Secreted and Transmembrane polypeptides and Nucleic Acids Encoding the Same

专利摘要:
The present invention relates to novel polypeptides and nucleic acid molecules encoding the polypeptides. Further, herein, a vector comprising a nucleic acid sequence and a host cell, a chimeric polypeptide molecule comprising a polypeptide of the invention fused with a heterologous polypeptide sequence, an antibody binding to a polypeptide of the invention, and a method of producing a polypeptide of the invention Is provided.
公开号:KR20040053176A
申请号:KR10-2004-7005574
申请日:2000-03-01
公开日:2004-06-23
发明作者:루크 데스노이어스；댄 엘. 이튼；오드리 고다드；폴 제이. 고다우스키；오스틴 엘. 거니；제임스 팬；티모시 에이. 스튜어트；콜린 케이. 와따나베；윌리엄 아이. 우드；제민 장
申请人:제넨테크, 인크.；
IPC主号:

专利说明:

Secreted and transmembrane polypeptides and nucleic acids encoding them {Secreted and Transmembrane polypeptides and Nucleic Acids Encoding the Same}
[47] The present invention relates to methods of identifying and isolating novel DNA and recombinant production of novel polypeptides.
[48] Background
[49] Extracellular proteins play an important role, among other things, in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation or interaction with other cells, is typically dictated by information received from other cells and / or adjacent environments. Often, this information is delivered by secretory polypeptides (eg, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides and hormones) to be accepted and translated by various cellular receptors or membrane binding proteins. These secretory polypeptides or signaling molecules generally reach their site of action in the extracellular environment via the secretory pathway of the cell.
[50] Secretory proteins have a variety of industrial uses, including pharmaceuticals, diagnostics, biosensors, and bioreactors. Most protein drugs currently available, such as thrombolytics, interferons, interleukins, erythropoietins, colony stimulating factors and various other cytokines, are secreted proteins. In addition, membrane proteins that are their receptors also have potential as therapeutic or diagnostic agents. Efforts to find new natural secreted proteins are being made by both industry and academia. Much effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. Examples of screening methods and techniques are described in, eg, Klein et al ., Proc. Natl. Acad. Sci. , 93: 7108-7113 (1996) and US Pat. No. 5,536,637.
[51] Membrane binding proteins and receptors may play an important role, among other things, in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, eg, proliferation, migration, differentiation or interaction with other cells, is typically dependent on information received from other cells and / or adjacent environments. Often, this information is delivered by secretory polypeptides (eg, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides and hormones) to be accepted and translated by various cellular receptors or membrane binding proteins. Such membrane-binding proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatase, receptors involved in cell-cell interactions, and cellular adhesin molecules such as selectin and integrin. For example, the transmission of signals that regulate cell growth and differentiation is in part regulated by phosphorylation of several cellular proteins. Protein tyrosine kinases, enzymes that accelerate this process, can also act as growth factor receptors. Examples include fibroblast growth factor receptors and nerve growth factor receptors.
[52] Membrane binding proteins and receptor molecules have a variety of industrial uses, including pharmaceuticals and diagnostics. For example, receptor immunoadhesin can be used as a therapeutic agent to block receptor-ligand interactions. Membrane binding proteins can also be used to screen for potential peptide or small molecule inhibitors for related receptor / ligand interactions.
[53] Efforts to find new natural receptors or membrane-bound proteins have been made by both industry and academia. Much effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for new receptors or membrane binding proteins.
[54] 1.PRO1484
[55] After the differentiation of fat, the morphology of the cell changes, a lot of lipids accumulate in the cell, and a specific gene expression program is activated (Liang et al., J. Biol. Chem. 271: 10697-10703 (1996)). Complement-related proteins of fat are highly induced during adipocyte differentiation and have significant homology with the subunit of complement factor C1q, collagen alpha 1 (x) and the brain-specific factor cerebellin. Covalent protein (Scherer et al., J. Biol. Chem. 270: 26746-26749 (1995)). The function of complement-related proteins in adipocytes is currently unknown, but their tissue-specific expression suggests that these proteins act as new signaling molecules in adipose tissue. As such, the identification and characterization of novel polypeptides homologous to the complement-related proteins of adipocytes is of considerable interest. Here we describe the identification and characterization of a novel polypeptide (designated herein as PRO1484 polypeptide) having homology with the complement-related proteins of adipocytes.
[56] 2.PRO4334
[57] PC-1, the glycoprotein of plasma cell membranes, is of interest. Cloning of PC-1 is described in the technical literature by Buckley, et al., J. Biol. Chem., 265 (29): 17506-11 (1990) and WO9519570-A. WO9519570-A describes PC-1, a human insulin receptor tyrosine kinase inhibitor. PC-1 has been reported to be useful in the diagnosis and treatment of diseases involving expression of inappropriate insulin receptor tyrosine kinase inhibitors, such as insulin-independent diabetes. Thus, proteins having homology with PC-1 are of interest.
[58] 3.PRO1122
[59] Cytokine interleukin 17 (IL-17) has been reported to stimulate epithelial, endothelial and fibroblasts to secrete prostaglandin E2 as well as cytokine and granulocyte-colony-stimulating factors such as IL-6 and IL-8. . It is also known that when cultured in the presence of IL-17, fibroblasts can continue to increase CD34 + and mature mainly into neutrophils. This suggests that IL-17 is one of the early initiators of T cell-dependent inflammatory responses and / or a member of the cytokine network that is hematopoietic in the immune system. See, Yao, et al., J Immunol., 155 (12): 5483-5486 (1995); Fossiez, et al., J. Exp. Med., 183 (6): 2593-2603 (1996); Kennedy, et al., J. Interferon Cytokine Res., 16 (8): 611-617 (1996). Thus, proteins related to IL-17, including CTLA-8 (misleadingly known as IL-17), are of interest.
[60] 4.PRO1889
[61] E48 antigenic proteins are cysteine-rich GPI-anchored membrane proteins belonging to the human protein group LY-6 (see, eg, WO 96/35808). The E48 antigen acts as a marker for squamous cells, exhibits the biological activity of cell-cell / cell-epilepsy adhesions, and is targeted for antibody-based immunotherapy. The amino acid sequence of the E48 antigen protein was already estimated from cDNA clones obtained from squamous cell carcinoma of the head and neck. As such, the E48 antigen serves as a potential target in the treatment of squamous cell carcinoma.
[62] We herein describe the identification and characterization of a novel polypeptide (designated herein as a PRO1889 polypeptide) having homology with the E48 antigen protein.
[63] 5. PRO1890
[64] Recognition of carbohydrates by lectins is known to play an important role in many physiological aspects of eukaryotes. Although there are many lectin families of several animals and plants, recently discovered calcium dependence or type C lectins are of greatest interest. For example, the recognition of carbohydrate moieties on endothelial cells or leukocytes by the selectin family of calcium dependent lectins has been found to be very important for the migration of leukocytes to sites of inflammation [Lasky, L., Ann. Rev. Biochem., 64 113-139 (1995)]. Biophysical analysis of this adherent interaction suggests that lectin-carbohydrate bonds formed in such cases may allow leukocytes to attach to the endothelium in the vasculature under high shear conditions. As such, the rate at which lectins recognize carbohydrates is so fast that they can quickly obtain the ligands needed in the bloodstream under high shear conditions. In this case, the physiological use of type C lectins is also supported by this relatively low affinity interaction, which is a requirement for the circulatory phenomenon of leukocytes observed at the site of acute inflammation. Mannose binding protein (Weis et al., Science 254, 1608-1615 [1991]; Weis et al., Nature 360127-134 [1992]) and E-selectin (Graves et al., Nature 367 (6463), 532- 538 [1994] with several crystallization assays (Erbe et al., J. Cell. Biol. 119 (1), 215-227 [1992]; Drickamer, Nature 360, 183-186 [1992]; Iobst et al., J. Biol. Chem. 169 (22), 15505-15511 [1994]; Kogan et al., J. Biol. Chem. 270 (23), 14047-14055 [1995]. In general, it is consistent with the assumption that Type C lectins are involved in the rapid recognition of dense carbohydrates. These data also suggest that type C lectins can perform many important physiological phenomena by recognizing carbohydrates with fast and relatively low affinity.
[65] Since lectin proteins are clearly important in many physiological reactions, efforts are currently being made to find new lectin proteins or proteins having sequence homology with lectin proteins. We herein describe the identification and characterization of novel polypeptides, herein designated PRO1890 polypeptides, which are homologous to the lectin proteins.
[66] 6.PRO1887
[67] Enzyme proteins play an important role in chemical reactions involving food digestion, biosynthesis of macromolecules, controlled release and utilization of chemical energy, and other reactions necessary to sustain life. Enzymes are also known to play an important role in combating various diseases and disorders. For example, hepatic carboxyesterases have been reported to help human tumor cells susceptibility to cancer prodrugs. Dans et al. Reported that, upon stable expression of cDNA encoding carboxyesterase in Rh30 human rhabdomyosarcoma cells, the cell's susceptibility to CPT-11 cancer prodrugs increased 8.1-fold [Cancer. Res. (1998) 58 (1): 20-22. The authors can use this prodrug / enzyme combination for treatment in a manner similar to the current approach of investigating the combination of ganciclovir / herpes simplex virus thymidine kinase and 5-fluorocytosine / cytosine deaminase. It is proposed. Van Pelt et al. Inhibited the infiltration of Plasmodium falciparum malaria sporozoite into human early hepatocytes by 55 kD sized human liver carboxylase in culture. J Hepatol (1997) 27 (4): 688-698].
[68] Carboxysterases have also been found to be important in the detoxification of drugs, pesticides and other xenobiotics. Carboxysterases of purified human liver are known to be involved in the metabolism of various drugs, including cocaine and heroin. Prindel et al. Promote the hydrolysis of cocaine and heroin and for the purification and cloning of carboxyl esterases between humans of a broad range of substrate specificities that may play an important role in the degradation of these drugs in human tissues. [J. Biol. Chem. (1997) 6: 272 (23): 14769-14775. Brzenzinski et al. Describe a competitive inhibition assay by spectrophotometry used to identify esters of drugs or the environment that are metabolized by carboxyl esterases [Drug Metab Dispos (1997) 25 (9). ): 1089-1096].
[69] Further background on carboxyesterases [Kroetz et al. Biochemistry, (1993) 32 (43): 11606-17, reports on cDNA cloning and characterization of carboxyesterases in human liver. Aida et al. (Biochim Biophys Acta (1993) 1174 (1): 72-4) reports cDNA cloning and characterization of significant carboxyesterases in males among carboxyesterases in mouse liver.
[70] In light of the important physiological role played by carboxyl esterases, both industry and academia are working to find new natural carboxyl ester analogs. We herein describe the identification and characterization of novel polypeptides having homology with carboxyesterases.
[71] 7.PRO1785
[72] Antioxidant enzymes are thought to play a critical role in survival while the parasite Schistosoma mansoni moves through the tissues of a given host. Recently, one of these enzymes, glutathione peroxidase, has been cloned (Roche, et al., Gene, 138: 149-152 (1994), accession number GSHC_SCHMA). Glutathione peroxidase is further described in FR2689906-A. Thus, glutathione peroxidase and nucleic acid encoding the same are useful as diagnostic agents and vaccines, and also in assays to find modulators of antioxidant enzymes.
[73] 8.PRO4353
[74] Semaphorin is a member of the protein cohort that is involved in the induction of axons during development. Semaphorin Y can be used to inhibit peripheral nerve growth. Semaphorin Z is useful as an inhibitor for central nervous system dilation. Semaphorin Z inhibitors can be used as enhancers of central nerve regeneration. Therefore, semaphorin and semaphorin modulators are of great interest [Kikuchi, et al., Brain Res Mol Brain Res., 51 (1-2): 229-37 (1997); Shoji, et al., Development, 125 (7): 1275-83 (1998).
[75] 9. PRO4357
[76] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4357.
[77] 10.PRO4405
[78] Both industry and academia are working to find new natural transmembrane receptor proteins. Much effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel transmembrane receptor proteins. We herein describe the identification and characterization of novel transmembrane polypeptides, designated herein as PRO4405.
[79] 11.PRO4356
[80] Glycosylphosphatidylinositol (GPI) anchored proteoglycans are generally located on the surface of cells and are therefore known to be involved in regulating the response of cells to many growth factors, cell adhesion molecules and extracellular interstitial components. GPI-anchored proteins associated with metastasis (MAGPIAP) are one of the cell surface proteins that appear to be involved in metastasis. Metastasis is a form of cancer in which transformed or malignant cells migrate to expand cancer from one site to another. Thus, identifying MAGPIAP and polypeptides involved in metastasis is of interest.
[81] 12.PRO4352
[82] Cadherin is a large family of transmembrane proteins. Cadherin actually constitutes a calcium-dependent glycoprotein family that functions to regulate cell-cell adhesion in all solid tissues of multicellular organisms. At least Cadherin 1-13 and Types B, E, EP, M, N, P and R have been identified and characterized. Among the functions of Cadherin, with a few exceptions, Cadherin is known to participate in cell aggregation and bind to cell-cell attachment sites. Recently, all Cadherin share several repeats of Cadherin specific motifs that are thought to correspond to the folding of the extracellular domain, but members of the Cadherin giant family have been shown to have different structures and, in some cases, different functions. Reported. In particular, members of the Caherin giant have been reported to be involved in signal transduction (see Suzuki, J. Cell Biochem., 61 (4): 531-542 (1996)). For cadrine, Tanihara et al., J. Cell Sci., 107 (6): 1697-1704 (1994), Aberle et al., J. Cell Biochem., 61 (4): 514-523 (1996 and Tanihara et al., Cell Adhes. Commun., 2 (1): 15-26 (1994).
[83] Protocadherin is a member of the Kadherin giant that is highly expressed in the brain. In some studies, protocadherin exhibited cell adhesion activity. See Sano, et al., EMBO J., 12 (6): 2249-2256 (1993). However, research has also shown that some protocadherins, such as protocadherin 3 (also known as Pcdh3 or pc3), do not exhibit strong calcium dependent cell aggregation activity. For this study and additional features of Pcdh3, see Sago, et al., Genomics, 29 (3): 631-640 (1995). Thus, molecules associated with pc3 are of great interest. Also of great interest are the subtypes of dessomsomal cadrine described in Koch, et al., Differentiation, 47 (1): 29-36 (1991).
[84] Of particular interest are proteins having sequences homologous to the proteins described in Amagai, et al., Cell, 67 (5): 869-77 (1991). The study describes antibodies to a novel epithelial cadrine in the cell adhesion disease pemphigus vulgaris. In addition, the battlefield card herrin is of interest. Also of interest are novel cadherins, proteins that have homology with fat tumor suppressor genes.
[85] 13.PRO4380
[86] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4380 polypeptides.
[87] 14.PRO4354
[88] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4354 polypeptides.
[89] 15.PRO4408
[90] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4408 polypeptides.
[91] 16.PRO5737
[92] Interleukin-1 is two proteins that play important roles early in the inflammatory response (IL-1α and IL-1β) (for review see Dinarello, Blood, 87: 2095-2147 (1996) and references). do]. These two proteins are made into precursor proteins in the cell and then cleaved when secreted to produce biologically active mature carboxy-terminal 17 kDa sized fragments. In the case of IL-1β, this cleavage involves intracellular cysteine protease known as ICE, which is required to release the active fragment from the inactive precursor. The precursor of IL-1α is active.
[93] These two proteins bind to cell surface receptors present in virtually all types of cells and act to initiate a variety of reactions, either alone or in combination with other secretory factors. These proteins may include proliferation (eg, fibroblasts, T cells), apoptosis (eg, A375 melanoma cells), induction of cytokines (eg, induction of TNF, IL-1, IL-8), Activation of receptors (eg E-selectin), production of eicosanoids (eg PGE2) and secretion of degradation enzymes (eg collagenase). To achieve this effect, IL-1 activates transcription factors such as NF-KB and AP-1. Several of the actions of IL-1 on target cells are thought to be mediated through the activation of kinase cascades associated with cell stress, such as the activation of stress-activated MAP kinases JNK / SAPK and p38.
[94] A third member of the IL-1 family was discovered later, acting as a natural antagonist of IL-1α and IL-1β by binding to the IL-1 receptor but not introducing intracellular signals or biological responses. This protein is called IL-1Ra (for IL-1 receptor antagonist) or IRAP (for IL-1 receptor antagonist protein). IL-1Ra exists in three or more selectively spliced forms, one of which encodes a secreted protein and the other two encode intracellular proteins. IL-1α, IL-1β, and IL-1Ra exhibit approximately 25-30% sequence identity with each other, and a similar 3 consisting of 12 β-strands folded into β-barrels with structural motifs repeated three times therein. Share the dimensional structure.
[95] There are three known IL-1 receptor subunits. This active receptor conjugate consists of a type I receptor and an IL-1 accessory protein (IL-1RAcP). Type I receptors act to bind IL-1α, IL-1β and IL-1Ra ligands, and can bind even in the absence of IL-1RAcP. However, signal transduction requires the interaction of IL-1α or IL-1β with IL-1RAcP. Since IL-1Ra does not interact with IL-1RAcP, it cannot induce signal transduction. Type II receptors, the third receptor subunit, bind IL-1α and IL-1β but are unable to transmit signals due to the lack of intracellular domains. Instead, type II receptors act as inducers in their membrane-bound form or as IL-1 antagonists in the processed secretory form to inhibit the activity of IL-1. Type II receptors bind weakly with IL-1Ra.
[96] Numerous studies using IL-1Ra, soluble IL-1R derived from the extracellular domain of the type I IL-1 receptor, antibodies against IL-1α or IL-1β, and transgenic knockout mice regarding their genes, IL -1 has been found to work in many pathophysiology [see Dinarello, Blood, 87: 2095-2147 (1996) for review). For example, IL-1Ra is known to be effective in animal models of sepsis shock, rheumatoid arthritis, graft-versus-host disease (GVHD), stroke, cardiac ischemia, psoriasis, inflammatory bowel disease and asthma. IL-1Ra has also been shown to be effective in clinical trials for rheumatoid arthritis and GVHD and in clinical trials for inflammatory bowel disease, asthma and psoriasis.
