys Ala Ser Ser Met Ser His Leu Gln Ser Leu Arg Glu Val Lys Leu Asn Asn Asn Glu Leu Glu Thr Ile Pro Asn Leu Gly Pro Val Ser Ala Asn Ile Thr Leu Leu Ser Leu Ala Gly Asn Arg Ile Val Glu Ile Leu Pro Glu His Leu Lys Glu Phe Gln Ser Leu Glu Thr Leu Asp Leu Ser Ser Asn Asn Ile Ser Glu Leu Gln Thr Ala Phe Pro Ala Leu Gln Leu Lys Tyr Leu Tyr Leu Asn Ser Asn Arg Val Thr Ser Met Glu Pro Gly Tyr Phe Asp Asn Leu Ala Asn Thr Leu Leu Val Leu Lys Leu Asn Arg Asn Arg Ile Ser Ala Ile Pro Pro Lys Met Phe Lys Leu Pro Gln Leu Gln His Leu Glu Leu Asn Arg Asn Lys Ile Lys Asn Val Asp Gly Leu Thr Phe Gln Gly Leu Gly Ala Leu Lys Ser Leu Lys Met Gln Arg Asn Gly Val Thr Lys Leu Met Asp Gly Ala Phe Trp Gly Leu Ser Asn Met Glu Ile Leu Gln Leu Asp His Asn Asn Leu Thr Glu Ile Thr Lys Gly Trp Leu Tyr Gly Leu Leu Met Leu Gln Glu Leu His Leu Ser Gln Asn Ala Ile Asn Arg Ile Ser Pro Asp Ala Trp Glu Phe Cys Gln Lys Leu Ser Glu Leu Asp Leu Thr Phe Asn His Leu Ser Arg Leu Asp Asp Ser Ser Phe Leu Gly Leu Ser Leu Leu Asn Thr Leu His Ile Gly Asn Asn Arg Val Ser Tyr Ile Ala Asp Cys Ala Phe Arg Gly Leu Ser Ser Leu Lys Thr Leu Asp Leu Lys Asn Asn Glu Ile Ser Trp Thr Ile Glu Asp Met Asn Gly Ala Phe Ser Gly Leu Asp Lys Leu Arg Arg Leu Ile Leu Gln Gly Asn Arg Ile Arg Ser Ile Thr Lys Lys Ala Phe Thr Gly Leu Asp Ala Leu Glu His Leu Asp Leu Ser Asp Asn Ala Ile Met Ser Leu Gln Gly Asn Ala Phe Ser Gln Met Lys Lys Leu Gln Gln Leu His Leu Asn Thr Ser Ser Leu Leu Cys Asp Cys Gln Leu Lys Trp Leu Pro Gln Trp Val AIa Glu Asn Asn Phe Gln Ser Phe Val Asn Ala Ser Cys Ala His Pro Gln Leu Leu Lys Gly Arg Ser Ile Phe Ala Val Ser Pro Asp Gly Phe Val Cys Asp Asp Phe Pro Lys Pro Gln Ile Thr Val Gln Pro Glu Thr Gln Ser Ala Ile Lys Gly Ser Asn Leu Ser Phe Ile Cys Ser Ala Ala Ser Ser Ser Asp Ser Pro Met Thr Phe Ala Trp Lys Lys Asp Asn Glu Leu Leu His Asp Ala Glu Met Glu Asn Tyr Ala His Leu Arg Ala Gln Gly Gly Glu Val Met Glu Tyr Thr Thr Ile Leu Arg Leu Arg Glu Val Glu Phe Ala Ser Glu Gly Lys Tyr Gln Cys Val Ile Ser Asn His Phe Gly Ser Ser Tyr Ser Val Lys Ala Lys Leu Thr Val Asn Met Leu Pro Ser Phe Thr Lys Thr Pro Met Asp Leu Thr Ile Arg Ala Gly Ala Met Ala Arg Leu Glu Cys Ala Ala Val Gly His Pro Ala Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro Ala Ala Arg Glu Arg Arg Met His Val Met Pro Glu Asp Asp Val Phe Phe Ile Val Asp Val Lys Ile Glu Asp Ile Gly Val Tyr Ser Cys Thr Ala Gln Asn Ser Ala Gly Ser Ile Ser Ala Asn Ala Thr Leu Thr Val Leu Glu Thr Pro Ser Phe Leu Arg Pro Leu Leu Asp Arg Thr Val Thr Lys Gly Glu Thr Ala Val Leu Gln Cys Ile Ala Gly Gly Ser Pro Pro Pro Lys Leu Asn Tzp Thr Lys Asp Asp Ser Pro Leu Val Val Thr Glu Arg His Phe Phe Ala Ala Gly Asn Gln Leu Leu Ile Ile Val Asp Ser Asp Val Ser Asp Ala Gly Lys Tyr Thr Cys Glu Met Ser Asn Thr Leu Gly Thr Glu Arg Gly Asn Val 1~

Arg Leu Ser Val Ile Pro Thr Pro Thr Cys Asp Ser Pro Gln Met Thr Ala Pro Ser Leu Asp Asp Asp Gly Trp A:La Thr Val Gly Val Val Ile Ile Ala Val Val Cys Cys Val Val Gly Thr Ser Leu Val Trp Val Val Ile Ile Tyr His Thr Arg Arg Arg Asn Glu Asp Cys Ser Ile Thr Asn Thr Asp Glu Thr Asn Leu Pro Ala Asp Ile Pro Ser Tyr Leu Ser Ser Gln Gly Thr Leu Ala Asp Arg Gln Asp Gly Tyr Val Ser Ser Glu Ser Gly Ser His His Gln Phe Val Thr Ser Ser Gly Ala Gly Phe Phe Leu Pro Gln His Asp Ser Ser Gly Thr Cys His Ile Asp Asn Ser Ser Glu Ala Asp Val Glu Ala Ala Thr Asp Leu Phe Leu Cys Pro Phe Leu Gly Ser Thr Gly Pro Met Tyr Leu Lys Gly Asn Val Tyr Gly Ser Asp Pro Phe Glu Thr Tyr His Thr Gly Cys Ser Pro Asp Pro Arg Thr Val Leu Met Asp His Tyr Glu Pro Ser Tyr Ile Lys Lys Lys Glu Cys Tyr Pro Cys Ser His Pro Ser Glu Glu Ser Cys Glu Arg Ser Phe Ser Asn Ile Ser Trp Pro Ser His Val Arg Lys Leu Leu Asn Thr Ser Tyr Ser His Asn Glu Gly Pro Gly Met Lys Asn Leu Cys Leu Asn Lys Ser Ser Leu Asp Phe Ser Ala Asn Pro Glu Pro Ala Ser Val Ala Ser Ser Asn Sex Phe Met Gly Thr Phe Gly Lys Ala Leu Arg Arg Pro His Leu Asp Ala Tyr Ser Ser Phe Gly Gln Pro Ser Asp Cys Gln Pro Arg Ala Phe Tyr Leu Lys Ala His Ser Ser Pro Asp Leu Asp Ser Gly Ser Glu Glu Asp Gly Lys Glu Arg Thr Asp Phe Gln Glu Glu Asn Ig His Ile Cys Thr Phe Lys Gln Thr Leu Glu Asn Tyr Arg Thr Pro Asn Phe Gln Ser Tyr Asp Leu Asp Thr <210> 11 <211> 2906 <212> DNA
<213> artificial sequence <400> 11 ggggagagga attgaccatg taaaaggaga cttttttttt tggtggtggt 50 ggctgttggg tgccttgcaa aaatgaagga tgcaggacgc agctttctcc 100 tggaaccgaa cgcaatggat aaactgattg tgcaagagag aaggaagaac 150 gaagcttttt cttgtgagcc ctggatctta acacaaatgt gtatatgtgc 200 acacagggag cattcaagaa tgaaataaac cagagttaga cccgcggggg 250 ttggtgtgtt ctgacataaa taaataatct taaagcagct gttcccctcc 300 ccacccccaa aaaaaaggat gattggaaat gaagaaccga ggattcacaa 350 agaaaaaagt atgttcattt ttctctataa aggagaaagt gagccaagga 400 gatatttttg gaatgaaaag tttggggctt ttttagtaaa gtaaagaact 450 ggtgtggtgg tgttttcctt tctttttgaa tttcccacaa gaggagagga 500 aattaataat acatctgcaa agaaatttca gagaagaaaa gttgaccgcg 550 gcagattgag gcattgattg ggggagagaa accagcagag cacagttgga 600 tttgtgccta tgttgactaa aattgacgga taattgcagt tggatttttc 650 ttcatcaacc tccttttttt taaattttta ttccttttgg tatcaagatc 700 atgcgttttc tcttgttctt aaccacctgg atttccatct ggatgttgct 750 gtgatcagtc tgaaatacaa ctgtttgaat tccagaagga ccaacaccag 800 ataaattatg aatgttgaac aagatgacct tacatccaca gcagataatg 850 ataggtccta ggtttaacag ggccctattt gaccccctgc ttgtggtgct 900 gctggctctt caacttcttg tggtggctgg tctggtgcgg gctcagacct 950 gcccttctgt gtgctcctgc agcaaccagt tcagcaaggt gatttgtgtt 1000 cggaaaaacc tgcgtgaggt tccggatggc atctccacca acacacggct 1050 gctgaacctc catgagaacc aaatccagat catcaaagtg aacagcttca 1100 agcacttgag gcacttggaa atcctacagt tgagtaggaa ccatatcaga 1150 accattgaaa ttggggcttt caatggtctg gcgaacctca acactctgga 1200 actctttgac aatcgtctta ctaccatccc gaatggagct tttgtatact 1250 tgtctaaact gaaggagctc tggttgcgaa acaaccccat tgaaagcatc 1300 ccttcttatg cttttaacag aattccttct ttgcgccgac tagacttagg 1350 ggaattgaaa agactttcat acatctcaga aggtgccttt gaaggtctgt 1400 ccaacttgag gtatttgaac cttgccatgt gcaaccttcg ggaaatccct 1450 aacctcacac cgctcataaa actagatgag ctggatcttt ctgggaatca 1500 tttatctgcc atcaggcctg gctctttcca gggtttgatg caccttcaaa 1550 aactgtggat gatacagtcc cagattcaag tgattgaacg gaatgccttt 1600 gacaaccttc agtcactagt ggagatcaac ctggcacaca ataatctaac 1650 attactgcct catgacctct