[97] More recently, interleukin-18 (IL-18) has been found to be included in the IL-1 family (for review see Dinarello et al, J. Leukocyte Biol., 63: 658-664 (1998)). do]. IL-18 shares the β-pleated, barrel-like form of IL-1α and IL-1β. In addition, IL-18 is a natural ligand for members of the family of IL-1 receptors, conventionally known as IL-1R-related protein (IL-1Rrp) (now known as IL-18 receptor (IL-18R)). IL-18 is known to initiate an inflammatory cytokine cascade in a mixed group of peripheral blood mononuclear cells (PBMCs) by inducing TNF production in activated cells by initiating constitutive IL-18 receptors on lymphocytes and NK cells. That is, TNF stimulates production of IL-1 and IL-8 in CD14 + cells. IL-18 induces both TNF, IL-1, and CC and CXC chemokines and induces Fas ligand as well as in-nuclear localization of nuclear factor 6B (NF-6B) It is classified as another proinflammatory cytokine that may contribute to inflammation.
[98] 17.PRO4425
[99] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4425 polypeptides.
[100] 18.PRO5990
[101] Secretogranin proteins (eg, secretoranin I and II) are present in secretory granules of various endocrine and neurons [Schimmel, A. et al., FEBS Lett. 314 (3): 375-80 (1992); Gerdes, H. H. et al., J. Biol. Chem. 264 (20): 12009-15]. Secretographin proteins may act to surround secretion products, including regulatory peptides [Chanat, E. et al., FEBS Lett. 351 (2): 225-30 (1994); Rosa, P. et al., J. Cell. Biol. 101 (5): 1999-2011 (1985); Gorr, S. U. et al., Am. J. Physiol. 257 (2): E247-54 (1989). Secretogranin has been successfully used as a biological marker in many cases. For example, secretogranin II is important as an immunohistochemical marker for endocrine neoplasia [Fischer-Colbrie, R. et al., J. Biol. Chem. 265 (16): 9208-13 (1990). Eder et al. Found that the ratio of secretoranin II to chromogranin was very constant in the patient population, which stabilized the CSF levels of other peptides such as neuropeptides. Suggests that it can be used as a parameter (Eder, U., et al., J. Neural Transm., 105 (1): 39-51 (1998)).
[102] We herein describe the identification and characterization of novel polypeptides (designated herein as PRO5990 polypeptides) having sequence similarity with secretoranin.
[103] 19.PRO6030
[104] Both industry and academia are working to find new natural transmembrane receptor proteins. Much effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel transmembrane receptor proteins. We herein describe the identification and characterization of novel transmembrane polypeptides, designated herein as PRO6030 polypeptides.
[105] 20.PRO4424
[106] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4424 polypeptides.
[107] 21.PRO4422
[108] Lysozyme is a protein that is widely distributed in various tissues and secretions of humans, including milk, tears, and saliva. It has been demonstrated that lysozyme hydrolyzes the bond between N-acetylglucosamine. Lysozyme is an inhibitor of chemotactic and toxic oxygen free radical production and has been shown to play several roles in the calcification reaction. As such, the discovery of novel polypeptides homologous to lysozyme is of practical interest [Nakano and Graf, Biochim. Biophys Acta, 1090 (2): 273-6 (1991)].
[109] 22.PRO4430
[110] Both industry and academia are working to find new natural secreted proteins. Much of this effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel secreted proteins. We herein describe the identification and characterization of novel secreted polypeptides, designated herein as PRO4430 polypeptides.
[111] 23.PRO4499
[112] Both industry and academia are working to find new natural transmembrane receptor proteins. Much effort is focused on screening mammalian recombinant DNA libraries to find coding sequences for novel transmembrane receptor proteins. We herein describe the identification and characterization of novel transmembrane polypeptides, designated herein as PRO4499 polypeptides.
[113] <Overview of invention>
[114] 1.PRO1484
[115] We have found a cDNA clone (DNA44686-1653) that is homologous to a nucleic acid encoding the complement-related protein of an adipocyte and encodes a novel polypeptide (designated herein as "PRO1484").
[116] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1484 polypeptide.
[117] In one aspect, the isolated nucleic acid comprises (a) a DNA molecule encoding a PRO1484 polypeptide having a sequence of approximately amino acid residues 1 or 23 in Figure 2 (SEQ ID NO: 2) or 246, or (b) the DNA molecule of (a) above. Complementary DNA with at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% DNA.
[118] In another aspect, the present invention relates to an isolated nucleic acid molecule encoding a PRO1484 polypeptide comprising DNA hybridizing with approximately nucleotide 77 of Figure 1 (SEQ ID NO: 1) or the complement of a nucleic acid between 143 and 814. Preferably, hybridization occurs under stringent hybridization and washing conditions.
[119] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203581 (DNA44686-1653) or (b) a nucleic acid molecule of (a). An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203581 (DNA44686-1653).
[120] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85% and more preferably at least about 90% of the amino acid residue 1 or the sequence of about 23 to about 246 of FIG. 2 (SEQ ID NO: 2) Above, most preferably, it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having sequence identity of at least about 95% or (b) the complement of the DNA of (a).
[121] In another aspect, the invention provides a DNA that encodes a PRO1484 polypeptide having at least 600 nucleotides and, under stringent conditions, a test DNA molecule having (a) amino acid residue 1 of FIG. 2 (SEQ ID NO: 2) or a sequence from about 23 to about 246. Molecule or (b) hybridize with the complement of the DNA molecule of (a) and the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably about (a) or (b) If it has at least 90%, most preferably at least about 95% sequence identity, it relates to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[122] In certain aspects, the invention provides an isolated nucleic acid molecule comprising or complementary to DNA encoding a PRO1484 polypeptide with or without an N-terminal signal sequence and / or starting methionine. By experiment, the signal peptide was found to span about 22 from amino acid position about 1 in the sequence of FIG. 2 (SEQ ID NO: 2).
[123] In another aspect, the invention provides (a) at least about 80%, preferably at least about 85%, more preferably about 90 when compared to residue 1 of FIG. 2 (SEQ ID NO: 2) or the amino acid sequence of about 23 to about 246 At least%, most preferably at least about 95%, of DNA encoding a polypeptide that is recorded as positive or (b) an isolated nucleic acid molecule comprising the complement of the DNA of (a).
[124] Another embodiment relates to fragments of the PRO1484 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length, It can be derived from the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1).
[125] In another embodiment, the present invention provides an isolated PRO1484 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[126] In certain aspects, the invention provides an isolated native sequence PRO1484 polypeptide which, in some embodiments, comprises the amino acid sequence comprising residue 1 or about 23 to about 246 of FIG. 2 (SEQ ID NO: 2).
[127] In another aspect, the invention provides an amino acid residue 1 of FIG. 2 (SEQ ID NO: 2) or at least about 80%, preferably at least about 85%, more preferably at least about 90%, Preferably it relates to an isolated PRO1484 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[128] In another aspect, the invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, as compared to residue 1 of Figure 2 (SEQ ID NO: 2) or the amino acid sequence of about 23 to about 246, Most preferably, it is directed to an isolated PRO1484 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[129] In another aspect, the invention provides an isolated PRO1484 polypeptide comprising the amino acid residue 1 of Figure 2 (SEQ ID NO: 2) or a sequence from about 23 to about 246, or a fragment thereof sufficient to provide a binding site for an anti-PRO1484 antibody It is about. Preferably, the PRO1484 fragment retains the qualitative biological activity of the native PRO1484 polypeptide.
[130] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO1484 polypeptide having a sequence of about 1 or about 23 to about 246 amino acid residues in FIG. 2 (SEQ ID NO: 3) or (b) hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, if at least 90%, most preferably at least about 95%, of sequence identity, and (iii) the polypeptide from the cell culture It provides a polypeptide produced by the step of recovering.
[131] 2.PRO4334
[132] We have found a cDNA clone (DNA59608-2577) that encodes a novel polypeptide (named “PRO4334” herein) having homology with PC-1.
[133] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4334 polypeptide.
[134] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding the amino acid residue 1 of Figure 4 (SEQ ID NO: 9) or a PRO4334 polypeptide having a sequence from about 23 to about 440, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[135] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4334 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 150 and about 1403 of FIG. 3 (SEQ ID NO: 8). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[136] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203870 (DNA59608-2577) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203870 (DNA59608-2577).
[137] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the amino acid residues of Figure 4 (SEQ ID NO: 9) with the sequence of about 23 to about 440; Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[138] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 23 to about 440 of Figure 4 (SEQ ID NO: 9). A DNA molecule encoding a PRO4334 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[139] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 23 to about 440 of FIG. 4 (SEQ ID NO: 9), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[140] Another embodiment relates to fragments of the PRO4334 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[141] In another embodiment, the present invention provides an isolated PRO4334 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[142] In certain aspects, the invention provides an isolated native sequence PRO4334 polypeptide, which in one embodiment comprises an amino acid sequence comprising residues 23-440 of FIG. 4 (SEQ ID NO: 9).
[143] In another aspect, the invention relates to the sequence of amino acid residues 23 to about 440 of Figure 4 (SEQ ID NO: 9) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4334 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[144] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 23 to about 440 of Figure 4 (SEQ ID NO: 9). Relates to an isolated PRO4334 polypeptide comprising an amino acid sequence wherein at least about 95% are recorded as positive.
[145] In another aspect, the invention relates to an isolated PRO4334 polypeptide comprising the sequence of amino acid residues 23 to about 440 of FIG. 4 (SEQ ID NO: 9), or a fragment thereof sufficient to provide a binding site for an anti-PRO4334 antibody. . Preferably, the PRO4334 fragment retains the qualitative biological activity of the native PRO4334 polypeptide.
[146] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4334 polypeptide having a sequence from about 23 to about 440 amino acid residues in FIG. 4 (SEQ ID NO: 9) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[147] 3.PRO1122
[148] We have found cDNA clones (DNA62377-1381) that have sequence identity with CTLA-8 encoding novel polypeptides (named "PRO1122" herein).
[149] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1122 polypeptide.
[150] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding the amino acid residue 1 of Figure 6 (SEQ ID NO: 11) or a PRO1122 polypeptide having a sequence from about 19 to about 197, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[151] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO1122 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 104 and about 640 of FIG. 5 (SEQ ID NO: 10). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[152] In another aspect, the invention provides a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203552 (DNA62377-1381) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203552 (DNA62377-1381).
[153] In another aspect, the present invention provides a composition comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, amino acid residues of the sequences of about 19 to about 197 of the amino acid residues of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[154] In another aspect, the invention provides, under stringent conditions, a test DNA molecule comprising (a) a DNA molecule encoding a PRO1122 polypeptide having a sequence from about 19 to about 197 amino acid residues in FIG. 6 (SEQ ID NO: 11), or (b) above (a) At least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 80% with the complement of the DNA molecule of (a) or (b). If it has 95% or more of sequence identity, it relates to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[155] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 19 to about 197 of FIG. 6 (SEQ ID NO: 11), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[156] In another embodiment, the present invention provides an isolated PRO1122 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[157] In certain aspects, the invention provides an isolated native sequence PRO1122 polypeptide, which in one embodiment comprises an amino acid sequence comprising residues 19-197 of FIG. 6 (SEQ ID NO: 11).
[158] In another aspect, the invention relates to the sequence of amino acid residues 19 to about 197 of Figure 6 (SEQ ID NO: 11) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO1122 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[159] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 19 to 197 of FIG. 6 (SEQ ID NO: 11). An isolated PRO1122 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[160] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under stringent conditions (a) a PRO1122 polypeptide having a sequence from about 19 to about 197 amino acid residues in FIG. 6 (SEQ ID NO: 11), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[161] 4.PRO1889
[162] We have found a cDNA clone (DNA77623-2524) that encodes a novel polypeptide (designated herein as "PRO1889") that is homologous to the nucleic acid encoding the E48 antigen protein.
[163] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1889 polypeptide.
[164] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1889 polypeptide having a sequence of about 1 or about 21 to about 97 amino acid residues in FIG. 8 (SEQ ID NO: 16) or (b) the DNA of (a) A DNA having at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with the complement of the molecule.
[165] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO1889 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between about 39 or about 99 and about 329 of the nucleotide of FIG. 7 (SEQ ID NO: 15). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[166] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203546 (DNA77623-2524) or (b) a nucleic acid molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203546 (DNA77623-2524).
[167] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90% of the amino acid residue 1 of Figure 8 (SEQ ID NO: 16) or the sequence of about 21 to about 97 Above, most preferably, it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having sequence identity of at least about 95% or (b) the complement of the DNA of (a).
[168] In another aspect, the present invention provides a DNA that encodes a PRO1889 polypeptide having at least 315 nucleotides and, under stringent conditions, a test DNA molecule (a) having amino acid residue 1 of FIG. 8 (SEQ ID NO: 16) or a sequence from about 21 to about 97. Molecule or (b) hybridize with the complement of the DNA molecule of (a) and the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably about (a) or (b) If it has at least 90%, most preferably at least about 95% sequence identity, it relates to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[169] In certain aspects, the invention provides an isolated nucleic acid molecule comprising or complementary to DNA encoding a PRO1889 polypeptide with or without an N-terminal signal sequence and / or starting methionine. By experiment, the signal peptide was found to span about 20 from amino acid position about 1 in the sequence of FIG. 8 (SEQ ID NO: 16).
[170] In another aspect, the invention provides (a) at least about 80%, preferably at least about 85%, more preferably about 90 when compared to residue 1 of Figure 8 (SEQ ID NO: 16) or the amino acid sequence of about 21 to about 97 At least%, most preferably at least about 95%, of DNA encoding a polypeptide that is recorded as positive or (b) an isolated nucleic acid molecule comprising the complement of the DNA of (a).
[171] Another embodiment relates to fragments of the PRO1889 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length, Can be derived from the nucleotide sequence shown in FIG. 7 (SEQ ID NO: 15).
[172] In another embodiment, the present invention provides an isolated PRO1889 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[173] In certain aspects, the invention provides an isolated native sequence PRO1889 polypeptide, which in some embodiments, comprises the amino acid sequence comprising residue 1 or about 21 to about 97 of FIG. 8 (SEQ ID NO: 16).
[174] In another aspect, the invention provides an amino acid residue 1 of Figure 8 (SEQ ID NO: 16) or at least about 80%, preferably at least about 85%, more preferably at least about 90%, Preferably it relates to an isolated PRO1889 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[175] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most compared to residue 1 of Figure 8 (SEQ ID NO: 16) or the amino acid sequence of about 21 to 97 Preferably, it relates to an isolated PRO1889 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[176] In another aspect, the invention provides an isolated PRO1889 polypeptide comprising the amino acid residue 1 of Figure 8 (SEQ ID NO: 16) or a sequence from about 21 to about 97, or a fragment thereof sufficient to provide a binding site for an anti-PRO1889 antibody. It is about. Preferably, the PRO1889 fragment retains the qualitative biological activity of the native PRO1889 polypeptide.
[177] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO1889 polypeptide having a sequence of about 1 or about 21 to about 97 amino acid residues in FIG. 8 (SEQ ID NO: 16), or (b) hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, if at least 90%, most preferably at least about 95%, of sequence identity, and (iii) the polypeptide from the cell culture It provides a polypeptide produced by the step of recovering.
[178] In another embodiment, the invention confirms the presence of a PRO1889 polypeptide, comprising exposing a cell suspected of containing a PRO1889 polypeptide to an anti-PRO1889 antibody and confirming that the antibody binds to the cell. It is about how to.
[179] In another embodiment, the invention relates to detecting the expression level of a gene encoding a PRO1889 polypeptide in (a) a test sample of tissue cells obtained from a mammal and (b) a control sample of known normal tissue cells of the same cell type. A method of diagnosing the presence of cancer cells in a mammal wherein the expression of higher levels in the test sample indicates the presence of cancer cells, particularly cancerous squamous cells, in the mammal.
[180] Another embodiment of the present invention is directed to a method for identifying a compound capable of inhibiting the expression and / or activity of a PRO1889 polypeptide by contacting the candidate compound with the PRO1889 polypeptide for a period of time sufficient to allow the candidate compound and the PRO1889 polypeptide to interact. It is about. In certain aspects, the candidate compound or PRO1889 polypeptide is immobilized on a solid phase support. In another aspect, the non-fixed component comprises a detectable label.
[181] In another embodiment, the invention relates to detecting the expression level of a gene encoding a PRO1889 polypeptide in (a) a test sample of tissue cells obtained from a mammal and (b) a control sample of known normal tissue cells of the same cell type. Wherein the expression is at a higher level in the test sample, the method provides a method of diagnosing a tumor in a mammal in which the tumor is present in the mammal from which the test tissue cell was obtained.
[182] In another embodiment, the invention provides a method of contacting an anti-PRO1889 antibody with a test sample of tissue cells obtained from a mammal and (b) detecting the formation of the conjugate of the anti-PRO1889 and PRO1889 polypeptides in the test sample. It provides a method for diagnosing a tumor in a mammal. Detection can be qualitative or quantitative and can be performed compared to monitoring the formation of conjugates in a control sample of known normal tissue cells of the same cell type. The large amount of conjugate formed in the test sample indicates the presence of tumors in the mammal from which the test tissue cells were obtained. The antibody preferably comprises a detectable label. Formation of the conjugates can be monitored, for example, by optical microscopy, flow cytometry, fluorometry, or other techniques known in the art. Preferably, the test sample is obtained from a mammalian subject suspected of growing or proliferating tumor cells (eg, cancer cells).
[183] In another embodiment, the present invention provides a cancer diagnostic kit comprising an anti-PRO1889 antibody and a carrier (eg, a buffer) in a suitable container. This kit preferably contains instructions for using an antibody to detect the PRO1889 polypeptide.
[184] In another embodiment, the present invention provides a method of inhibiting growth of tumor cells comprising exposing cells overexpressing PRO1889 polypeptide to an effective amount of a substance that inhibits expression and / or activity of the PRO1889 polypeptide. do. This material is preferably an anti-PRO1889 polypeptide, small organic and inorganic peptides, phosphopeptides, antisense or ribozyme molecules, or triple helical molecules. In certain aspects, this substance, such as an anti-PRO1889 antibody, induces cell death. In another aspect, the tumor cells are further radiation treated and / or exposed to cytotoxic or chemotherapeutic agents.