tcactccctt gcatcatcta gagcggatac 1700 atttacatca caacccttgg aactgtaact gtgacatact gtggctcagc 1750 tggtggataa aagacatggc cccctcgaac acagcttgtt gtgcccggtg 1800 taacactcct cccaatctaa aggggaggta cattggagag ctcgaccaga 1850 attacttcac atgctatgct ccggtgattg tggagccccc tgcagacctc 1900 aatgtcactg aaggcatggc agctgagctg aaatgtcggg cctccacatc 1950 cctgacatct gtatcttgga ttactccaaa tggaacagtc atgacacatg 2000 gggcgtacaa agtgcggata gctgtgctca gtgatggtac gttaaatttc 2050 acaaatgtaa ctgtgcaaga tacaggcatg tacacatgta tggtgagtaa 2100 ttccgttggg aatactactg cttcagccac cctgaatgtt actgcagcaa 2150 ccactactcc tttctcttac ttttcaaccg tcacagtaga gactatggaa 2200 ccgtctcagg atgaggcacg gaccacagat aacaatgtgg gtcccactcc 2250 agtggtcgac tgggagacca ccaatgtgac cacctctctc acaccacaga 2300 gcacaaggtc gacagagaaa accttcacca tcccagtgac tgatataaac 2350 agtgggatcc caggaattga tgaggtcatg aagactacca aaatcatcat 2400 tgggtgtttt gtggccatca cactcatggc tgcagtgatg ctggtcattt 2450 tctacaagat gaggaagcag caccatcggc aaaaccatca cgccccaaca 2500 aggactgttg aaattattaa tgtggatgat gagattacgg gagacacacc 2550 catggaaagc cacctgccca tgcctgctat cgagcatgag cacctaaatc 2600 actataactc atacaaatct cccttcaacc acacaacaac agttaacaca 2650 ataaattcaa tacacagttc agtgcatgaa ccgttattga tccgaatgaa 2700 ctctaaagac aatgtacaag agactcaaat ctaaaacatt tacagagtta 2750 caaaaaacaa acaatcaaaa aaaaagacag tttattaaaa atgacacaaa 2800 tgactgggct aaatctactg tttcaaaaaa gtgtctttac aaaaaaacaa 2850 aaaagaaaag aaatttattt attaaaaatt ctattgtgat ctaaagcaga 2900 caaaaa 2906 <210> 12 <211> 640 <212> PRT
<213> artificial sequence <400> 12 Met Leu Asn Lys Met Thr Leu His Pro Gln Gln Ile Met Ile Gly Pro Arg Phe Asn Arg Ala Leu Phe Asp Pro Leu Leu Val Val Leu Leu Ala Leu Gln Leu Leu Val Val Ala Gly Leu Val Arg Ala Gln Thr Cys Pro Ser Val Cys Ser Cys Ser Asn Gln Phe Ser Lys Val Ile Cys Val Arg Lys Asn Leu Arg Glu Val Pro Asp Gly Ile Ser Thr Asn Thr Arg Leu Leu Asn Leu His Glu Asn Gln Ile Gln Ile Ile Lys Val Asn Ser Phe Lys His Leu Arg His Leu Glu Ile Leu Gln Leu Ser Arg Asn His Ile Arg Thr Ile Glu Ile Gly Ala Phe Asn Gly Leu Ala Asn Leu Asn Thr Leu Glu Leu Phe Asp Asn Arg Leu Thr Thr Ile Pro Asn Gly Ala Phe Val Tyr Leu Ser Lys Leu Lys Glu Leu Trp Leu Arg Asn Asn Pro Ile Glu Ser Ile Pro Ser Tyr Ala Phe Asn Arg Ile Pro Ser Leu Arg Arg Leu Asp Leu Gly Glu Leu Lys Arg Leu Ser Tyr Ile Ser Glu Gly Ala Phe Glu Gly Leu Ser Asn Leu Arg Tyr Leu Asn Leu Ala Met Cys Asn Leu Arg Glu Ile Pro Asn Leu Thr Pro Leu Ile Lys Leu Asp Glu Leu Asp Leu Ser Gly Asn His Leu Ser Ala Ile Arg Pro Gly Ser Phe Gln Gly Leu Met His Leu Gln Lys Leu Trp Met Ile Gln Ser Gln Ile Gln Val Ile Glu Arg Asn Ala Phe Asp Asn Leu Gln Ser Leu Val Glu Ile Asn Leu Ala His Asn Asn Leu Thr Leu Leu Pro His Asp Leu Phe Thr Pro Leu His His Leu Glu Arg Ile His Leu His His Asn Pro Trp Asn Cys Asn Cys Asp Ile Leu Trp Leu Ser Trp Trp Ile Lys Asp Met Ala Pro Ser Asn Thr Ala Cys Cys Ala Arg Cys Asn Thr Pro Pro Asn Leu Lys Gly Arg Tyr Ile Gly Glu Leu Asp Gln Asn Tyr Phe Thr Cys Tyr Ala Pro Val Ile Val Glu Pro Pro Ala Asp Leu Asn Val Thr Glu Gly Met Ala Ala Glu Leu Lys Cys Arg Ala Ser Thr Ser Leu Thr Ser Val Ser Trp Ile Thr Pro Asn Gly Thr Val Met Thr His Gly Ala Tyr Lys Val Arg Ile Ala Val Leu Ser Asp Gly Thr Leu Asn Phe Thr Asn Val Thr Val Gln Asp Thr Gly Met Tyr Thr Cys Met Val Ser Asn Ser Val Gly Asn Thr Thr Ala Ser Ala Thr Leu Asn Val Thr Ala Ala Thr Thr Thr Pro Phe Ser Tyr Phe Ser Thr Val Thr Val Glu Thr Met Glu Pro Ser Gln Asp Glu Ala Arg Thr Thr Asp Asn Asn Val Gly Pro Thr Pro Val Val Asp Trp Glu Thr Thr Asn Val Thr Thr Ser Leu Thr Pro Gln Ser Thr Arg Ser Thr Glu Lys Thr Phe Thr Ile Pro Val Thr Asp Ile Asn Ser Gly Ile Pro Gly Ile Asp Glu Val Met Lys Thr Thr Lys Ile Ile Ile Gly Cys Phe Val Ala Ile Thr Leu Met Ala Ala Val Met Leu Val Ile Phe Tyr Lys Met Arg Lys Gln His His Arg Gln Asn His His Ala Pro Thr Arg Thr Val Glu Ile Ile Asn Val Asp Asp Glu Ile Thr Gly Asp Thr Pro Met Glu Ser His Leu Pro Met Pro Ala Ile Glu His Glu His Leu Asn His Tyr Asn Ser Tyr Lys Ser Pro Phe Asn His Thr Thr Thr Val Asn Thr Ile Asn Ser Ile His Ser Ser Val His Glu Pro Leu Leu Ile Arg Met Asn Ser Lys Asp Asn Val Gln Glu Thr Gln Ile <210> 13 <211> 4051 <212> DNA
<213> artificial sequence <400> 13 agccgacgct gctcaagctg caactctgtt gcagttggca gttcttttcg 50 gtttccctcc tgctgtttgg gggcatgaaa gggcttcgcc gccgggagta 100 aaagaaggaa ttgaccgggc agcgcgaggg aggagcgcgc acgcgaccgc 150 gagggcgggc gtgcaccctc ggctggaagt ttgtgccggg ccccgagcgc 200 gcgccggctg ggagcttcgg gtagagacct aggccgctgg accgcgatga 250 gcgcgccgag cctccgtgcg cgcgccgcgg ggttggggct gctgctgtgc 300 gcggtgctgg ggcgcgctgg ccggtccgac agcggcggtc gcggggaact 350 cgggcagccc tctggggtag ccgccgagcg cccatgcccc actacctgcc 400 gctgcctcgg ggacctgctg gactgcagtc gtaagcggct agcgcgtctt 450 cccgagccac tcccgtcctg ggtcgctcgg ctggacttaa gtcacaacag 500 attatctttc atcaaggcaa gttccatgag ccaccttcaa agccttcgag 550 aagtgaaact gaacaacaat gaattggaga ccattccaaa tctgggacca 600 gtctcggcaa atattacact tctctccttg gctggaaaca ggattgttga 650 aatactccct gaacatctga aagagtttca gtcccttgaa actttggacc 700 ttagcagcaa caatatttca gagctccaaa ctgcatttcc agccctacag 750 ctcaaatatc tgtatctcaa cagcaaccga gtcacatcaa tggaacctgg 800 gtattttgac aatttggcca acacactcct tgtgttaaag ctgaacagga 850 accgaatctc agctatccca cccaagatgt ttaaactgcc ccaactgcaa 900 catctcgaat tgaaccgaaa caagattaaa aatgtagatg gactgacatt 950 ccaaggcctt ggtgctctga agtctctgaa aatgcaaaga aatggagtaa 1000 cgaaacttat ggatggagct ttttgggggc tgagcaacat ggaaattttg 1050 cagctggacc ataacaacct aacagagatt accaaaggct ggctttacgg 1100 cttgctgatg ctgcaggaac ttcatctcag ccaaaatgcc atcaacagga 1150 tcagccctga tgcctgggag ttctgccaga agctcagtga gctggaccta 1200 actttcaatc acttatcaag gttagatgat tcaagcttcc ttggcctaag 1250 cttactaaat acactgcaca ttgggaacaa cagagtcagc tacattgctg 1300 attgtgcctt ccgggggctt tccagtttaa agactttgga tctgaagaac 1350 aatgaaattt cctggactat tgaagacatg aatggtgctt tctctgggct 1400 tgacaaactg aggcgactga tactccaagg aaatcggatc cgttctatta 1450 ctaaaaaagc cttcactggt ttggatgcat tggagcatct agacctgagt 1500 acaacgcaat catgtcttta caaggcaatg cattttcaca aatgaagaaa 1550 ctgcaacaat tgcatttaaa tacatcaagc cttttgtgcg attgccagct 1600 aaaatggctc ccacagtggg tggcggaaaa caactttcag agctttgtaa 1650 atgccagttg tgcccatcct cagctgctaa aaggaagaag catttttgct 1700 gttagcccag atggctttgt gtgtgatgat tttcccaaac cccagatcac 1750 ggttcagcca gaaacacagt cggcaataaa aggttccaat ttgagtttca 1800 tctgctcagc tgccagcagc agtgattccc caatgacttt tgcttggaaa 1850 aaagacaatg aactactgca tgatgctgaa atggaaaatt atgcacacct 1900 ccgggcccaa ggtggcgagg tgatggagta taccaccatc cttcggctgc 1950 gcgaggtgga atttgccagt gaggggaaat atcagtgtgt catctccaat 2000 cactttggtt catcctactc tgtcaaagcc aagcttacag taaatatgct 2050 tccctcattc accaagaccc ccatggatct caccatccga gctggggcca 2100 tggcacgctt ggagtgtgct gctgtggggc acccagcccc ccagatagcc 2150 tggcagaagg atgggggcac agacttccca gctgcacggg agagacgcat 2200 gcatgtgatg