[185] In another embodiment, the present invention provides a container comprising: a container; Label on container; And a composition comprising an active substance contained in a container, wherein the composition is effective to inhibit the growth of tumor cells, wherein the label on the container is characterized by overexpression of the PRO1889 polypeptide using the composition. The active substance in the composition is a substance that inhibits the expression and / or activity of the PRO1889 polypeptide. In a preferred aspect, the active agent is an anti-PRO1889 antibody.
[186] 5. PRO1890
[187] We have found a cDNA clone (DNA79230-2525) that encodes a novel polypeptide (designated herein as "PRO1890") that is homologous to a nucleic acid encoding a lectin protein.
[188] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1890 polypeptide.
[189] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1890 polypeptide having a sequence of about 1 or about 22 to about 273 amino acid residues in FIG. 10 (SEQ ID NO: 18) or (b) the DNA of (a) A DNA having at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with the complement of the molecule.
[190] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO1890 polypeptide comprising DNA hybridizing with the nucleotide of about 378 or the complement of a nucleic acid between about 441 and about 1196 of FIG. 9 (SEQ ID NO: 17). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[191] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203549 (DNA79230-2525) or (b) a nucleic acid molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203549 (DNA79230-2525).
[192] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90% of the amino acid residue 1 of FIG. 10 (SEQ ID NO: 18) or the sequence of about 22 to about 273 Above, most preferably, it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having sequence identity of at least about 95% or (b) the complement of the DNA of (a).
[193] In another aspect, the invention provides DNA that encodes a PRO1890 polypeptide having at least 475 nucleotides and, under stringent conditions, a test DNA molecule (a) having amino acid residue 1 of FIG. 10 (SEQ ID NO: 18) or a sequence from about 22 to about 273. Molecule or (b) hybridize with the complement of the DNA molecule of (a) and the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably about (a) or (b) If it has at least 90%, most preferably at least about 95% sequence identity, it relates to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[194] In certain aspects, the present invention includes or may include DNA encoding a PRO1890 polypeptide and its soluble variant (ie, the transmembrane domain is deleted or inactivated) with or without an N-terminal signal sequence and / or initiating methionine. An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 21 to about 21 amino acid positions in the sequence of FIG. 10 (SEQ ID NO: 18). By experiment, the transmembrane domain was found to span the amino acid position about 214 to about 235 in the PRO1890 amino acid sequence (FIG. 10, SEQ ID NO: 18).
[195] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90 when compared to residue 1 of FIG. 10 (SEQ ID NO: 18) or the amino acid sequence of about 22 to about 273 At least%, most preferably at least about 95%, of DNA encoding a polypeptide that is recorded as positive or (b) an isolated nucleic acid molecule comprising the complement of the DNA of (a).
[196] Another embodiment is directed to a fragment of the PRO1890 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length, Can be derived from the nucleotide sequence shown in FIG. 9 (SEQ ID NO: 17).
[197] In another embodiment, the present invention provides an isolated PRO1890 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[198] In certain aspects, the invention provides an isolated native sequence PRO1890 polypeptide which, in some embodiments, comprises an amino acid sequence comprising residue 1 or about 22 to about 273 of FIG. 10 (SEQ ID NO: 18).
[199] In another aspect, the invention relates to the amino acid residue 1 of Figure 10 (SEQ ID NO: 18) or to the sequence of about 22 to about 273 and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most Preferably it relates to an isolated PRO1890 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[200] In another aspect, the invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, as compared to residue 1 of Figure 10 (SEQ ID NO: 18) or the amino acid sequence of about 22 to about 273, Most preferably, it is directed to an isolated PRO1890 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[201] In another aspect, the invention provides an isolated PRO1890 polypeptide comprising the amino acid residue 1 of Figure 10 (SEQ ID NO: 18) or a sequence from about 22 to about 273, or a fragment thereof sufficient to provide a binding site for an anti-PRO1890 antibody. It is about. Preferably, the PRO1890 fragment retains the qualitative biological activity of the native PRO1890 polypeptide.
[202] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO1890 polypeptide having a sequence of about 1 or about 22 to about 273 amino acid residues in FIG. 10 (SEQ ID NO: 18) or (b) hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, if at least 90%, most preferably at least about 95%, of sequence identity, and (iii) the polypeptide from the cell culture It provides a polypeptide produced by the step of recovering.
[203] 6.PRO1887
[204] We have found a cDNA clone (DNA79862-2522) that encodes a novel polypeptide (named "PRO1887" herein) having homology with carboxyesterases.
[205] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1887 polypeptide.
[206] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO1887 polypeptide having amino acid residue 1 of Figure 12 (SEQ ID NO: 23) or a sequence from about 28 to about 571, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[207] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO1887 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 87 and about 1718 of FIG. 11 (SEQ ID NO: 22). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[208] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203550 (DNA79862-2522) or (b) a nucleic acid molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203550 (DNA79862-2522).
[209] In another aspect, the invention provides a composition comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, amino acid residues of the sequences of about 28 to about 571 of the amino acid residues of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[210] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 28 to about 571 in FIG. A DNA molecule encoding a PRO1887 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[211] In certain aspects, the invention includes, or is, DNA encoding the PRO1887 polypeptide and its soluble variant (ie, the transmembrane domain is deleted or inactivated) with or without an N-terminal signal sequence and / or initiating methionine. An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 27 from amino acid position 1 in the sequence of FIG. 12 (SEQ ID NO: 23). By experiment, the transmembrane domain was found to span the amino acid position about 226 to about 245 in the PRO1887 amino acid sequence (FIG. 12, SEQ ID NO: 23).
[212] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 28 to about 571 of FIG. 12 (SEQ ID NO: 23), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[213] Another embodiment is directed to a fragment of a PRO1887 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[214] In another embodiment, the present invention provides an isolated PRO1887 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[215] In certain aspects, the invention provides an isolated native sequence PRO1887 polypeptide which, in one embodiment, comprises the amino acid sequence comprising residues 28 to about 571 of FIG. 12 (SEQ ID NO: 23).
[216] In another aspect, the invention relates to the sequence of amino acid residues 28 to about 571 of Figure 12 (SEQ ID NO: 23) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO1887 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[217] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 28 to 571 of Figure 12 (SEQ ID NO: 23). An isolated PRO1887 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[218] In another aspect, the invention relates to an isolated PRO1887 polypeptide comprising the sequence of amino acid residues 28 to about 571 of FIG. 12 (SEQ ID NO: 23), or fragments thereof sufficient to provide a binding site for an anti-PRO1887 antibody. . Preferably, the PRO1887 fragment retains the qualitative biological activity of the native PRO1887 polypeptide.
[219] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO1887 polypeptide having a sequence from about 28 to about 571 amino acid residues in FIG. 12 (SEQ ID NO: 23), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[220] 7.PRO1785
[221] We have found a cDNA clone (DNA80136-2503) encoding a novel polypeptide (designated herein as "PRO1785") with sequence identity to peroxidase.
[222] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO1785 polypeptide.
[223] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 4 (SEQ ID NO: 29) or a PRO1785 polypeptide having a sequence from about 32 to about 209, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[224] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO1785 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 95 and about 628 of FIG. 13 (SEQ ID NO: 28). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[225] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203541 (DNA80136-2503) or (b) a nucleic acid molecule of (a) above. It relates to an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203541 (DNA80136-2503).
[226] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the amino acid residues of FIG. 14 (SEQ ID NO: 29) with the sequence of about 32 to about 209; Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[227] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 32 to about 209 in Figure 14 (SEQ ID NO: 29). A DNA molecule encoding a PRO1785 polypeptide having or (b) a complement of the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[228] In certain aspects, the invention includes or is capable of encoding DNA that encodes a PRO1785 polypeptide and its soluble variant (ie, the transmembrane domain is deleted or inactivated) with or without an N-terminal signal sequence and / or initiating methionine. An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 31 from amino acid position 1 in the sequence of FIG. 14 (SEQ ID NO: 29). By experiment, the transmembrane domain was found to span the amino acid position about 18 to about 37 in the PRO1785 amino acid sequence (FIG. 14, SEQ ID NO: 29).
[229] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 32 to about 209 of FIG. 14 (SEQ ID NO: 29), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[230] Another embodiment relates to fragments of the PRO1785 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[231] In another embodiment, the present invention provides an isolated PRO1785 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[232] In certain aspects, the invention provides an isolated native sequence PRO1785 polypeptide which, in one embodiment, comprises an amino acid sequence comprising residues 32-209 of FIG. 14 (SEQ ID NO: 29).
[233] In another aspect, the invention relates to the sequence of amino acid residues 32 to about 209 of Figure 14 (SEQ ID NO: 29) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO1785 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[234] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 32 to 209 of Figure 14 (SEQ ID NO: 29). An isolated PRO1785 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[235] In another aspect, the invention relates to an isolated PRO1785 polypeptide comprising the sequence of amino acid residues 32 to about 209 of FIG. 14 (SEQ ID NO: 29), or a fragment thereof sufficient to provide a binding site for an anti-PRO1785 antibody. . Preferably, the PRO1785 fragment retains the qualitative biological activity of the native PRO1785 polypeptide.
[236] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO1785 polypeptide having a sequence from about 32 to about 209 amino acid residues in FIG. 14 (SEQ ID NO: 29), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[237] 8.PRO4353
[238] We have found a cDNA clone (DNA80145-2594) encoding a novel polypeptide (named herein "PRO4353") that is homologous to semaphorin Z.
[239] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4353 polypeptide.
[240] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 16 (SEQ ID NO: 35) or a PRO4353 polypeptide having a sequence from about 26 to about 888, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[241] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4353 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 94 and about 2682 in FIG. 15 (SEQ ID NO: 34). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[242] In another aspect, the invention provides a DNA molecule encoding (a) the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 204-PTA (DNA80145-2594) or (b) the DNA of (a) To an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% . In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 204-PTA (DNA80145-2594).
[243] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the amino acid residues of FIG. 16 (SEQ ID NO: 35) with the sequence of about 26 to about 888; Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[244] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) a sequence of about 26 to about 888 amino acid residues in FIG. 16 (SEQ ID NO: 35). A DNA molecule encoding a PRO4353 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[245] In certain aspects, the invention includes or is capable of encoding DNA that encodes a PRO4353 polypeptide with or without an N-terminal signal sequence and / or initiating methionine and a soluble variant thereof (ie, the transmembrane domain is deleted or inactivated). An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 25 from amino acid position 1 in the sequence of FIG. 16 (SEQ ID NO: 35). Experiments have shown that the transmembrane domain spans amino acid positions about 318 to about 339 and about 598 to about 617 (FIG. 16, SEQ ID NO: 35).
[246] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 26 to about 888 of FIG. 16 (SEQ ID NO: 35), Most preferably it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[247] Another embodiment relates to fragments of the PRO4353 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[248] In another embodiment, the present invention provides an isolated PRO4353 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[249] In certain aspects, the invention provides an isolated native sequence PRO4353 polypeptide which, in one embodiment, comprises the amino acid sequence comprising residues 26-888 of FIG. 16 (SEQ ID NO: 35).
[250] In another aspect, the invention relates to the sequence of amino acid residues 26 to about 888 of Figure 16 (SEQ ID NO: 35) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4353 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[251] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 26 to 888 of Figure 16 (SEQ ID NO: 35). An isolated PRO4353 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[252] In another aspect, the invention relates to an isolated PRO4353 polypeptide comprising the sequence of amino acid residues 26 to about 888 of FIG. 16 (SEQ ID NO: 35), or a fragment thereof sufficient to provide a binding site for an anti-PRO4353 antibody. . Preferably, the PRO4353 fragment retains the qualitative biological activity of the native PRO4353 polypeptide.
[253] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under strict conditions (a) a PRO4353 polypeptide having a sequence of about 26 to about 888 amino acid residues in FIG. 16 (SEQ ID NO: 35), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[254] 9. PRO4357
[255] We have found a cDNA clone (DNA84917-2597) that encodes a novel polypeptide (named herein "PRO4357") having homology with "BK158_1".
[256] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4357 polypeptide.
[257] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 18 (SEQ ID NO: 40) or a PRO4357 polypeptide having a sequence from about 18 to about 502, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[258] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4357 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 337 and about 1791 of FIG. 17 (SEQ ID NO: 39). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[259] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203863 (DNA84917-2597) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203863 (DNA84917-2597).
[260] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 18 to about 502 of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[261] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, the test DNA molecule is (a) sequenced from about 18 to about 502 amino acid residues in FIG. 18 (SEQ ID NO: 40). A DNA molecule encoding a PRO4357 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[262] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 18 to about 502 of FIG. 18 (SEQ ID NO: 40), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[263] Another embodiment relates to fragments of the PRO4357 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[264] In another embodiment, the present invention provides an isolated PRO4357 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[265] In certain aspects, the invention provides an isolated native sequence PRO4357 polypeptide, which in one embodiment comprises the amino acid sequence comprising residues 18-502 of FIG. 18 (SEQ ID NO: 40).
[266] In another aspect, the invention relates to the sequence of amino acid residues 18 to about 502 of Figure 18 (SEQ ID NO: 40) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4357 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[267] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 18 to 502 of FIG. 18 (SEQ ID NO: 40). An isolated PRO4357 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[268] In another aspect, the invention relates to an isolated PRO4357 polypeptide comprising the sequence of amino acid residues 18 to about 502 of FIG. 18 (SEQ ID NO: 40), or a fragment thereof sufficient to provide a binding site for an anti-PRO4357 antibody. . Preferably, the PRO4357 fragment retains the qualitative biological activity of the native PRO4357 polypeptide.
[269] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under strict conditions (a) a PRO4357 polypeptide having a sequence from about 18 to about 502 amino acid residues in FIG. 18 (SEQ ID NO: 40), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[270] 10.PRO4405
[271] We have found a cDNA clone (DNA84920-2614) encoding a novel polypeptide (designated herein as "PRO4405").
[272] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4405 polypeptide.
[273] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 20 (SEQ ID NO: 45) or a PRO4405 polypeptide having a sequence from about 35 to about 310, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[274] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4405 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 181 and about 1008 of FIG. 19 (SEQ ID NO: 44). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[275] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203966 (DNA84920-2614) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203966 (DNA84920-2614).
[276] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 35 to about 310 of FIG. 20 (SEQ ID NO: 45), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[277] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is (a) sequenced from about 35 to about 310 amino acid residues in FIG. 20 (SEQ ID NO: 45). A DNA molecule encoding a PRO4405 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[278] In certain aspects, the present invention includes or may include DNA encoding a PRO4405 polypeptide and its soluble variant (ie, the transmembrane domain is deleted or inactivated) with or without an N-terminal signal sequence and / or initiating methionine. An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 34 from amino acid position 1 in the sequence of FIG. 20 (SEQ ID NO: 45). By experiment, the transmembrane domain was found to span the amino acid position about 58 to about 76 in the PRO4405 amino acid sequence (FIG. 20, SEQ ID NO: 45), and may be a type II transmembrane domain.
[279] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 35 to about 310 of FIG. 20 (SEQ ID NO: 45), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[280] Another embodiment is directed to a fragment of the PRO4405 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[281] In another embodiment, the present invention provides an isolated PRO4405 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[282] In certain aspects, the invention provides an isolated native sequence PRO4405 polypeptide, which in one embodiment comprises an amino acid sequence comprising residues 35-310 of FIG. 20 (SEQ ID NO: 45).
[283] In another aspect, the invention relates to the sequence of amino acid residues 35 to about 310 of Figure 20 (SEQ ID NO: 45) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4405 polypeptide comprising amino acid sequence having at least about 95% sequence identity.
[284] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 35 to 310 of FIG. 20 (SEQ ID NO: 45). An isolated PRO4405 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[285] In another aspect, the invention relates to an isolated PRO4405 polypeptide comprising the sequence of amino acid residues 35 to about 310 of FIG. 20 (SEQ ID NO: 45), or a fragment thereof sufficient to provide a binding site for an anti-PRO4405 antibody. . Preferably, the PRO4405 fragment retains the qualitative biological activity of the native PRO4405 polypeptide.
[286] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under stringent conditions (a) a PRO4405 polypeptide having a sequence from about 35 to about 310 amino acid residues in FIG. 20 (SEQ ID NO: 45), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[287] 11.PRO4356
[288] We have found a cDNA clone (DNA86576-2595) that encodes a novel polypeptide (named "PRO4356" herein) that is homologous to MAGPIAP.
[289] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4356 polypeptide.
[290] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 22 (SEQ ID NO: 50) or a PRO4356 polypeptide having a sequence from about 20 to about 251, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[291] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4356 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 112 and about 807 of FIG. 21 (SEQ ID NO: 49). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[292] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203868 (DNA86576-2595) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203868 (DNA86576-2595).
[293] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 20 to about 251 of Figure 22 (SEQ ID NO: 50), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[294] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides, and, under stringent conditions, the test DNA molecule is (a) sequenced from about 20 to about 251 amino acid residues in FIG. A DNA molecule encoding a PRO4356 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[295] In certain aspects, the present invention includes or may include DNA encoding a PRO4356 polypeptide with or without an N-terminal signal sequence and / or initiating methionine and soluble variants thereof (ie, the transmembrane domain is deleted or inactivated). An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 19 from amino acid position 1 in the sequence of FIG. 22 (SEQ ID NO: 50). Experiments have shown that the transmembrane domain spans amino acid positions from about 233 to about 251 in the PRO4356 amino acid sequence (FIG. 22, SEQ ID NO: 50).
[296] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 20 to about 251 of FIG. 22 (SEQ ID NO: 50), Most preferably it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[297] Another embodiment is directed to a fragment of a PRO4356 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[298] In another embodiment, the present invention provides an isolated PRO4356 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[299] In certain aspects, the invention provides an isolated native sequence PRO4356 polypeptide which, in one embodiment, comprises the amino acid sequence comprising residues 20-251 of FIG. 22 (SEQ ID NO: 50).
[300] In another aspect, the invention relates to the sequence of amino acid residues 20 to about 251 of Figure 22 (SEQ ID NO: 50) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4356 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[301] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 20 to 251 of Figure 22 (SEQ ID NO: 50). An isolated PRO4356 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[302] In another aspect, the invention relates to an isolated PRO4356 polypeptide comprising the sequence of amino acid residues 20 to about 251 of FIG. 22 (SEQ ID NO: 50), or a fragment thereof sufficient to provide a binding site for an anti-PRO4356 antibody. . Preferably, the PRO4356 fragment retains the qualitative biological activity of the native PRO4356 polypeptide.
[303] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4356 polypeptide having a sequence of about 20 to about 251 amino acid residues in FIG. 22 (SEQ ID NO: 50) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[304] 12.PRO4352
[305] We have found a cDNA clone (DNA87976-2593) that encodes a novel polypeptide (designated herein as "PRO4352") that is homologous to protocadherin pc3.
[306] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4352 polypeptide.
[307] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 24 (SEQ ID NO: 52) or a PRO4352 polypeptide having a sequence from about 27 to about 800, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[308] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4352 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 257 and about 2578 of FIG. 23 (SEQ ID NO: 51). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[309] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203888 (DNA87976-2593) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203888 (DNA87976-2593).
[310] In another aspect, the invention provides a composition comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, amino acid residues from about 27 to about 800 of the amino acid residues of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[311] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 27 to about 800 in FIG. DNA molecule encoding a PRO4352 polypeptide having or (b) a hybridization with the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[312] In certain aspects, the invention includes or is capable of encoding DNA that encodes a PRO4352 polypeptide with or without an N-terminal signal sequence and / or initiating methionine and a soluble variant thereof (ie, the transmembrane domain is deleted or inactivated). An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 26 from amino acid position 1 in the sequence of FIG. 24 (SEQ ID NO: 52). Experiments have shown that the transmembrane domain spans amino acid positions from about 687 to about 711 in the PRO4352 amino acid sequence (FIG. 24, SEQ ID NO: 52).
[313] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 27 to about 800 of FIG. 24 (SEQ ID NO: 52), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[314] Another embodiment is directed to a fragment of a PRO4352 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[315] In another embodiment, the present invention provides an isolated PRO4352 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[316] In certain aspects, the invention provides an isolated native sequence PRO4352 polypeptide, in one embodiment, the polypeptide comprises an amino acid sequence comprising residues 27-800 of FIG. 24 (SEQ ID NO: 52).
[317] In another aspect, the present invention provides the sequence of amino acid residues 27 to about 800 of Figure 24 (SEQ ID NO: 52) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4352 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[318] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 27 to 800 of FIG. 24 (SEQ ID NO: 52). An isolated PRO4352 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[319] In another aspect, the invention relates to an isolated PRO4352 polypeptide comprising the sequence of amino acid residues 27 to about 800 of FIG. 24 (SEQ ID NO: 52), or a fragment thereof sufficient to provide a binding site for an anti-PRO4352 antibody. . Preferably, the PRO4352 fragment retains the qualitative biological activity of the native PRO4352 polypeptide.
[320] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under stringent conditions (a) a PRO4352 polypeptide having a sequence from about 27 to about 800 amino acid residues in FIG. 24 (SEQ ID NO: 52), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[321] 13.PRO4380
[322] We have found a cDNA clone (DNA92234-2602) that encodes a novel polypeptide (designated herein as "PRO4380").
[323] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4380 polypeptide.
[324] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 26 (SEQ ID NO: 57) or a PRO4380 polypeptide having a sequence from about 27 to about 507, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[325] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4380 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 279 and about 1721 in FIG. 25 (SEQ ID NO: 56). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[326] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203948 (DNA92234-2602) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203948 (DNA92234-2602).
[327] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 27 to about 507 of Figure 26 (SEQ ID NO: 57), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[328] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 27 to about 507 of Figure 26 (SEQ ID NO: 57). A DNA molecule encoding a PRO4380 polypeptide having or (b) a complement of the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[329] In certain aspects, the invention includes, or is, DNA encoding a PRO4380 polypeptide with or without an N-terminal signal sequence and / or initiating methionine and a soluble variant thereof (ie, the transmembrane domain is deleted or inactivated). An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 26 from amino acid position 1 in the sequence of FIG. 26 (SEQ ID NO: 57). By experiment, the transmembrane domain was found to span the amino acid position about 273 to about 292 in the PRO4380 amino acid sequence (FIG. 26, SEQ ID NO: 57).
[330] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, as compared to the amino acid sequence of residues 27 to about 507 of Figure 26 (SEQ ID NO: 57), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[331] Another embodiment is directed to a fragment of a PRO4380 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[332] In another embodiment, the present invention provides isolated PRO4380 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[333] In certain aspects, the invention provides an isolated native sequence PRO4380 polypeptide, which in one embodiment comprises an amino acid sequence comprising residues 27 to 507 of FIG. 26 (SEQ ID NO: 57).
[334] In another aspect, the invention relates to the sequence of amino acid residues 27 to about 507 of Figure 26 (SEQ ID NO: 57) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4380 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[335] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 27 to 507 of Figure 26 (SEQ ID NO: 57). An isolated PRO4380 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[336] In another aspect, the invention relates to an isolated PRO4380 polypeptide comprising the sequence of amino acid residues 27 to about 507 of Figure 26 (SEQ ID NO: 57), or a fragment thereof sufficient to provide a binding site for an anti-PRO4380 antibody. . Preferably, the PRO4380 fragment retains the qualitative biological activity of the native PRO4380 polypeptide.
[337] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4380 polypeptide having a sequence from about 27 to about 507 amino acid residues in FIG. 26 (SEQ ID NO: 57), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[338] 14.PRO4354
[339] We have found a cDNA clone (DNA92256-2596) that encodes a novel polypeptide (designated herein as "PRO4354").
[340] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4354 polypeptide.
[341] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO4354 polypeptide having a sequence of about 22 to about 248 amino acid residues in FIG. 28 (SEQ ID NO: 59) or (b) a complement of the DNA molecule of (a) At least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with the sieve.
[342] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4354 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 171 and about 851 of FIG. 27 (SEQ ID NO: 58). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[343] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203071 (DNA92256-2596) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203071 (DNA92256-2596).
[344] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the amino acid residues of Figure 28 (SEQ ID NO: 59) with the sequence of about 22 to about 248; Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[345] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 22 to about 248 in FIG. 28 (SEQ ID NO: 59). A DNA molecule encoding a PRO4354 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[346] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 22 to about 248 of FIG. 28 (SEQ ID NO: 59), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[347] Another embodiment is directed to a fragment of the PRO4354 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[348] In another embodiment, the present invention provides an isolated PRO4354 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[349] In certain aspects, the invention provides an isolated native sequence PRO4354 polypeptide, in one embodiment, the polypeptide comprises an amino acid sequence comprising residues 22-248 of FIG. 28 (SEQ ID NO: 59).
[350] In another aspect, the invention relates to the sequence of amino acid residues 22 to about 248 of Figure 28 (SEQ ID NO: 59) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4354 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[351] In another aspect, the invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 22 to 248 of FIG. 28 (SEQ ID NO: 59). An isolated PRO4354 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[352] In another aspect, the invention relates to an isolated PRO4354 polypeptide comprising the sequence of amino acid residues 22 to about 248 of FIG. 28 (SEQ ID NO: 59), or a fragment thereof sufficient to provide a binding site for an anti-PRO4354 antibody. . Preferably, the PRO4354 fragment retains the qualitative biological activity of the native PRO4354 polypeptide.
[353] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4354 polypeptide having a sequence from about 22 to about 248 amino acid residues in FIG. 28 (SEQ ID NO: 59) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[354] 15.PRO4408
[355] We have found cDNA clones (DNA92274-2617) encoding novel polypeptides (named herein "PRO4408").
[356] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4408 polypeptide.
[357] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding a PRO4408 polypeptide having a sequence of about 23 to about 223 amino acid residues in FIG. 30 (SEQ ID NO: 61) or (b) a complement of the DNA molecule of (a) At least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with the sieve.
[358] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4408 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 155 and about 757 of FIG. 29 (SEQ ID NO: 60). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[359] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by the human protein cDNA of (a) ATCC Accession No. 203971 (DNA92274-2617) or (b) a DNA molecule of (a). An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203171 (DNA92274-2617).
[360] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 30 to about 223 of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[361] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 23 to about 223 of FIG. 30 (SEQ ID NO: 61). A DNA molecule encoding a PRO4408 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[362] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 23 to about 223 of FIG. 30 (SEQ ID NO: 61), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[363] Another embodiment relates to fragments of the PRO4408 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[364] In another embodiment, the present invention provides an isolated PRO4408 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[365] In certain aspects, the invention provides an isolated native sequence PRO4408 polypeptide which, in one embodiment, comprises the amino acid sequence comprising residues 23-223 of FIG. 30 (SEQ ID NO: 61).
[366] In another aspect, the invention relates to the sequence of amino acid residues 23 to about 223 of Figure 30 (SEQ ID NO: 61) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4408 polypeptide comprising amino acid sequence having at least about 95% sequence identity.
[367] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 23 to 223 of FIG. 30 (SEQ ID NO: 61). An isolated PRO4408 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[368] In another aspect, the invention relates to an isolated PRO4408 polypeptide comprising the sequence of amino acid residues 23 to about 223 of FIG. 30 (SEQ ID NO: 61), or a fragment thereof sufficient to provide a binding site for an anti-PRO4408 antibody. . Preferably, the PRO4408 fragment retains the qualitative biological activity of the native PRO4408 polypeptide.
[369] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4408 polypeptide having a sequence from about 23 to about 223 amino acid residues in FIG. 30 (SEQ ID NO: 61), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[370] 16.PRO5737
[371] We have found a cDNA clone (DNA92929-2534) that encodes a novel polypeptide (named "PRO5737" herein) having homology with IL-1.
[372] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO5737 polypeptide.
[373] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 32 (SEQ ID NO: 63) or a PRO5737 polypeptide having a sequence from about 18 to about 134, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[374] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO5737 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between about 96 or about 147 and about 497 residues of FIG. 31 (SEQ ID NO: 62). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[375] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203586 (DNA92929-2534) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203586 (DNA92929-2534).
[376] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 18 to about 134 of Figure 32 (SEQ ID NO: 63), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[377] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, the test DNA molecule is (a) sequenced from about 18 to about 134 amino acid residues in FIG. 32 (SEQ ID NO: 63). A DNA molecule encoding a PRO5737 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[378] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 18 to about 134 of FIG. 32 (SEQ ID NO: 63), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[379] Another embodiment relates to a fragment of the PRO5737 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[380] In another embodiment, the present invention provides isolated PRO5737 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[381] In certain aspects, the invention provides an isolated native sequence PRO5737 polypeptide which, in one embodiment, comprises the amino acid sequence comprising residues 18-134 of FIG. 32 (SEQ ID NO: 63).
[382] In another aspect, the invention relates to the sequence of amino acid residues 18 to about 134 of Figure 32 (SEQ ID NO: 63) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO5737 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[383] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 18 to 134 of Figure 32 (SEQ ID NO: 63). An isolated PRO5737 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[384] In another aspect, the invention relates to an isolated PRO5737 polypeptide comprising the sequence of amino acid residues 18 to about 134 of FIG. 32 (SEQ ID NO: 63), or a fragment thereof sufficient to provide a binding site for an anti-PRO5737 antibody. . Preferably, the PRO5737 fragment retains the qualitative biological activity of the native PRO5737 polypeptide.
[385] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO5737 polypeptide having a sequence from about 18 to about 134 amino acid residues in FIG. 32 (SEQ ID NO: 63) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[386] 17.PRO4425
[387] We have found a cDNA clone (DNA93011-2637) that encodes a novel polypeptide (named "PRO4425" herein) having homology with the protein of Genbank Accession No. HGS_RE295.
[388] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4425 polypeptide.
[389] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 34 (SEQ ID NO: 65) or a PRO4425 polypeptide having a sequence from about 20 to about 136, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[390] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4425 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 84 and about 434 of FIG. 33 (SEQ ID NO: 64). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[391] In another aspect, the invention provides a DNA molecule encoding (a) the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 20-PTA (DNA93011-2637) or (b) the DNA of (a) above. To an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% . In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 20-PTA (DNA93011-2637).
[392] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 20 to about 136 of Figure 34 (SEQ ID NO: 65), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[393] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides, and, under stringent conditions, a test DNA molecule may comprise (a) a sequence of about 20 to about 136 amino acid residues in FIG. 34 (SEQ ID NO: 65). A DNA molecule encoding a PRO4425 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[394] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, when compared to the amino acid sequence of residues 20 to about 136 of FIG. 34 (SEQ ID NO: 65), Most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[395] Another embodiment is directed to a fragment of the PRO4425 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[396] In another embodiment, the present invention provides an isolated PRO4425 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[397] In certain aspects, the invention provides an isolated native sequence PRO4425 polypeptide, in one embodiment, the polypeptide comprises an amino acid sequence comprising residues 20-136 of FIG. 34 (SEQ ID NO: 65).
[398] In another aspect, the invention relates to the sequence of amino acid residues 20 to about 136 of Figure 34 (SEQ ID NO: 65) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4425 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[399] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 20 to 136 of Figure 34 (SEQ ID NO: 65) An isolated PRO4425 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[400] In another aspect, the invention relates to an isolated PRO4425 polypeptide comprising the sequence of amino acid residues 20 to about 136 of FIG. 34 (SEQ ID NO: 65), or a fragment thereof sufficient to provide a binding site for an anti-PRO4425 antibody. . Preferably, the PRO4425 fragment retains the qualitative biological activity of the native PRO4425 polypeptide.
[401] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4425 polypeptide having a sequence from about 20 to about 136 amino acid residues in FIG. 34 (SEQ ID NO: 65) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[402] 18.PRO5990
[403] We have found a cDNA clone (designated herein as DNA96042-2682) that encodes a novel polypeptide (designated herein as "PRO5990") that is homologous to a nucleic acid encoding to secreto granine.
[404] In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO5990 polypeptide.
[405] In one aspect, an isolated nucleic acid molecule is (a) a DNA molecule encoding a PRO5990 polypeptide having a sequence of about 1 or about 22 to about 468 amino acid residues in FIG. 36 (SEQ ID NO: 67) or (b) the DNA of (a) At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 80% with the complement of the molecule At least 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more Preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably about 96 At least%, more preferably at least about 97%, more preferably 98% or more, and more preferably comprises a nucleotide sequence having a sequence identity of at least about 99%.
[406] In another aspect, an isolated nucleic acid molecule is (a) a nucleotide sequence encoding a PRO5990 polypeptide having a sequence of about 1 or about 22 to about 468 amino acid residues in FIG. 36 (SEQ ID NO: 67) or (b) of (a) above. Complements of nucleotide sequences.
[407] In another aspect, an isolated nucleic acid molecule is (a) a DNA molecule having a sequence of about 265 or about 328 to about 1668 of the nucleotides of FIG. 35 (SEQ ID NO: 66) or (b) a complement of the DNA molecule of (a). At least 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, even more preferred Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, even more preferably Is at least about 97%, more preferably at least about 98%, more preferably It comprises a nucleotide sequence having a sequence identity of at least about 99%.
[408] In another aspect, an isolated nucleic acid molecule comprises (a) the nucleotide sequence of about 265 or about 328 to about 1668 of Figure 35 (SEQ ID NO: 66) or (b) the complement of the nucleotide sequence of (a).
[409] In a further aspect, the present invention provides a DNA encoding the same mature polypeptide encoded by human protein cDNA deposited with ATCC Accession No. 382-PTA (DNA96042-2682) to ATCC on July 20, 1999. At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 80% with the complement of the molecule or (b) the DNA molecule of (a) Is at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89% , More preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably At least about 95%, more preferably at least about 96%, more preferably 97%, more preferably at least about 98%, and more preferably relates to an isolated nucleic acid molecule comprising a nucleotide sequence having a sequence identity of at least about 99%. In a preferred embodiment, the isolated nucleic acid molecule encodes the same mature polypeptide encoded by (a) a human protein cDNA deposited with ATCC Accession No. 382-PTA (DNA96042-2682) to ATCC on July 20, 1999. Nucleotide sequence or (b) the complement of nucleotide sequence of (a) above.
[410] In another aspect, the invention provides an antibody comprising (a) the full-length polypeptide coding sequence of human protein cDNA deposited with ATCC on July 20, 1999 under ATCC Accession No. 382-PTA (DNA96042-2682) or (b) the (a) At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably with the complement of the nucleotide sequence of Preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90% At least about 91%, more preferably at least about 92%, more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95%, even more preferably Is at least about 96%, more preferably at least about 97%, more preferably It relates to an isolated nucleic acid molecule comprising a nucleotide sequence having about 98%, more preferably at least about 99% sequence identity. In a preferred embodiment, the isolated nucleic acid molecule is (a) a full-length polypeptide coding sequence of DNA deposited with ATCC Accession No. 382-PTA (DNA96042-2682) on July 20, 1999, or (b) the (a) ), The complement of the nucleotide sequence.
[411] In another aspect, the invention encodes an active PRO5990 polypeptide as defined below, comprising a nucleotide sequence that hybridizes with the complement of a nucleic acid sequence encoding amino acid 1 or about 22 to about 468 of FIG. 36 (SEQ ID NO: 67). To an isolated nucleic acid molecule. Preferably, hybridization occurs under stringent hybridization and washing conditions.
[412] In another aspect, the invention encodes an active PRO5990 polypeptide as defined below, comprising a nucleotide sequence that hybridizes with the complement of the nucleic acid sequence between about 265 or about 328 and about 1668 of nucleotide of FIG. 35 (SEQ ID NO: 66). To an isolated nucleic acid molecule. Preferably, hybridization occurs under stringent hybridization and washing conditions.
[413] In a further aspect, the present invention provides a test DNA molecule having at least about 1301 nucleotides and, under stringent conditions, (a) a PRO5990 polypeptide having a sequence of about 1 or about 22 to about 468 amino acid residues in FIG. 36 (SEQ ID NO: 67). Or (b) hybridizes with the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably at least about 81%, more preferably with (a) or (b) Preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87% At least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably Is at least about 93%, more preferably at least about 94%, More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99% An isolated nucleic acid molecule produced by isolating a test DNA molecule.
[414] In another aspect, the invention provides (a) at least about 80%, preferably at least about 81%, and more preferably about 80% of the residues of FIG. 36 (SEQ ID NO: 67) or the amino acid sequence of about 22 to 468 At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably about 93 At least%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, even more preferred Preferably, at least about 99% of the polypeptides are recorded as positive Nucleotide sequence, or (b) encoding relates to an isolated nucleic acid molecule comprising the complement of the nucleotide sequence of (a).