cccgaggatg acgtgttctt tatcgtggat gtgaagatag 2250 aggacattgg ggtatacagc tgcacagctc agaacagtgc aggaagtatt 2300 tcagcaaatg caactctgac tgtcctagaa acaccatcat ttttgcggcc 2350 actgttggac cgaactgtaa ccaagggaga aacagccgtc ctacagtgca 2400 ttgctggagg aagccctccc cctaaactga actggaccaa agatgatagc 2450 ccattggtgg taaccgagag gcactttttt gcagcaggca atcagcttct 2500 gattattgtg gactcagatg tcagtgatgc tgggaaatac acatgtgaga 2550 tgtctaacac ccttggcact gagagaggaa acgtgcgcct cagtgtgatc 2600 cccactccaa cctgcgactc ccctcagatg acagccccat cgttagacga 2650 tgacggatgg gccactgtgg gtgtcgtgat catagccgtg gtttgctgtg 2700 tggtgggcac gtcactcgtg tgggtggtca tcatatacca cacaaggcgg 2750 aggaatgaag attgcagcat taccaacaca gatgagacca acttgccagc 2800 agatattcct agttatttgt catctcaggg aacgttagct gacaggcagg 2850 atgggtacgt gtcttcagaa agtggaagcc accaccagtt tgtcacatct 2900 tcaggtgctg gatttttctt accacaacat gacagtagtg ggacctgcca 2950 tattgacaat agcagtgaag ctgatgtgga agctgccaca gatctgttcc 3000 ttgtccgttt ttgggatcca caggccctat gtatttgaag ggaaatgtgt 3050 atggctcaga tccttttgaa acatatcata caggttgcag tcctgaccca 3100 agaacagttt taatggacca ctatgagccc agttacataa agaaaaagga 3150 gtgctaccca tgttctcatc cttcagaaga atcctgcgaa cggagcttca 3200 gtaatatatc gtggccttca catgtgagga agctacttaa cactagttac 3250 tctcacaatg aaggacctgg aatgaaaaat ctgtgtctaa acaagtcctc 3300 tttagatttt agtgcaaatc cagagccagc gtcggttgcc tcgagtaatt 3350 ctttcatggg tacctttgga aaagctctca ggagacctca cctagatgcc 3400 tattcaagct ttggacagcc atcagattgt cagccaagag ccttttattt 3450 gaaagctcat tcttccccag acttggactc tgggtcagag gaagatggga 3500 aagaaaggac agattttcag gaagaaaatc acatttgtac ctttaaacag 3550 actttagaaa actacaggac tccaaatttt cagtcttatg acttggacac 3600 atagactgaa tgagaccaaa ggaaaagctt aacatactac ctcaagtgaa 3650 cttttattta aaagagagag aatcttatgt tttttaaatg gagttatgaa 3700 ttttaaaagg ataaaaatgc tttatttata cagatgaacc aaaattacaa 3750 aaagttatga aaatttttat actgggaatg atgctcatat aagaatacct 3800 ttttaaacta ttttttaact ttgttttatg caaaaaagta tcttacgtaa 3850 attaatgata taaatcatga ttattttatg tatttttata atgccagatt 3900 tctttttatg gaaaatgagt tactaaagca ttttaaataa tacctgcctt 3950 gtaccatttt ttaaatagaa gttacttcat tatattttgc acattatatt 4000 taataaaatg tgtcaatttg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4050 a 4051 <210> 14 <211> 1119 <212> PRT
<213> artificial sequence <400> 14 Met Ser Ala Pro Ser Leu Arg Ala Arg Ala Ala Gly Leu Gly Leu Leu Leu Cys Ala Val Leu Gly Arg Ala Gly Arg Ser Asp Ser Gly Gly Arg Gly Glu Leu Gly Gln Pro Ser Gly Val Ala Ala Glu Arg Pro Cys Pro Thr Thr Cys Arg Cys Leu Gly Asp Leu Leu Asp Cys Ser Arg Lys Arg Leu Ala Arg Leu Pro Glu Pro Leu Pro Ser Trp Val Ala Arg Leu Asp Leu Ser His Asn Arg Leu Ser Phe Ile Lys Ala Ser Ser Met Ser His Leu Gln Ser Leu Arg Glu Val Lys Leu Asn Asn Asn Glu Leu Glu Thr Ile Pro Asn Leu Gly Pro Val Ser Ala Asn Ile Thr Leu Leu Ser Leu Ala Gly Asn Arg Ile Val Glu Ile Leu Pro Glu His Leu Lys Glu Phe Gln Ser Leu Glu Thr Leu Asp Leu Ser Ser Asn Asn Ile Ser Glu Leu Gln Thr Ala Phe Pro Ala Leu Gln Leu Lys Tyr Leu Tyr Leu Asn Ser Asn Arg Val Thr Ser Met Glu Pro Gly Tyr Phe Asp Asn Leu Ala Asn Thr Leu Leu Val Leu Lys Leu Asn Arg Asn Arg Ile Ser Ala Ile Pro Pro Lys Met Phe Lys Leu Pro Gln Leu Gln His Leu Glu Leu Asn Arg Asn Lys Ile Lys Asn Val Asp Gly Leu Thr Phe Gln Gly Leu Gly Ala Leu Lys Ser Leu Lys Met Gln Arg Asn Gly Val Thr Lys Leu Met Asp Gly Ala Phe Trp Gly Leu Ser Asn Met Glu Ile Leu Gln Leu Asp His Asn Asn Leu