[415] In certain aspects, the invention provides an isolated nucleic acid molecule comprising or complementary to DNA encoding a PRO5990 polypeptide with or without an N-terminal signal sequence and / or starting methionine. By experiment, the signal peptide was found to span about 21 to about 21 amino acid positions in the sequence of FIG. 36 (SEQ ID NO: 67). Although the C-terminal boundary of the signal peptide may be different, most are only at about 5 amino acids on either side of the C-terminal boundary of the signal peptide, where the C-terminal boundary of the signal peptide is known in the art. Can be identified according to criteria commonly used to identify the type of amino acid sequence member in Nielsen et al ., Prot. Eng. 10: 1-6 (1997) and Heinje et al. Nucl. Acids. Res. 14: 4683-4690 (1986)]. In addition, in some cases, it is believed that the cleavage of the signal sequence of the secreted polypeptides is not entirely identical, resulting in one or more secreted polypeptides. The present invention includes such polypeptides and polynucleotides encoding them. As such, in the present application, the signal peptide of the PRO5990 polypeptide shown in FIG. 36 (SEQ ID NO: 67) spans amino acids 1 to X in FIG. 36 (SEQ ID NO: 67), wherein X is amino acids 16-26 of FIG. 36 (SEQ ID NO: 67). Any amino acid from. Thus, as a mature form of the PRO5990 polypeptide encompassed by the present invention, a polypeptide comprising amino acids X to 468 of Figure 36 (SEQ ID NO: 67), wherein X is amino acid 16 of Figure 36 (SEQ ID NO: 67) To any amino acid from to 26), and variants thereof. Also included in the present invention are isolated nucleic acid molecules encoding such polypeptides.
[416] Another embodiment is a PRO5990 polypeptide coding sequence that can be used, for example, as a hybridization probe or optionally to encode a fragment of a PRO5990 polypeptide that can encode a polypeptide comprising a binding site for an anti-PRO5990 antibody. Is about a fragment of. The length of such nucleic acid fragments is generally at least about 20 nucleotides, preferably at least about 30, more preferably at least about 40, more preferably at least about 50, more preferably at least about 60, more Preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 100, more preferably at least about 110, more preferably about 120 At least about 130, more preferably at least about 140, more preferably at least about 140, more preferably at least about 150, more preferably at least about 160, more preferably at least about 170, more preferred Preferably at least about 180, more preferably at least about 190, more preferably at least about 200, more preferably at least about 250, more preferably at least about 300, and more preferably about 350 More preferably about 400 , More preferably about 450 or more, more preferably about 500 or more, more preferably about 600 or more, more preferably about 700 or more, more preferably about 800 or more, more preferably At least about 900, more preferably at least about 1000, wherein the term "about" refers to the length of the nucleotide sequence mentioned above plus or minus 10% of this length. In a preferred embodiment, the nucleotide sequence fragment is derived from a specific coding region of the nucleotide sequence shown in FIG. 35 (SEQ ID NO: 66). New fragments of the PRO5990 polypeptide-encoding nucleotide sequence can be used to align the PRO5990 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well-known sequence alignment programs to determine whether the PRO5990 polypeptide-encoding nucleotide sequence fragment is new. It can be determined by the usual way of determining. All such PRO5990 polypeptide-encoding nucleotide sequences are considered herein and can be determined without undue experimentation. Also contemplated are PRO5990 polypeptide fragments encoded by this nucleotide molecule fragment, preferably PRO5990 polypeptide fragments comprising a binding site for an anti-PRO5990 antibody.
[417] In another embodiment, the invention provides an isolated PRO5990 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[418] In certain aspects, the invention provides an isolated native sequence PRO5990 polypeptide which, in some embodiments, comprises the amino acid sequence comprising about 1 or about 22 to about 468 residues of FIG. 36 (SEQ ID NO: 67).
[419] In another aspect, the present invention provides a sequence of about 1 or about 22 to about 468 amino acid residues of FIG. 36 (SEQ ID NO: 67) and at least about 80%, preferably at least about 81%, more preferably at least about 82%, More preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably about At least 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more Preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably about 99 Comprising amino acid sequences having at least% sequence identity It relates to an isolated PRO5990 polypeptide.
[420] In another aspect, the present invention provides an amino acid sequence that is encoded by ATCC Accession No. 382-PTA (DNA96042-2682) to ATCC on July 20, 1999, at least about 80%, preferably with an amino acid sequence encoded by human protein cDNA. At least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, More preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably about At least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more Preferably at least about 98%, more preferably at least about 99% The isolated PRO5990 comprising amino acid sequence having a sequence identity relates to the polypeptide. In a preferred embodiment, the isolated PRO5990 polypeptide comprises an amino acid sequence encoded by human protein cDNA deposited with ATCC Accession No. 382-PTA (DNA96042-2682) to ATCC on July 20, 1999.
[421] In another aspect, the invention provides at least about 80%, preferably at least about 81%, more preferably about 82% when compared to the residue about 1 or about 22 to about 468 amino acid sequence of FIG. 36 (SEQ ID NO: 67). Or more, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably Is at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93% , More preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, even more preferably Amino acid standing at about 99% positive The isolated PRO5990 relates to a polypeptide comprising a.
[422] In certain aspects, the invention provides an isolated PRO5990 polypeptide encoded by a nucleotide sequence encoding an amino acid sequence as described above, and having no N-terminal signal sequence and / or initiating methionine. Also described herein is a method of preparing the isolated PRO5990 polypeptide, wherein the method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the PRO5990 polypeptide and from the cell culture. Recovering the polypeptide.
[423] In another aspect, the invention provides an isolated PRO5990 polypeptide comprising a sequence of about 1 or about 22 to about 468 amino acid residues in FIG. 36 (SEQ ID NO: 67), or a binding site for a biologically active or anti-PRO5990 antibody. A fragment thereof sufficient to provide, wherein a PRO5990 polypeptide fragment that retains biological activity or provides a binding site for an anti-PRO5990 antibody can be identified by conventional methods using techniques well known in the art. Preferably, the PRO5990 fragment retains the qualitative biological activity of the native PRO5990 polypeptide.
[424] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO5990 polypeptide having a sequence of about 1 or about 22 to about 468 amino acid residues in FIG. 36 (SEQ ID NO: 67) or (b) hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 81%, more preferably at least about (a) or (b) At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably about 93 At least%, more preferably at least about 94%, more preferably at least about 95% And, more preferably, at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%, of sequence identity (ii) comprising a test DNA molecule Culturing the host cell under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
[425] Another embodiment of the present invention provides for the preparation of a PRO5990 polypeptide, an agonist or antagonist thereof, or a drug useful for the treatment of symptoms that respond to an anti-PRO5990 antibody, wherein the PRO5990 polypeptide, an agonist or antagonist thereof as described above, Or to the use of an anti-PRO5990 antibody.
[426] 19.PRO6030
[427] We have found a cDNA clone (designated herein as DNA96850-2705) that encodes a novel polypeptide (designated herein as "PRO6030").
[428] In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO6030 polypeptide.
[429] In one aspect, an isolated nucleic acid molecule is (a) a DNA molecule encoding a PRO6030 polypeptide having a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72) or (b) the DNA of (a) At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 80% with the complement of the molecule At least 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more Preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably about 96 At least%, more preferably at least about 97%, more preferably 98% or more, and more preferably comprises a nucleotide sequence having a sequence identity of at least about 99%.
[430] In another aspect, an isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a PRO6030 polypeptide having a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72), or (b) of (a) Complements of nucleotide sequences.
[431] In another aspect, an isolated nucleic acid molecule may comprise (a) a DNA molecule having a sequence of about 60 or about 138 to about 1025 nucleotides of FIG. 37 (SEQ ID NO: 71) or (b) a complement of the DNA molecule of (a). At least 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, even more preferred Preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91% At least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, even more preferably Is at least about 97%, more preferably at least about 98%, more preferably It comprises a nucleotide sequence having a sequence identity of at least about 99%.
[432] In another aspect, an isolated nucleic acid molecule comprises (a) the nucleotide sequence of about 60 or about 138 to about 1025 of FIG. 37 (SEQ ID NO: 71) or (b) the complement of the nucleotide sequence of (a).
[433] In a further aspect, the present invention provides a DNA encoding the same mature polypeptide encoded by human protein cDNA deposited with ATCC Accession No. 479-PTA (DNA96850-2705) to ATCC on August 3, 1999. At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 80% with the complement of the molecule or (b) the DNA molecule of (a) Is at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89% , More preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably About 95% or more, more preferably about 96% or more, more preferably about An isolated nucleic acid molecule comprising a nucleotide sequence having at least 97%, more preferably at least about 98%, more preferably at least about 99% sequence identity. In a preferred embodiment, the isolated nucleic acid molecule (a) encodes the same mature polypeptide encoded by the human protein cDNA deposited with ATCC Accession No. 479-PTA (DNA96850-2705) to ATCC on August 3, 1999. Nucleotide sequence or (b) the complement of nucleotide sequence of (a) above.
[434] In another aspect, the invention provides an antibody comprising (a) the full length polypeptide coding sequence of a human protein cDNA deposited with ATCC on August 3, 1999 under ATCC Accession No. 479-PTA (DNA96850-2705) or (b) the (a) At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably with the complement of the nucleotide sequence of Preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90% At least about 91%, more preferably at least about 92%, more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95%, even more preferably Is at least about 96%, more preferably at least about 97%, more preferably Crab relates to an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 98%, more preferably at least about 99% sequence identity. In a preferred embodiment, the isolated nucleic acid molecule is (a) a full-length polypeptide coding sequence of DNA deposited with ATCC on August 3, 1999 under ATCC Accession No. 479-PTA (DNA96850-2705) or (b) the (a) ), The complement of the nucleotide sequence.
[435] In another aspect, the invention encodes an active PRO6030 polypeptide as defined below, comprising a nucleotide sequence that hybridizes with the complement of a nucleic acid sequence encoding amino acid 1 or about 27 to about 322 of FIG. 38 (SEQ ID NO: 72). To an isolated nucleic acid molecule. Preferably, hybridization occurs under stringent hybridization and washing conditions.
[436] In another aspect, the invention encodes an active PRO6030 polypeptide as defined below, comprising a nucleotide sequence that hybridizes with the complement of a nucleic acid sequence between about 60 or about 138 and about 1025 of FIG. 37 (SEQ ID NO: 71). To an isolated nucleic acid molecule. Preferably, hybridization occurs under stringent hybridization and washing conditions.
[437] In a further aspect, the present invention provides a test DNA molecule having at least about 528 nucleotides and, under stringent conditions, (a) a PRO6030 polypeptide having a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72). Or (b) hybridizes with the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably at least about 81%, more preferably with (a) or (b) Preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87% At least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably Is at least about 93%, more preferably at least about 94%, More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99% An isolated nucleic acid molecule produced by isolating a test DNA molecule.
[438] In another aspect, the invention provides (a) at least about 80%, preferably at least about 81%, more preferably as compared to the amino acid sequence of about 1 or about 27 to about 322 of the residues of FIG. 38 (SEQ ID NO: 72) At least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, More preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably about At least 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more Preferably, at least about 99% of the polypeptides are recorded as positive To an isolated nucleic acid molecule comprising the nucleotide sequence encoding de or (b) the complement of the nucleotide sequence of (a).
[439] In certain aspects, the invention provides an isolated nucleic acid molecule comprising or complementary to DNA encoding a PRO6030 polypeptide with or without an N-terminal signal sequence and / or starting methionine. By experiment, the signal peptide was found to span about 26 from amino acid position about 1 in the sequence of FIG. 38 (SEQ ID NO: 72). Although the C-terminal boundary of the signal peptide may be different, most are only at about 5 amino acids on either side of the C-terminal boundary of the signal peptide, where the C-terminal boundary of the signal peptide is known in the art. Can be identified according to criteria commonly used to identify the type of amino acid sequence member in Nielsen et al ., Prot. Eng. 10: 1-6 (1997) and Heinje et al. Nucl. Acids. Res. 14: 4683-4690 (1986)]. In addition, in some cases, it is believed that the cleavage of the signal sequence of the secreted polypeptides is not entirely identical, resulting in one or more secreted polypeptides. The present invention includes such polypeptides and polynucleotides encoding them. As such, in the present application, the signal peptide of the PRO6030 polypeptide shown in FIG. 38 (SEQ ID NO: 72) spans from amino acid 1 to X in FIG. 38 (SEQ ID NO: 72), where X is amino acids 21-31 of FIG. 38 (SEQ ID NO: 72). Any amino acid from. Thus, a mature form of the PRO6030 polypeptide encompassed by the present invention includes a polypeptide comprising amino acids X to 322 of FIG. 38 (SEQ ID NO: 72), wherein X is amino acid 21 to SEQ ID NO: Any amino acid from 31) and variants thereof. Also included in the present invention are isolated nucleic acid molecules encoding such polypeptides.
[440] Another aspect of the invention provides an isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a PRO6030 polypeptide in which the transmembrane domain is deleted or inactivated, wherein the transmembrane domain is shown in FIG. 38 ( It was experimentally confirmed that the amino acid position spans from about 142 to about 158 in the sequence of SEQ ID NO: 72). Thus, the soluble extracellular domain of the PRO6030 polypeptides described herein is under consideration.
[441] In this regard, another aspect of the present invention relates to (a) a DNA molecule encoding amino acids 1 to X of Figure 38 (SEQ ID NO: 72), wherein X is any amino acid from amino acids 137 to 147 of Figure 38 (SEQ ID NO: 72) Or (b) at least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably with the complement of the DNA molecule of (a) Preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89% At least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, even more preferably Is at least about 95%, more preferably at least about 96%, more preferably about 97 An isolated nucleic acid molecule comprising a nucleotide sequence having at least%, more preferably at least about 98%, more preferably at least about 99% sequence identity. In certain aspects, an isolated nucleic acid molecule is (a) a nucleotide sequence encoding amino acids 1-X of FIG. 38 (SEQ ID NO: 72), where X is any amino acid from amino acids 137-147 of FIG. 38 (SEQ ID NO: 72). Or (b) the complement of the DNA molecule of (a).
[442] In another aspect of the invention, the isolated nucleic acid molecule is (a) at least about 80%, preferably at least about 81%, more preferably compared to the amino acid sequence of residues about 1 to X of FIG. 38 (SEQ ID NO: 72) Is at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87% , More preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, even more preferably At least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, More preferably, at least about 99% of the polls are recorded as positive Peptide is the complement of the DNA molecule of (where, X is any amino acid from amino acid 137 to 147 of Figure 38 (SEQ ID NO: 72)) coding, or (b) wherein (a) the.
[443] Another embodiment encodes a PRO6030 polypeptide that can be used to encode a fragment of a PRO6030 polypeptide, for example, which can be used as a hybridization probe or, optionally, a polypeptide comprising a binding site for an anti-PRO6030 antibody. To a fragment of the sequence. The length of such nucleic acid fragments is generally at least about 20 nucleotides, preferably at least about 30, more preferably at least about 40, more preferably at least about 50, more preferably at least about 60, more Preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 100, more preferably at least about 110, more preferably about 120 At least about 130, more preferably at least about 140, more preferably at least about 140, more preferably at least about 150, more preferably at least about 160, more preferably at least about 170, more preferred Preferably at least about 180, more preferably at least about 190, more preferably at least about 200, more preferably at least about 250, more preferably at least about 300, and more preferably about 350 More preferably about 400 , More preferably about 450 or more, more preferably about 500 or more, more preferably about 600 or more, more preferably about 700 or more, more preferably about 800 or more, more preferably At least about 900, more preferably at least about 1000, wherein the term "about" refers to the length of the nucleotide sequence mentioned above plus or minus 10% of this length. In a preferred embodiment, the nucleotide sequence fragment is derived from a specific coding region of the nucleotide sequence shown in FIG. 37 (SEQ ID NO: 71). New fragments of the PRO6030 polypeptide-encoding nucleotide sequence align the PRO6030 polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well-known sequence alignment programs to determine whether the PRO6030 polypeptide-encoding nucleotide sequence fragment is new. It can be determined by the usual way of determining. All such PRO6030 polypeptide-coding nucleotide sequences are considered herein and can be determined without undue experimentation. Also contemplated are PRO6030 polypeptide fragments encoded by this nucleotide molecule fragment, preferably PRO6030 polypeptide fragments comprising a binding site for an anti-PRO6030 antibody.
[444] In another embodiment, the present invention provides an isolated PRO6030 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[445] In certain aspects, the invention provides an isolated native sequence PRO6030 polypeptide, in some embodiments, the polypeptide comprises an amino acid sequence comprising about 1 or about 27 to about 322 residues of FIG. 38 (SEQ ID NO: 72).
[446] In another aspect, the present invention provides a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72) and at least about 80%, preferably at least about 81%, more preferably at least about 82%, More preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably about At least 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more Preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably about 99 Comprising amino acid sequences having at least% sequence identity Ridoen relates to PRO6030 polypeptides.
[447] In another aspect, the invention provides an amino acid sequence that is encoded by the human protein cDNA deposited with ATCC Accession No. 479-PTA (DNA96850-2705) to ATCC on August 3, 1999, preferably at least about 80%. At least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, More preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably about At least 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more Preferably at least about 98%, more preferably at least about 99% The isolated PRO6030 comprising amino acid sequence having a sequence identity relates to the polypeptide. In a preferred embodiment, the isolated PRO6030 polypeptide comprises an amino acid sequence encoded by human protein cDNA deposited with ATCC Accession No. 479-PTA (DNA96850-2705) to ATCC on August 3, 1999.
[448] In another aspect, the invention provides at least about 80%, preferably at least about 81%, more preferably about 82% when compared to the residue about 1 or about 27 to about 322 amino acid sequence of FIG. 38 (SEQ ID NO: 72). Or more, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably Is at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93% , More preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, even more preferably Amino acid standing at about 99% positive It relates to an isolated PRO6030 polypeptide comprising a row.