Thr Glu Ile Thr Lys Gly Trp Leu Tyr Gly Leu Leu Met Leu Gln Glu Leu His Leu Ser Gln Asn Ala Ile Asn Arg Ile Ser Pro Asp Ala Trp Glu Phe Cys Gln Lys Leu Ser Glu Leu Asp Leu Thr Phe Asn His Leu Ser Arg Leu Asp Asp Ser Ser Phe Leu Gly Leu Ser Leu Leu Asn Thr Leu His Ile Gly Asn Asn Arg Val Ser Tyr Ile Ala Asp Cys Ala Phe Arg Gly Leu Ser Ser Leu Lys Thr Leu Asp Leu Lys Asn Asn Glu Ile Ser Trp Thr Ile Glu Asp Met Asn Gly Ala Phe Ser Gly Leu Asp Lys Leu Arg Arg Leu Ile Leu Gln Gly Asn Arg Ile Arg Ser Ile Thr Lys Lys Ala Phe Thr Gly Leu Asp Ala Leu Glu His Leu Asp Leu Ser Asp Asn Ala Ile Met Ser Leu Gln Gly Asn Ala Phe Ser Gln Met Lys Lys Leu Gln Gln Leu His Leu Asn Thr Ser Ser Leu Leu Cys Asp Cys Gln Leu Lys Trp Leu Pro Gln Trp Val Ala Glu Asn Asn Phe Gln Ser Phe Val Asn Ala Ser Cys Ala His Pro Gln Leu Leu Lys Gly Arg Ser Ile Phe Ala Val Ser Pro Asp Gly Phe Val Cys Asp Asp Phe Pro Lys Pro Gln Ile Thr Val Gln Pro Glu Thr Gln Ser Ala Ile Lys Gly Ser Asn Leu Ser Phe Ile Cys Ser Ala Ala Ser Ser Ser Asp Ser Pro Met Thr Phe Ala Tzp Lys Lys Asp Asn Glu Leu Leu His Asp Ala Glu Met Glu Asn Tyr Ala His Leu Arg Ala Gln Gly Gly Glu Val Met Glu Tyr Thr Thr Ile Leu Arg Leu Arg Glu Val Glu Phe Ala Ser Glu Gly Lya Tyr Gln Cys Val Ile Ser Asn His Phe Gly Ser Ser Tyr Ser Val Lys Ala Lys Leu Thr Val Asn Met Leu Pro Ser Phe Thr Lys Thr Pro Met Asp Leu Thr Ile Arg Ala Gly Ala Met Ala Arg Leu Glu Cys Ala Ala Val Gly His Pro Ala Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro Ala Ala Arg Glu Arg Arg Met His Val Met Pro Glu Asp Asp Val Phe Phe Ile Val Asp Val Lys Ile Glu Asp Ile Gly Val Tyr Ser Cys Thr Ala Gln Asn Ser Ala Gly Ser Ile Ser Ala Asn Ala Thr Leu Thr Val Leu Glu Thr Pro Ser Phe Leu Arg Pro Leu Leu Asp Arg Thr Val Thr Lys Gly Glu Thr Ala Val Leu Gln Cys Ile Ala Gly Gly Ser Pro Pro Pro Lys Leu Asn Trp Thr Lys Asp Asp Ser Pro Leu Val Val Thr Glu Arg His Phe Phe Ala Ala Gly Asn Gln Leu Leu Ile Ile Val Asp Ser Asp Val Ser Asp Ala Gly Lys Tyr Thr Cys Glu Met Ser Asn Thr Leu Gly Thr Gl.u Arg Gly Asn Val Arg Leu Ser Val Ile Pro Thr Pro Thr Cys Asp Ser Pro Gln Met Thr Ala Pro Ser Leu Asp Asp Asp Gly Trp Ala Thr Val Gly Val Val Ile Ile Ala Val Val Cys Cys Val Val Gly Thr Ser Leu Val Trp Val Val Ile Ile Tyr His Thr Arg Arg Arg Asn Glu Asp Cys Ser Ile Thr Asn Thr Asp Glu Thr Asn Leu Pro Ala Asp Ile Pro Ser Tyr Leu Ser Ser Gln Gly Thr Leu Ala Asp Arg Gln Asp Gly Tyr Val Ser Ser Glu Ser Gly Ser His His Gln Phe Val Thr Ser Ser Gly Ala Gly Phe Phe Leu Pro Gln His Asp Ser Ser Gly Thr Cys His Ile Asp Asn Ser Ser Glu Ala Asp Val Glu Ala Ala Thr Asp Leu Phe Leu Cys Pro Phe Leu Gly Ser Thr Gly Pro Met Tyr Leu Lys Gly Asn Val Tyr Gly Ser Asp Pro Phe Glu Thr Tyr His Thr Gly Cys Ser Pro Asp Pro Arg Thr Val Leu Met Asp His Tyr Glu Pro Ser Tyr Ile Lys Lys Lys Glu Cys Tyr Pro Cys Ser His Pro Ser Glu Glu Ser Cys Glu Arg Ser Phe Ser Asn Ile Ser Tzp Pro Ser His Val Arg Lys Leu Leu Asn Thr Ser Tyr Ser His Asn Glu Gly Pro Gly Met Lys Asn Leu Cys Leu Asn Lys Ser Ser Leu Asp Phe Ser Ala Asn Pro Glu Pro Ala Ser Val Ala Ser Ser Asn Ser Phe Met Gly Thr Phe Gly Lys Ala Leu Arg Arg Pro His Leu Asp Ala Tyr Ser Ser Phe Gly Gln Pro Ser Asp Cys Gln Pro Arg Ala Phe Tyr Leu Lys Ala His Ser Ser Pro Asp Leu Asp Ser Gly Ser Glu Glu Asp Gly Lys Glu Arg Thr Asp Phe Gln Glu Glu Asn His Ile Cys Thr Phe Lys Gln Thr Leu Glu Asn Tyr Arg Thr Pro Asn Phe Gln Ser Tyr Asp Leu Asp Thr <210> 15 <211> 22 <212> DNA