[449] In certain aspects, the invention provides an isolated PRO6030 polypeptide encoded by a nucleotide sequence encoding an amino acid sequence as described above, and having no N-terminal signal sequence and / or initiating methionine. Also described herein is a method of preparing the isolated PRO6030 polypeptide, which method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the PRO6030 polypeptide, from the cell culture. Recovering the polypeptide.
[450] In another aspect, the invention provides an isolated PRO6030 polypeptide in which the transmembrane domain is deleted or inactivated. Also described herein is a method of making such a polypeptide, wherein the method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the PRO6030 polypeptide, and the PRO6030 polypeptide from the cell culture. Recovery is included.
[451] As such, one aspect of the invention is at least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably about 83% with amino acids 1-X of FIG. 38 (SEQ ID NO: 72). Or more, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably Is at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94% , More preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99% Isolated soluble PRO6030 poly comprising sequence Suited relates to (wherein, X is any amino acid from amino acid 137 to 147 of Figure 38 (SEQ ID NO: 72)). In a preferred aspect, the isolated soluble PRO6030 polypeptide comprises amino acids 1-X of FIG. 38 (SEQ ID NO: 72), wherein X is any amino acid from amino acids 137-147 of FIG. 38 (SEQ ID NO: 72).
[452] In another aspect of the invention, the isolated soluble PRO6030 polypeptide is at least about 80%, preferably at least about 81%, and more preferably about 80% as compared to the amino acid sequence of residues about 1 to X of FIG. 38 (SEQ ID NO: 72). At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably about 93 At least%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, even more preferred More than 99% are positive It comprises the amino acid sequence of the lock (where, X is any amino acid from amino acid 137 to 147 of Figure 38 (SEQ ID NO: 72)).
[453] In another aspect, the invention provides an isolated PRO6030 polypeptide comprising a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72), or a binding site for a biologically active or anti-PRO6030 antibody. To fragments thereof sufficient to provide, wherein PRO6030 polypeptide fragments retaining biological activity or providing a binding site for an anti-PRO6030 antibody can be identified by conventional methods using techniques well known in the art. Preferably, the PRO6030 fragment retains the qualitative biological activity of the native PRO6030 polypeptide.
[454] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO6030 polypeptide having a sequence of about 1 or about 27 to about 322 amino acid residues in FIG. 38 (SEQ ID NO: 72) or (b) hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 81%, more preferably about (a) or (b) At least 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more Preferably at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably about 93 At least%, more preferably at least about 94%, more preferably at least about 95% And, more preferably, at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably at least about 99%, of sequence identity (ii) comprising a test DNA molecule Culturing the host cell under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
[455] In another embodiment, the present invention relates to a composition comprising, together with a carrier, an agonist or antagonist of a PRO6030 polypeptide or a PRO6030 polypeptide as described herein. Optionally, the carrier is a pharmaceutically acceptable carrier.
[456] Another embodiment of the present invention provides for the preparation of a PRO6030 polypeptide, an agonist or antagonist thereof, or a drug useful for the treatment of symptoms that respond to an anti-PRO6030 antibody, the PRO6030 polypeptide, an agonist or antagonist thereof as described above, Or to the use of an anti-PRO6030 antibody.
[457] 20.PRO4424
[458] We have found a cDNA clone (DNA96857-2636) that encodes a novel polypeptide (named "PRO4424" herein) having homology with the protein of Genbank Accession No. HGS_A135.
[459] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4424 polypeptide.
[460] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 40 (SEQ ID NO: 74) or a PRO4424 polypeptide having a sequence from about 29 to about 221, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[461] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4424 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 136 and about 714 of FIG. 39 (SEQ ID NO: 73). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[462] In another aspect, the invention provides a DNA molecule encoding (a) the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 17-PTA (DNA96857-2636) or (b) the DNA of (a) above. To an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95% . In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 17-PTA (DNA96857-2636).
[463] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 29 to about 221 of FIG. 40 (SEQ ID NO: 74), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[464] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides, and, under stringent conditions, a test DNA molecule is (a) sequenced from about 29 to about 221 amino acid residues in FIG. 40 (SEQ ID NO: 74). A DNA molecule encoding a PRO4424 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[465] In another aspect, the invention provides (a) at least about 80%, preferably at least about 85%, more preferably about 90% as compared to the amino acid sequence of residues about 29 to about 221 of FIG. 40 (SEQ ID NO: 74). Above, most preferably, it relates to an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[466] Another embodiment is directed to a fragment of the PRO4424 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[467] In another embodiment, the present invention provides an isolated PRO4424 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[468] In certain aspects, the invention provides an isolated native sequence PRO4424 polypeptide, which in one embodiment comprises an amino acid sequence comprising residues 29-221 of FIG. 40 (SEQ ID NO: 74).
[469] In another aspect, the invention relates to the sequence of amino acid residues 29 to about 221 of Figure 40 (SEQ ID NO: 74) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4424 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[470] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 29 to 221 of FIG. 40 (SEQ ID NO: 74). An isolated PRO4424 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[471] In another aspect, the invention relates to an isolated PRO4424 polypeptide comprising the sequence of amino acid residues 29 to about 221 of FIG. 40 (SEQ ID NO: 74), or a fragment thereof sufficient to provide a binding site for an anti-PRO4424 antibody. . Preferably, the PRO4424 fragment retains the qualitative biological activity of the native PRO4424 polypeptide.
[472] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4424 polypeptide having a sequence from about 29 to about 221 amino acid residues in FIG. 40 (SEQ ID NO: 74), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[473] 21.PRO4422
[474] We have found a cDNA clone (DNA96867-2620) that encodes a novel polypeptide (designated herein as "PRO4422") that is homologous to lysozyme g.
[475] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4422 polypeptide.
[476] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 42 (SEQ ID NO: 76) or a PRO4422 polypeptide having a sequence from about 20 to about 194, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[477] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4422 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 375 and about 899 of FIG. 41 (SEQ ID NO: 75). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[478] In another aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by (a) the human protein cDNA of ATCC Accession No. 203972 (DNA96867-2620) or (b) a DNA molecule of (a) above. An isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 95%. In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 203972 (DNA96867-2620).
[479] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the sequence of amino acid residues from about 20 to about 194 of FIG. Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[480] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) a sequence of about 20 to about 194 amino acid residues in FIG. A DNA molecule encoding a PRO4422 polypeptide having or (b) a complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[481] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90% when compared to the amino acid sequence of residues 20 to about 194 in FIG. 42 (SEQ ID NO: 76). , And most preferably, an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[482] Another embodiment relates to fragments of the PRO4422 polypeptide coding sequence that can be used as hybridization probes. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[483] In another embodiment, the present invention provides an isolated PRO4422 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[484] In certain aspects, the invention provides an isolated native sequence PRO4422 polypeptide, in one embodiment, the polypeptide comprises an amino acid sequence comprising residues 20 to about 194 of FIG. 42 (SEQ ID NO: 76).
[485] In another aspect, the present invention provides the sequence of amino acid residues 20-194 of Figure 42 (SEQ ID NO: 76) with at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably about An isolated PRO4422 polypeptide comprising an amino acid sequence having at least 95% sequence identity.
[486] In another aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequence of residues 20-194 of Figure 42 (SEQ ID NO: 76). An isolated PRO4422 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[487] In another aspect, the invention relates to an isolated PRO4422 polypeptide comprising the sequence of amino acid residues 20 to about 194 of FIG. 42 (SEQ ID NO: 76), or a fragment thereof sufficient to provide a binding site for an anti-PRO4422 antibody. . Preferably, the PRO4422 fragment retains the qualitative biological activity of the native PRO4422 polypeptide.
[488] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4422 polypeptide having a sequence from about 20 to about 194 amino acid residues in FIG. 42 (SEQ ID NO: 76), or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[489] 22.PRO4430
[490] We have found a cDNA clone (DNA96878-2626) encoding a novel polypeptide (named "PRO4430" herein) having homology with the protein of Genbank Accession No. MMHC213L3_9.
[491] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4430 polypeptide.
[492] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 44 (SEQ ID NO: 78) or a PRO4430 polypeptide having a sequence from about 19 to about 125, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[493] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4430 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 110 and about 430 of FIG. 43 (SEQ ID NO: 77). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[494] In another aspect, the invention provides a DNA molecule encoding (a) the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 23-PTA (DNA96878-2626) or (b) the DNA of (a) above. To an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% . In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 23-PTA (DNA96878-2626).
[495] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, amino acid residues from about 19 to about 125 of the amino acid residues of FIG. 44 (SEQ ID NO: 78), Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[496] In another aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, a test DNA molecule is selected from (a) the sequence of amino acid residues from about 19 to about 125 in FIG. 44 (SEQ ID NO: 78). A DNA molecule encoding a PRO4430 polypeptide having or (b) a complement of the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85%, with (a) or (b) Or more preferably, at least about 90%, most preferably at least about 95%, to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[497] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90% when compared to the amino acid sequence of residues 19 to about 125 of FIG. 44 (SEQ ID NO: 78). , And most preferably, an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[498] Another embodiment is directed to a fragment of the PRO4430 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[499] In another embodiment, the present invention provides an isolated PRO4430 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[500] In certain aspects, the invention provides an isolated native sequence PRO4430 polypeptide, in one embodiment, the polypeptide comprises an amino acid sequence comprising residues 19-125 of FIG. 44 (SEQ ID NO: 78).
[501] In another aspect, the invention relates to the sequence of amino acid residues 19 to about 125 of Figure 44 (SEQ ID NO: 78) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4430 polypeptide comprising an amino acid sequence having at least about 95% sequence identity.
[502] In a further aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably, as compared to the amino acid sequence of residues 19 to about 125 of Figure 44 (SEQ ID NO: 78). Preferably, it relates to an isolated PRO4430 polypeptide comprising an amino acid sequence wherein at least about 95% is recorded as positive.
[503] In another aspect, the invention relates to an isolated PRO4430 polypeptide comprising the sequence of amino acid residues 19 to about 125 of FIG. 44 (SEQ ID NO: 78), or a fragment thereof sufficient to provide a binding site for an anti-PRO4430 antibody. . Preferably, the PRO4430 fragment retains the qualitative biological activity of the native PRO4430 polypeptide.
[504] In another aspect, the invention provides an antibody comprising (i) a DNA molecule encoding a test DNA molecule under stringent conditions (a) a PRO4430 polypeptide having a sequence from about 19 to about 125 amino acid residues in Figure 44 (SEQ ID NO: 78) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[505] 23.PRO4499
[506] We have found a cDNA clone (DNA96889-2641) encoding a novel polypeptide (designated herein as "PRO4499").
[507] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO4499 polypeptide.
[508] In one aspect, the isolated nucleic acid is (a) a DNA molecule encoding amino acid residue 1 of Figure 46 (SEQ ID NO: 80) or a PRO4499 polypeptide having a sequence from about 31 to about 339, or (b) the DNA molecule of (a) above. And at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% of DNA with complement.
[509] In another aspect, the invention relates to an isolated nucleic acid molecule encoding a PRO4499 polypeptide comprising DNA that hybridizes with the complement of a nucleic acid between residues about 275 and about 1201 of FIG. 45 (SEQ ID NO: 79). Preferably, hybridization occurs under stringent hybridization and washing conditions.
[510] In a further aspect, the invention provides a DNA molecule encoding (a) the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 119-PTA (DNA96889-2641) or (b) the DNA of (a) above. To an isolated nucleic acid molecule comprising DNA having a sequence identity of at least about 80%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% . In a preferred embodiment, the nucleic acid comprises DNA encoding the same mature polypeptide encoded by the human protein cDNA of ATCC Accession No. 119-PTA (DNA96889-2641).
[511] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90%, of the amino acid residues of FIG. 46 (SEQ ID NO: 80) with the sequence of about 31 to about 339 Most preferably it relates to an isolated nucleic acid molecule comprising DNA encoding a polypeptide having at least about 95% sequence identity or (b) the complement of the DNA of (a).
[512] In a further aspect, the present invention has at least about 50, preferably at least about 100 nucleotides and, under stringent conditions, the test DNA molecule (a) has a sequence of about 31 to about 339 amino acid residues in FIG. A DNA molecule encoding a PRO4499 polypeptide having: or (b) a complement of the complement of the DNA molecule of (a), wherein the test DNA molecule is at least about 80%, preferably about 85, with (a) or (b) When it has at least%, more preferably at least about 90%, most preferably at least about 95% sequence identity, it relates to an isolated nucleic acid molecule produced by isolating test DNA molecules.
[513] In certain aspects, the invention includes, or is, DNA encoding a PRO4499 polypeptide and its soluble variant (ie, the transmembrane domain is deleted or inactivated) with or without an N-terminal signal sequence and / or initiating methionine. An isolated nucleic acid molecule is provided that is complementary to a coding nucleic acid molecule. By experiment, the signal peptide was found to span about 30 from amino acid position 1 in the sequence of FIG. 46 (SEQ ID NO: 80). By experiment, the transmembrane domain was found to span the amino acid position about 171 to about 190 in the PRO4499 amino acid sequence (FIG. 46, SEQ ID NO: 80).
[514] In another aspect, the invention provides an antibody comprising (a) at least about 80%, preferably at least about 85%, more preferably at least about 90% as compared to the amino acid sequence of residues 31 to about 339 in FIG. 46 (SEQ ID NO: 80). , And most preferably, an isolated nucleic acid molecule comprising a DNA encoding a polypeptide wherein at least about 95% is recorded as positive or (b) the complement of the DNA of (a).
[515] Another embodiment is directed to a fragment of the PRO4499 polypeptide coding sequence that can be used as a hybridization probe. The nucleic acid fragment is about 20 to about 80 nucleotides, preferably about 20 to about 60 nucleotides, more preferably about 20 to about 50 nucleotides, and most preferably about 20 to about 40 nucleotides in length.
[516] In another embodiment, the present invention provides an isolated PRO4499 polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[517] In certain aspects, the invention provides an isolated native sequence PRO4499 polypeptide, which in one embodiment comprises the amino acid sequence comprising residues 31 to 339 of FIG. 46 (SEQ ID NO: 80).
[518] In another aspect, the invention relates to the sequence of amino acid residues 31 to about 339 of Figure 46 (SEQ ID NO: 80) and at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably An isolated PRO4499 polypeptide comprising amino acid sequence having at least about 95% sequence identity.
[519] In a further aspect, the present invention provides at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably as compared to the amino acid sequences of residues 31 to 339 in FIG. 46 (SEQ ID NO: 80). Relates to an isolated PRO4499 polypeptide comprising an amino acid sequence wherein at least about 95% are recorded as positive.
[520] In another aspect, the invention relates to an isolated PRO4499 polypeptide comprising the sequence of amino acid residues 31 to about 339 of FIG. 46 (SEQ ID NO: 80), or a fragment thereof sufficient to provide a binding site for an anti-PRO4499 antibody. . Preferably, the PRO4499 fragment retains the qualitative biological activity of the native PRO4499 polypeptide.
[521] In another aspect, the invention provides an antibody comprising (i) a test DNA molecule under stringent conditions (a) a DNA molecule encoding a PRO4499 polypeptide having a sequence from about 31 to about 339 amino acid residues in FIG. 46 (SEQ ID NO: 80) or (b) Hybridizing with the complement of the DNA molecule of (a), and wherein the test DNA molecule is at least about 80%, preferably at least about 85%, more preferably at least about 90% with (a) or (b) (Ii) culturing the host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, most preferably having at least about 95% sequence identity, and (iii) recovering the polypeptide from the cell culture. It provides a polypeptide produced by the step.
[522] 24. Additional Embodiments
[523] In another embodiment of the invention, the invention provides a vector comprising a DNA encoding any of the polypeptides herein. Also provided are host cells comprising such vectors. By way of example, the host cell may be a CHO cell, E. coli. E. coli or yeast. Also provided is a method of producing any of the polypeptides described herein, which method comprises culturing the host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
[524] In another embodiment, the present invention provides chimeric molecules comprising any polypeptide described herein that is fused with a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include any polypeptide described herein that is fused with an epitope tag sequence or an F _c region of an immunoglobulin.
[525] In another embodiment, the present invention provides antibodies that specifically bind to any of the above or below polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single chain antibody.
[526] In another embodiment, the present invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences or as antisense probes, which probes can be derived from any of the above or below nucleotide sequences.
[527] In another embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide.
[528] In one aspect, an isolated nucleic acid molecule comprises (a) an extracellular amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, or with or without a signal peptide as disclosed herein. At least about 80%, or about 81%, of a DNA molecule encoding a PRO polypeptide having a domain or any other fragment specifically defined as a full-length amino acid sequence as disclosed herein; or (b) the complement of the DNA molecule of (a). At least%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89% Or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or At least about 98%, or at least about 99% sequence It comprises a nucleotide sequence having a sealability.
[529] In another aspect, an isolated nucleic acid molecule comprises (a) a coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, a coding sequence of a PRO polypeptide lacking a signal peptide as disclosed herein, or a signal peptide as disclosed herein A DNA molecule comprising a coding sequence of an extracellular domain of a transmembrane PRO polypeptide that is absent or of any other fragment specifically defined as a full-length amino acid sequence as disclosed herein or (b) complementary to the DNA molecule of (a) above At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or At least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or about 96 At least%, or at least about 97%, It comprises a nucleotide sequence having about 98% or more, or at least about 99% nucleic acid sequence identity.
[530] In a further aspect, the invention relates to a DNA molecule encoding the same mature polypeptide encoded by any of (a) any of the human protein cDNA deposited with the ATCC as disclosed herein, or (b) a DNA molecule of (a) above. At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, with the complement, Or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or about An isolated nucleic acid molecule comprising a nucleotide sequence having at least 96%, or at least about 97%, or at least about 98%, or at least about 99% nucleic acid sequence identity.
[531] In another aspect, the invention provides an isolated nucleic acid molecule comprising or complementary to a nucleotide sequence encoding a PRO polypeptide in which a transmembrane domain is deleted or inactivated, wherein the transmembrane of the polypeptide is provided herein. The domain is disclosed. Thus, the soluble extracellular domain of the PRO polypeptides described herein is under consideration.
[532] Another embodiment is for example an antisense oligonucleotide probe, or for use as a hybridization probe, optionally for encoding a fragment of a PRO polypeptide that can encode a polypeptide comprising a binding site for an anti-PRO antibody. It relates to a fragment of the PRO polypeptide coding sequence or the complement thereof that can be used as. Such nucleic acid fragments generally have a length of at least about 20 nucleotides, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, Or at least about 90, or at least about 100, or at least about 110, or at least about 120, or at least about 130, or at least about 140, or at least about 150, or at least about 160, or about At least 170, or at least about 180, or at least about 190, or at least about 200, or at least about 250, or at least about 300, or at least about 350, or at least about 400, or about 450 Or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, wherein the term "about" refers to a nucleotide sequence The length plus or minus 10% of this length. A novel fragment of a PRO polypeptide-encoding nucleotide sequence can be used to align the PRO polypeptide-coding nucleotide sequence with other known nucleotide sequences using any of a number of well-known sequence alignment programs to determine whether the PRO polypeptide-coding nucleotide sequence fragment is new. It can be determined by the usual way of determining. All such PRO polypeptide-coding nucleotide sequences are considered herein. Also contemplated are PRO polypeptide fragments encoded by this nucleotide molecule fragment, preferably PRO polypeptide fragments comprising a binding site for an anti-PRO antibody.
[533] In another embodiment, the present invention provides an isolated PRO polypeptide encoded by any of the isolated nucleic acid sequences identified above.
[534] In certain aspects, the invention provides a full length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, an extracellular domain of a transmembrane protein with or without a signal peptide as disclosed herein, or as disclosed herein. At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or about 85, with a PRO polypeptide having any other fragment specifically defined as the full length amino acid sequence At least%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93% Or an isolated PRO polypeptide comprising an amino acid sequence having at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.One will.
[535] In a further aspect, the invention provides at least about 80%, or at least about 81%, or at least about 82%, or about 83 amino acid sequences encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein At least%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91% Or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity An isolated PRO polypeptide comprising an amino acid sequence having
[536] In a further aspect, the invention provides a full length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, an extracellular domain of a transmembrane protein with or without a signal peptide as disclosed herein or At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or about as compared to the amino acid sequence of a PRO polypeptide having any other fragment specifically defined as a full length amino acid sequence as disclosed in At least 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or about 92% At least, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% are recorded amino acid sequences It relates to an isolated PRO polypeptide comprising.
[537] In certain aspects, the invention provides an isolated PRO polypeptide encoded by a nucleotide sequence encoding an amino acid sequence as described above, and having no N-terminal signal sequence and / or initiating methionine. Also described herein is a method of making the isolated PRO polypeptide, wherein the method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and removing the PRO from the cell culture. Recovering the polypeptide.
[538] In another aspect, the invention provides an isolated PROpolypeptide having a deletion or inactivation of a transmembrane domain. Also described herein is a method of making the isolated PRO polypeptide, wherein the method comprises culturing a host cell comprising a vector comprising a suitable coding nucleic acid molecule under conditions suitable for expression of the PRO polypeptide and removing the PRO from the cell culture. Recovering the polypeptide.
[539] In another embodiment, the present invention relates to agonists and antagonists of natural PRO polypeptides as defined above. In certain embodiments, the agonist or antagonist is an anti-PRO antibody or small molecule.
[540] In a further embodiment, the invention relates to a method of identifying an agonist or antagonist for a PRO polypeptide, comprising contacting the PRO polypeptide with a candidate molecule and monitoring the biological activity mediated by the PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
[541] In another embodiment, the invention relates to a composition comprising an agonist or antagonist of a PRO polypeptide or a PRO polypeptide as described herein, or an anti-PRO antibody with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
[542] Another embodiment of the present invention provides for the manufacture of a PRO polypeptide, an agonist or antagonist thereof, or a drug useful for the treatment of a condition responsive to an anti-PRO antibody, wherein the PRO polypeptide, an agonist or antagonist as described above, Or to the use of an anti-PRO antibody.
[1] 1 shows the nucleotide sequence of the native sequence PRO1484 cDNA (SEQ ID NO: 1), wherein SEQ ID NO: 1 is a clone designated herein as "DNA44686-1653".
[2] 2 shows an amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: 1 shown in FIG.
[3] 3 shows the nucleotide sequence of the native sequence PRO4334 cDNA (SEQ ID NO: 8), wherein SEQ ID NO: 8 is a clone designated herein as “DNA59608-2577”.
[4] 4 shows an amino acid sequence (SEQ ID NO: 9) derived from the coding sequence of SEQ ID NO: 8 shown in FIG.
[5] 5 shows the nucleotide sequence of the native sequence PRO1122 cDNA (SEQ ID NO: 10), wherein SEQ ID NO: 10 is a clone designated herein as "DNA62377-1381".
[6] FIG. 6 shows an amino acid sequence (SEQ ID NO: 11) derived from the coding sequence of SEQ ID NO: 10 shown in FIG.
[7] FIG. 7 shows the nucleotide sequence of the native sequence PRO1889 cDNA (SEQ ID NO: 15), wherein SEQ ID NO: 15 is a clone designated herein as “DNA77623-2524”.
[8] 8 shows an amino acid sequence (SEQ ID NO: 16) derived from the coding sequence of SEQ ID NO: 15 shown in FIG.
[9] 9 shows the nucleotide sequence of the native sequence PRO1890 cDNA (SEQ ID NO: 17), wherein SEQ ID NO: 17 is a clone designated herein as "DNA79230-2525".
[10] FIG. 10 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 17 shown in FIG. 9 (SEQ ID NO: 18).
[11] 11 shows the nucleotide sequence of the native sequence PRO1887 cDNA (SEQ ID NO: 22), wherein SEQ ID NO: 22 is a clone designated herein as "DNA79862-2522".
[12] FIG. 12 shows an amino acid sequence (SEQ ID NO: 23) derived from the coding sequence of SEQ ID NO: 22 shown in FIG.
[13] FIG. 13 shows the nucleotide sequence of the native sequence PRO1785 cDNA (SEQ ID NO: 28), wherein SEQ ID NO: 28 is a clone designated herein as “DNA80136-2503”.
[14] FIG. 14 shows an amino acid sequence (SEQ ID NO: 29) derived from the coding sequence of SEQ ID NO: 28 shown in FIG.
[15] 15 shows the nucleotide sequence of the native sequence PRO4353 cDNA (SEQ ID NO: 34), wherein SEQ ID NO: 34 is a clone designated herein as "DNA80145-2594".
[16] FIG. 16 shows an amino acid sequence (SEQ ID NO: 35) derived from the coding sequence of SEQ ID NO: 34 shown in FIG.
[17] FIG. 17 shows the nucleotide sequence of the native sequence PRO4357 cDNA (SEQ ID NO: 39), wherein SEQ ID NO: 39 is a clone designated herein as “DNA84917-2597”.
[18] FIG. 18 shows an amino acid sequence (SEQ ID NO: 40) derived from the coding sequence of SEQ ID NO: 39 shown in FIG.
[19] FIG. 19 shows the nucleotide sequence of the native sequence PRO4405 cDNA (SEQ ID NO: 44), wherein SEQ ID NO: 44 is a clone designated herein as “DNA84920-2614”.
[20] FIG. 20 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 44 shown in FIG. 19 (SEQ ID NO: 45).
[21] 21 shows the nucleotide sequence of the native sequence PRO4356 cDNA (SEQ ID NO: 49), wherein SEQ ID NO: 49 is a clone designated herein as "DNA86576-2595".
[22] FIG. 22 shows an amino acid sequence (SEQ ID NO: 50) derived from the coding sequence of SEQ ID NO: 49 shown in FIG. 21.
[23] FIG. 23 shows the nucleotide sequence of the native sequence PRO4352 cDNA (SEQ ID NO: 51), wherein SEQ ID NO: 51 is a clone designated herein as “DNA87976-2593”.
[24] FIG. 24 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 51 shown in FIG. 23 (SEQ ID NO: 52).
[25] 25 shows the nucleotide sequence of the native sequence PRO4380 cDNA (SEQ ID NO: 56), wherein SEQ ID NO: 56 is a clone designated herein as "DNA92234-2602".
[26] FIG. 26 shows an amino acid sequence (SEQ ID NO: 57) derived from the coding sequence of SEQ ID NO: 56 shown in FIG.
[27] FIG. 27 shows the nucleotide sequence of the native sequence PRO4354 cDNA (SEQ ID NO: 58), wherein SEQ ID NO: 58 is a clone designated herein as “DNA92256-2596”.
[28] FIG. 28 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 58 shown in FIG. 27 (SEQ ID NO: 59).
[29] 29 shows the nucleotide sequence of the native sequence PRO4408 cDNA (SEQ ID NO: 60), wherein SEQ ID NO: 60 is a clone designated herein as “DNA92274-2617”.
[30] FIG. 30 shows an amino acid sequence (SEQ ID NO: 61) derived from the coding sequence of SEQ ID NO: 60 shown in FIG. 29.
[31] FIG. 31 shows the nucleotide sequence of the native sequence PRO5737 cDNA (SEQ ID NO: 62), wherein SEQ ID NO: 62 is a clone designated herein as “DNA92929-2534”.
[32] FIG. 32 shows an amino acid sequence (SEQ ID NO: 63) derived from the coding sequence of SEQ ID NO: 62 shown in FIG. 31.
[33] FIG. 33 shows the nucleotide sequence of the native sequence PRO4425 cDNA (SEQ ID NO: 64), wherein SEQ ID NO: 64 is a clone designated herein as “DNA93011-2637”.
[34] FIG. 34 shows the amino acid sequence derived from the coding sequence of SEQ ID NO: 64 shown in FIG. 33 (SEQ ID NO: 65).
[35] 35 shows the nucleotide sequence of the native sequence PRO5990 cDNA (SEQ ID NO: 66), wherein SEQ ID NO: 66 is a clone designated herein as "DNA96042-2682".
[36] FIG. 36 shows the amino acid sequence derived from the coding sequence of SEQ ID NO: 66 shown in FIG. 35 (SEQ ID NO: 67).
[37] FIG. 37 shows the nucleotide sequence of the native sequence PRO6030 cDNA (SEQ ID NO: 71), wherein SEQ ID NO: 71 is a clone designated herein as "DNA96850-2705".
[38] FIG. 38 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 71 shown in FIG. 37 (SEQ ID NO: 72).
[39] 39 shows the nucleotide sequence of the native sequence PRO4424 cDNA (SEQ ID NO: 73), wherein SEQ ID NO: 73 is a clone designated herein as "DNA96857-2636".
[40] FIG. 40 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 73 shown in FIG. 39 (SEQ ID NO: 74).
[41] FIG. 41 shows the nucleotide sequence of the native sequence PRO4422 cDNA (SEQ ID NO: 75), wherein SEQ ID NO: 75 is a clone designated herein as “DNA96867-2620”.
[42] FIG. 42 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 75 shown in FIG. 41 (SEQ ID NO: 76).
[43] 43 shows the nucleotide sequence of the native sequence PRO4430 cDNA (SEQ ID NO: 77), wherein SEQ ID NO: 77 is a clone designated herein as "DNA96878-2626".
[44] FIG. 44 shows an amino acid sequence derived from the coding sequence of SEQ ID NO: 77 shown in FIG. 43 (SEQ ID NO: 78).
[45] 45 shows the nucleotide sequence of the native sequence PRO4499 cDNA (SEQ ID NO: 79), wherein SEQ ID NO: 79 is a clone designated herein as "DNA96889-2641".
[46] FIG. 46 shows the amino acid sequence derived from the coding sequence of SEQ ID NO: 79 shown in FIG. 45 (SEQ ID NO: 80).
[543] <Detailed Description of the Preferred Embodiments>
[544] I. Justice
[545] The terms "PRO polypeptide" and "PRO" as used herein refer to a variety of polypeptides, followed immediately by numbers, where the full name (ie, PRO / number) refers to the specific polypeptide sequence described herein. As used herein, "PRO / numeric polypeptide" and "PRO / numeric", where the numerical portion is represented by actual numbers, are terms that include native sequence polypeptides and polypeptide variants (as further defined herein). The PRO polypeptides described herein can be isolated from a variety of sources, such as human tissue types or other sources, or can be prepared by recombinant or synthetic methods.
[546] "Native sequence PRO polypeptide" includes polypeptides having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be prepared by recombinant or synthetic methods. The term “natural sequence PRO polypeptide” specifically refers to a naturally-occurring variant form of such polypeptide, in which the naturally-occurring terminus of the particular PRO polypeptide is truncated or secreted (eg, extracellular domain sequence). (Eg, alternatively spliced forms) and naturally-occurring allelic variants. In various embodiments of the invention, the native sequence PRO polypeptides described herein are mature or full length native sequence polypeptides comprising the full length amino acid sequences shown in the accompanying figures. Start and stop codons are indicated in bold and underlined in the figures. However, although the PRO polypeptides disclosed in the accompanying figures herein are shown to begin with methionine residues referred to in amino acid position 1 in the figures, other methionine residues located above or below amino acid position 1 in the figures may begin to the PRO polypeptide. It is conceivable that it can be used as an amino acid residue, and is possible.
[547] An “extracellular domain” or “ECD” of a PRO polypeptide refers to a form of PRO polypeptide that is essentially free of transmembrane and cytoplasmic domains. Typically, the PRO polypeptide ECD comprises less than 1% of the transmembrane and / or cytoplasmic domains, preferably less than 0.5% of these domains. All transmembrane domains identified in the PRO polypeptides of the present invention have been identified according to criteria commonly used to identify hydrophobic domain types in the art. The exact boundaries of the transmembrane domain may vary, but most are only present at about 5 amino acids at each end of the domain, as initially identified herein. Thus, the extracellular domain of the PRO polypeptide may optionally include about 5 or less amino acids on either side of the transmembrane domain / extracellular domain boundary identified in the Examples and the specification, with or without a signal peptide. Such polypeptides, and nucleic acids encoding them, are included in the present invention.
[548] The approximate location of the “signal peptides” of the various PRO polypeptides disclosed herein are shown in the accompanying figures. Although the C-terminal boundary of the signal peptide may be different, most are only at about 5 amino acids on either side of the C-terminal boundary of the signal peptide, where the C-terminal boundary of the signal peptide is known in the art. In accordance with criteria commonly used to identify the type of amino acid sequence member in Nielsen et al ., Prot. Eng. 10: 1-6 (1997) and Heinje et al. Nucl. Acids. Res. 14: 4683-4690 (1986)]. In addition, in some cases, it is believed that the cleavage of the signal sequence of the secreted polypeptides is not entirely identical, resulting in one or more secreted polypeptides. As identified herein, mature polypeptides in which the signal peptide is cleaved only at about 5 amino acids on either side of the C-terminal boundary of the signal peptide, and polynucleotides encoding them, are included in the present invention.
[549] A "PRO polypeptide variant" refers to a full-length native sequence PRO polypeptide sequence disclosed herein, a PRO polypeptide sequence lacking a signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without signal peptide as disclosed herein, or By active PRO polypeptide as defined above or below, having at least about 80% amino acid sequence identity with any other fragment of the disclosed full length PRO polypeptide sequence. Such PRO polypeptide variants include, for example, PRO polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the full-length natural amino acid sequence. Typically, a PRO polypeptide variant is a full length native sequence PRO polypeptide sequence disclosed herein, a PRO polypeptide sequence without a signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without signal peptide as disclosed herein, or At least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83%, more preferably with any other specifically defined fragment of the full-length PRO polypeptide sequence as disclosed Is at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, more preferably at least about 89% , More preferably at least about 90%, more preferably at least about 91%, more preferably at least 92%, more preferably at least about 93%, more preferably At least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, most preferably at least about 99% Has sequence identity. Typically, PRO variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids, more often at least about 30 amino acids, more often at least about 40 amino acids, more often at least about 50 amino acids, More often at least about 60 amino acids, more often at least about 70 amino acids, more often at least about 80 amino acids, more often at least about 90 amino acids, more often at least about 100 amino acids, more often about 150 At least about amino acids, more often at least about 200 amino acids, more often at least about 300 amino acids or more.
[550] “% Amino acid sequence identity” for a PRO polypeptide sequence as disclosed herein is intended to align candidate sequences that have identical amino acid residues to a particular PRO polypeptide sequence and, if necessary, introduce any gaps after introducing a gap to obtain a maximum of percent sequence identity. Proper substitution is also defined as the proportion of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular PRO polypeptide sequence without being considered part of the same sequence. Alignment to determine amino acid sequence identity can be accomplished using various methods in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine suitable parameters for measuring alignment, including any algorithms needed to maximally align over the entire length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, where the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 is kept as a user document of the U.S. Copyright Office (20559 Washington, DC). Registered under US Copyright Registration TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be edited from the source code described in Table 1. The ALIGN-2 program can be edited and used on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.
[551] When ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity (%) of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B, relative to a given amino acid sequence B (in a given amino acid sequence B For a given amino acid sequence B, or having a constant amino acid sequence identity (%) relative to a given amino acid sequence B, or otherwise represented by a given amino acid sequence A), is calculated as follows:
[552] X / Y × 100
[553] Where X is the number of amino acid residues recorded by the sequence alignment program ALIGN-2 as equally matched in the program alignment of A and B, and Y is the total number of amino acid residues of B. Of course, when 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 is not equal to the amino acid sequence identity of B to A to A. As an example of calculating the amino acid sequence identity (%) using this method, Tables 2 and 3 below calculate the amino acid sequence identity (%) of the amino acid sequence referred to as the "comparative protein" to the amino acid sequence referred to as "PRO". Method, wherein "PRO" refers to the amino acid sequence of the putative PRO polypeptide of interest, "comparative protein" refers to the amino acid sequence of the polypeptide to which the "PRO" polypeptide is compared, and "X," "Y" and "Z." Each represents a different putative amino acid residue.
[554] Unless specifically stated otherwise, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph. However, amino acid sequence identity values (%) may also be obtained using the WU-BLAST-2 computer program (Melts in Enzymology 266: 460-480 (1996)) as described below. have. Most of the WU-BLAST-2 search parameters are set to their default values. Those not set to the default values, ie adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the amino acid sequence identity value (%) is determined by (a) the match of the identical amino acid residue between the amino acid sequence of the PRO polypeptide having a sequence derived from the native PRO polypeptide and the comparative amino acid sequence. The number is determined by WU-BLAST-2 (ie, the sequence to which the PRO polypeptide is compared may be a PRO variant polypeptide) and is determined by dividing (b) the total number of amino acid residues of the PRO polypeptide. For example, the term "polypeptide comprising amino acid sequence A having at least 80% amino acid sequence identity with amino acid sequence B", wherein amino acid sequence A is the comparative amino acid sequence and amino acid sequence B is the amino acid sequence of the PRO polypeptide.