<213> artificial sequence <400> 15 atcgttgtga agttagtgcc cc 22 <210> 16 <211> 23 <212> DNA
<213> artificial sequence <400> 16 acctgcgata tccaacagaa ttg 23 <210> 17 <211> 48 <212> DNA
<213> artificial sequence <400> 17 ggaagaggat acagtcactc tggaagtatt agtggctcca gcagttcc 48 <210> 18 <211> 46 <212> DNA
<213> artificial sequence <400> 18 gggtacacct gctcctgcac cgacggatat tggcttctgg aaggcc 46 <210> 19 <211> 22 <212> DNA
<213> artificial sequence <400> 19 acagattccc accagtgcaa cc 22 <210> 20 <211> 21 <212> DNA
<213> artificial sequence <400> 20 cacactcgtt cacatcttgg c 21 <210> 21 <211> 45 <212> DNA
<213> artificial sequence <400> 21 agggagcacg gacagtgtgc agatgtggac gagtgctcac tagca 45 <210> 22 <211> 21 <212> DNA
<213> artificial sequence <400> 22 agagtgtatc tctggctacg c 21 <210> 23 <211> 22 <212> DNA
<213> artificial sequence <400> 23 taagtccggc acattacagg tc 22 <210> 24 <211> 49 <212> DNA
<213> artificial sequence <400> 24 cccacgatgt atgaatggtg gactttgtgt gactcctggt ttctgcatc 49 <210> 25 <211> 22 <212> DNA
<213> artificial sequence <400> 25 aaagacgcat ctgcgagtgt cc 22 <210> 26 <211> 23 <212> DNA
<213> artificial sequence <400> 26 tgctgatttc acactgctct ccc 23 <210> 27 <211> 24 <212> DNA
<213> artificial sequence <400> 27 tcgcggagct gtgttctgtt tccc 24 <210> 28 <211> 50 <212> DNA
<213> artificial sequence <400> 28 tgatcgcgat ggggacaaag gcgcaagctc gagaggaaac tgttgtgcct 50 <210> 29 <211> 20 <212> DNA
<213> artificial sequence <400> 29 acacctggtt caaagatggg 20 <210> 30 <211> 24 <212> DNA
<213> artificial sequence <400> 30 taggaagagt tgctgaaggc acgg 24 <210> 31 <211> 20 <212> DNA
<213> artificial sequence <400> 31 ttgccttact caggtgctac 20 <210> 32 <211> 20 <212> DNA
<213> artificial sequence <400> 32 actcagcagt ggtaggaaag 20 <210> 33 <211> 22 <212> DNA
<213> artificial sequence <400> 33 gttggatctg ggcaacaata ac 22 <210> 34 <211> 24 <212> DNA
<213> artificial sequence <400> 34 attgttgtgc aggctgagtt taag 24 <210> 35 <211> 45 <212> DNA
<213> artificial sequence <400> 35 ggtggctata catggatagc aattacctgg acacgctgtc ccggg 45 <210> 36 <211> 50 <212> DNA
<213> artificial sequence <400> 36 ggattctaat acgactcact atagggctgc ggcggctcag gtcttcagtt 50 <210> 37 <211> 50 <212> DNA
<213> artificial sequence <400> 37 ctatgaaatt aaccctcact aaagggagca tgggatgggg agggatacgg 50 <210> 38 <211> 48 <212> DNA
<213> artificial sequence <400> 38 ctatgaaatt aaccctcact aaagggaata gcaggccatc ccaggaca 48 <210> 39 <211> 47 <212> DNA
<213> artificial sequence <400> 39 ctatgaaatt aaccctcact aaagggatgt cttccatgcc aaccttc 47

Claims

What is claimed:

1. A composition, comprising a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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 PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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 PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide, antagonist or a fragment thereof and a earner or excipient, 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 in response to an antigen.

4. Use of a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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 in 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 in need thereof, comprising administering to the mammal an effective amount of a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial 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 and fibrotic lung diseases such as 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 eosinophilic 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 antibody is a monoclonal antibody.

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

9. The composition or use of any of the preceding claims, wherein the antibody has nonhuman complementarity determining region (CDR) residues and human framework region (FR) residues.

10. A method for determining the presence of a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide, comprising exposing a cell suspected of containing the PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide to an anti-PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 antibody and determining binding of the antibody to the cell.

11. A method of diagnosing an immune related disease in a mammal, comprising detecting the level of expression of a gene encoding a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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 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 anti-PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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.

13. An immune related disease diagnostic kit, comprising an anti-PRO245, PRO217, PRO3O1, PRO266, PRO335, PRO331 or PRO326 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 PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide.

15. 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 enhancing 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 of a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide.

16. The article of manufacture of claim 21 wherein said active agent is an anti-PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 antibody.

17. A method for identifying a compound capable of inhibiting the expression or activity of a PRO245 polypeptide, comprising contacting a candidate compound with a PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 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 PRO245, PRO217, PRO301, PRO266, PRO335, PRO331 or PRO326 polypeptide is immobilized on a solid support.

19. The method of claim 18, wherein the non-immobilized component carries a detectable label.