[555] Amino acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST2 [Altschul et al. Nucleic Acids Res. 25: 3389-3402 (1997)]. The NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.) Or obtained from the National Institutes of Health, Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, for example, unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for All search parameters including multi-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62 are set to default values.
[556] When NCBI-BLAST2 is used for amino acid sequence comparison, the amino acid sequence identity (%) of a given amino acid sequence A with a given amino acid sequence B or with respect to a given amino acid sequence B relative to a given amino acid sequence B (or a given amino acid sequence) For B, which may be represented by a given amino acid sequence A having or including some% amino acid sequence identity to a given amino acid sequence B or relative to a given amino acid sequence B, is calculated as follows:
[557] X / Y × 100
[558] Where X is the number of amino acid residues recorded by the sequence alignment program NCBI-BLAST2 as equally matched in the program alignment of A and B, and Y is the total number of amino acid residues of B. Of course, when 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 is not equal to the amino acid sequence identity of B to A to A.
[559] A “PRO variant polynucleotide” or “PRO variant nucleic acid sequence” is a nucleic acid molecule encoding an active PRO polypeptide as defined below, the full length native sequence PRO polypeptide sequence disclosed herein, the full length lacking a signal peptide as disclosed herein At least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding a native sequence PRO polypeptide sequence, an extracellular domain of a PRO polypeptide with or without a signal peptide as disclosed herein, or any other fragment of the full length PRO polypeptide sequence disclosed herein It is a nucleic acid molecule having a. Typically, PRO variant polynucleotides are cells of a full length native sequence PRO polypeptide sequence disclosed herein, a full length native sequence PRO polypeptide sequence lacking a signal peptide as disclosed herein, a PRO polypeptide with or without a signal sequence as disclosed herein At least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84% of the nucleic acid sequence encoding the foreign domain, or any other fragment of the full-length PRO polypeptide sequence disclosed herein, Or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or about At least 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, most preferably at least about 99% nucleic acid sequence identity Neunda. Variants do not include native nucleotide sequences.
[560] Typically, the PRO variant polynucleotide is at least about 30 nucleotides, or at least about 60 nucleotides, or at least about 90 nucleotides, or at least about 120 nucleotides, or at least about 150 nucleotides, or at least about 180 nucleotides. Or about 210 or more nucleotides, or about 240 or more nucleotides, or about 270 or more nucleotides, or about 300 or more nucleotides, or about 450 or more nucleotides, or about 600 or more nucleotides, or about 900 or more nucleotides, Or more.
[561] The "% nucleic acid sequence identity" for a PRO-encoding nucleic acid sequence identified herein refers to that PRO nucleic acid sequence after aligning the PRO nucleic acid sequence with the candidate sequence and introducing a gap if necessary to obtain a maximum of sequence identity. It is defined as the ratio of nucleotides of the same candidate sequence as nucleotide of. Alignment to determine percent nucleic acid sequence identity can be achieved using a variety of methods known in the art, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Can be. However, for purposes herein,% nucleic acid sequence identity values can be obtained using the sequence comparison computer program ALIGN-2, where the complete source code for the ALIGN-2 program is described in Table 1 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code shown in Table 1 is kept as a user document of the U.S. Copyright Office (20559 Washington, DC), which code is U.S. Copyright No. TXU510087 It is registered as a call. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be compiled from the source code described in Table 1 below. The ALIGN-2 program can be edited and used on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.
[562] When ALIGN-2 is used for nucleic acid sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C relative to a given nucleic acid sequence D or with respect to a given nucleic acid sequence D (or a given nucleic acid) For sequence D, which can be represented by a given nucleic acid sequence C having or including some percent nucleic acid sequence identity (%) relative to a given nucleic acid sequence D or relative to a given nucleic acid sequence D) is calculated as follows:
[563] W / Z × 100
[564] Where W is the number of nucleotides recorded as identical matches in the program alignment of C and D by the sequence alignment program ALIGN-2, and Z is the total number of nucleotides of D. Of course, if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the percent nucleic acid sequence identity of C to D is not equal to the percent nucleic acid sequence identity of D to C. As an example of nucleic acid sequence identity (%) calculations, Tables 4 and 5 below show methods for calculating nucleic acid sequence identity (%) of nucleic acid sequences referred to as "comparative DNA" and nucleic acid sequences referred to as "PRO-DNA". Wherein "PRO-DNA" refers to the putative PRO-coding nucleic acid sequence, "comparative DNA" refers to the nucleotide sequence of a nucleic acid molecule comparing the "PRO-DNA" nucleic acid molecule, and "N," "L "And" V "each represent a different putative nucleotide.
[565] Unless specifically stated otherwise, all percent nucleic acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph. However,% nucleic acid sequence identity values can also be obtained using the WU-BLAST-2 computer program as described below (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). . Most of the WU-BLAST-2 search parameters are set to default values. Those not set to the default values, ie adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the percent nucleic acid sequence identity value is (a) of the PRO polypeptide-encoding nucleic acid molecule having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid by WU-BLAST-2. Determine the number of identical identical nucleotides matched between the nucleic acid sequence and the comparative nucleic acid molecule (ie, the sequence to which the PRO polypeptide-encoding nucleic acid molecule is compared may be a variant PRO polynucleotide), and (b) the PRO polypeptide- Determine by dividing by the total number of nucleotides of the coding nucleic acid molecule. For example, in the term “isolated nucleic acid molecule comprising nucleic acid sequence A having at least 80% nucleic acid sequence identity with nucleic acid sequence B”, nucleic acid sequence A is the comparative nucleic acid molecule and nucleic acid sequence B is the PRO polypeptide-coding Nucleic acid sequence of a nucleic acid molecule.
[566] Nucleic acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST2 [Altschul et al. Nucleic Acids Res. 25: 3389-3402 (1997)]. The NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.) Or obtained from the National Institutes of Health, Bethesda, Maryland. NCBI-BLAST2 uses several search parameters, for example unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi- All search parameters including pass = 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62 are set to default values.
[567] When NCBI-BLAST2 is used for sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C relative to a given nucleic acid sequence D, or relative to a given nucleic acid sequence D (or given nucleic acid sequence D) For a given nucleic acid sequence D, or with or containing some% nucleic acid sequence identity relative to a given nucleic acid sequence D, can be expressed as:
[568] W / Z × 100
[569] Where W is the number of nucleotides recorded by the sequence alignment program NCBI-BLAST2 to match identically in the program alignment of C and D, and Z is the total number of nucleotides of D. Of course, if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity (C) of C for D is not the same as the nucleic acid sequence identity (D) of D for C.
[570] In other embodiments, the PRO variant polynucleotide is a nucleic acid molecule that encodes an active PRO polypeptide and is capable of hybridizing (preferably under stringent hybridization conditions and wash conditions) with the nucleotide sequence encoding the full length PRO polypeptide disclosed herein. The PRO variant polypeptide may be one encoded by a PRO variant polynucleotide.
[571] The term "positive" with respect to sequence comparisons performed as described above is meant to include residues of the compared sequences which are not identical but have similar properties (e.g., see Table 6 below for the results of conservative substitutions). ). For purposes herein, a positive value (%) is determined between (a) the PRO polypeptide amino acid sequence having a sequence derived from the native PRO polypeptide sequence and the comparative amino acid sequence (ie, the amino acid sequence with which the PRO polypeptide sequence is compared). The number of amino acid residues recording a positive value is determined in the BLOSUM 62 matrix of WU-BLAST-2, which is then divided by (b) the total number of amino acid residues of the PRO polypeptide.
[572] Unless stated otherwise, positive values (%) are calculated as described in the immediately preceding paragraph. However, comparisons of amino acid sequence identity performed as described for ALIGN-2 and NCBI-BLAST2 above include amino acid residues of the comparing sequences that are identical as well as having similar characteristics. An amino acid residue that records a positive value for that amino acid residue is a residue that is the same residue as the amino acid residue or a preferred substituent of that amino acid residue (defined in Table 6 below).
[573] For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the positive value (%) of a given amino acid sequence A against a given amino acid sequence B, or a given amino acid sequence B against a given amino acid sequence B (or given For amino acid sequence B, which can be represented by a given amino acid sequence A having or including some positive value (%) relative to a given amino acid sequence B, or relative to a given amino acid sequence B), is calculated as follows:
[574] X / Y × 100
[575] Where X is the number of amino acid residues that record the positive values defined above in the program alignment of A and B by the sequence alignment program ALIGN-2 or NCBI-BLAST2, and Y is the total number of amino acid residues of B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, of course, the positive value (%) of A for B is not equal to the positive value (%) of B for A.
[576] “Isolated,” as used to describe the various polypeptides disclosed herein, means polypeptides that have been identified, isolated, and / or recovered from their natural environmental elements. Contaminant elements of the polypeptide's natural environment are typically substances that interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is purified to a degree sufficient to (1) obtain 15 or more residues of the N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) Coomassie blue ( Coomassie blue) or preferably silver stain is used to purify such that only one band appears in SDS-PAGE under non-reducing or reducing conditions. Since at least one element of the PRO polypeptide will not be present in the natural environment, an isolated polypeptide includes a polypeptide present in a recombinant cell. However, isolated polypeptide is typically obtained by one or more purification steps.
[577] An “isolated” PRO polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that has been identified and separated from one or more contaminating nucleic acid molecules that are typically associated with a naturally occurring PRO polypeptide encoding nucleic acid. Isolated polypeptide-encoding nucleic acid molecules are different from the forms or states found in nature. Thus, an isolated polypeptide-encoding nucleic acid molecule is distinguished from certain polypeptide-encoding nucleic acid molecules present in natural cells. However, isolated polypeptide-encoding nucleic acid molecules include, for example, polypeptide-encoding nucleic acid molecules present at chromosomal sites different from the chromosomes of natural cells and typically contained in cells expressing said polypeptide.
[578] The term "regulatory sequence" is a DNA sequence necessary for the expression of a coding sequence operably linked in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
[579] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a preprotein that participates in the secretion of the polypeptide, and the promoter or enhancer If he affects the transcription of the coding sequence, it is operably linked to the coding sequence, and the ribosomal binding site is operably linked to the coding sequence if he is placed to facilitate translation. In general, "operably linked" means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, are contiguous and within the reading site. 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 adapters or linkers are used in accordance with conventional practice.
[580] The term “antibody” is used in its broadest sense and specifically includes, for example, one anti-PRO monoclonal antibody (including agonists, antagonists and neutralizing antibodies), anti-PRO antibody compositions with polyepitope specificity, single chain Anti-PRO antibodies, and anti-PRO antibody fragments (see below). As used herein, the term “monoclonal antibody” means in fact an antibody obtained from a population of homologous antibodies, ie, individual antibodies that make up a population, except for possible naturally occurring mutations that may be present in small amounts.
[581] The "stringency" of the hybridization reaction can be readily determined by one skilled in the art and is generally an experimental calculation that depends on probe length, wash temperature, salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. In general, hybridization is dependent on the renalling ability of the denatured DNA when the complementary strand is present in an environment below its melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature available. As a result, the reaction conditions are more stringent at higher relative temperatures, but the reaction conditions are less stringent at lower relative temperatures. For more detailed information and explanations on the stringency of the hybridization reaction, see Ausubel et al. Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).
[582] As defined herein, "strict conditions" or "high stringency conditions" means (1) 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl alcohol at low ionic concentration and high temperature at washing, for example at 50 ° C. Conditions using pate, (2) 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone with 750 mM sodium chloride, 75 mM sodium citrate, for example at 42 ° C. during hybridization Conditions using a denaturant such as 50% (v / v) formamide with / 50 mM sodium phosphate buffer (pH 6.5), or (3) 50% formamide at 42 ° C, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dex Using tran sulphate, 0.2 x SSC (sodium chloride / sodium citrate) at 42 ° C. and 50% foam at 55 ° C. Washing the amide and then, EDTA containing 0.1 x SSC at 55 to ℃ - to perform a rigorous cleaning.
[583] "Medium stringency conditions" are those described in Sambrook et al., Molecular Cloning: A Laboratory Manual , New York: Cold Spring Harbor Press 1989, and are less stringent than washing solutions and hybridization conditions (e.g., For example, temperature, ion concentration and the use of% SDS). Examples of medium stringency conditions include 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt solution, 10% dextran Incubated overnight at 37 ° C. in a solution containing 20 mg / ml of pate and denatured truncated salmon sperm DNA, followed by washing the filter with 1 × SSC at about 37-50 ° C. Those skilled in the art will know how to adjust the temperature and ion concentration required to meet factors such as probe length and the like.
[584] As used herein, the term “epitope tagged” refers to a chimeric polypeptide comprising a PRO polypeptide fused with a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope that allows the antibody to be made, but the tag polypeptide is short enough that it does not interfere with the activity of the polypeptide to which it is fused. It is also desirable that the tag polypeptide is very specific so that the antibody against it does not actually cross react with other epitopes. Suitable tag polypeptides generally have six or more amino acid residues, and typically have about 8-50 amino acid residues (preferably about 10-20 amino acid residues).
[585] The term “immunoadhesin” as used herein refers to an antibody-like molecule that combines the binding specificity of a heterologous protein (adhesin) with the effector function of an immunoglobulin constant domain. Structurally, immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having some binding specificity (ie, heterologous) that is not the antigen recognition site and binding site of the antibody. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of a receptor or ligand. Immunoglobulin constant domain sequences of immunoadhesin may be IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, such as IgA (including IgA-1 and IgA-2), IgE, IgD or IgM Can be obtained from any immunoglobulin.
[586] As used herein, the term "active" or "activity" refers to a form of a PRO polypeptide that retains the biological and / or immunological activity of a natural or naturally-occurring PRO, wherein "biological" activity means Or biological function (inhibitory or facilitating function) induced by a natural or naturally-occurring PRO, rather than the ability to induce the production of antibodies against antigenic epitopes possessed by a naturally-occurring PRO, and "immunological "Activity refers to the ability to induce the production of antibodies against antigenic epitopes possessed by natural or naturally-occurring PRO.
[587] The term “antagonist” is used in its broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the native PRO polypeptides disclosed herein. Likewise, the term "agonist" is used in its broadest sense and includes any molecule that mimics the biological activity of the native PRO polypeptides disclosed herein. Suitable agonist or antagonist molecules include, in particular, agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules and the like. Methods for identifying an agonist or antagonist of a PRO polypeptide may include contacting the PRO polypeptide with a candidate agonist molecule or a candidate antagonist molecule and measuring a detectable change in one or more biological activities, typically associated with the PRO polypeptide.
[588] "Treatment" means a therapeutic treatment and a preventive or preventive measure carried out for the purpose of preventing or slowing (mitigating) a pathological condition or disease in a subject. Subjects in need of treatment include those already afflicted with the disease, as well as those susceptible to disease or those seeking to prevent the disease.
[589] "Chronic" administration refers to the administration of the substance in a continuous manner as opposed to the acute manner, to maintain the initial therapeutic effect (activity) for a long time. "Intermittent" administration refers to treatment that is actually performed periodically, rather than continuing without interruption.
[590] “Mammals” for treatment are mammals, including humans, livestock and breeding animals, and zoos, competitions or pets, such as dogs, cats, cattle, horses, sheep, pigs, goats, and rabbits. Any animal that is classified. Preferably the mammal is a human.
[591] “Co-administering” with one or more other therapeutic agents includes simultaneous (in combination) or continuous administration in any order.
[592] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients or stabilizers which are nontoxic to the cell or mammal being exposed to the dosages and concentrations employed. Physiologically acceptable carriers are often pH buffered aqueous solutions. 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 10 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 dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or nonionic surfactants such as TWEEN, polyethylene glycol (PEG) and PLURONICS®.
[593] "Antibody fragments" include portions of intact antibodies, preferably antigen binding regions or variable regions of intact antibodies. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments, diabodies, linear antibodies [Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)], single chain antibody molecules, and multispecific antibodies formed from antibody fragments.
[594] Papain digestion of the antibody produces two identical antigen binding fragments, a "Fab" fragment with a single antigen-binding site, and the remaining "F _c " fragments, which reflect the ability to readily crystallize. Treatment with pepsin results in an F (ab ') ₂ fragment having two antigen binding sites and capable of crosslinking with the antigen.
[595] "Fv" is the minimum antibody fragment that includes a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain that is tightly covalently bonded. In this form three CDRs of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer. In conclusion, six CDRs confer antigen-binding specificity to antibodies. However, even a single variable domain (or half of the Fv comprising only three CDRs specific for the antigen), although less affinity than the entire binding site, has the ability to recognize and bind the antigen.
[596] The Fab fragment also comprises the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab 'fragments by the addition of several residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH herein refers to Fab 'in which the cysteine residues of the constant domains bear free thiol groups. F (ab ') ₂ antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines between them. In addition, other chemical couplings of antibody fragments are known.
[597] The “light chain” of an antibody (immunoglobulin) from any vertebrate species can belong to one of two distinctly different types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain. .
[598] Immunoglobulins can belong to different groups depending on the amino acid sequence of the constant domain of their heavy chains. There are five main groups of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further classified into subgroups (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Can be.
[599] "Single-chain Fv" or "sFv" antibody fragments comprise the V _H and V _L domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains that allows the sFv to form the structure required for binding of the antigen. For sFv, see Pluckthan's [ The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[600] The term "dia body (diabody)" in the same polypeptide chain in the (V _H -V _L) of the light chain variable domain (V _L) of a heavy chain associated with the variable domain (V _H), including a small having two antigen binding sites Refers to an antibody fragment. Linkers that are too short to link two domains on the same chain are used to create two antigen-binding sites by linking the domains with the complementary domains of the other chain. Diabodies are described, for example, in EP 404,097, WO 93/11611 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
[601] An “isolated” antibody is an antibody identified, isolated and / or recovered from elements of its natural environment. Antibody contaminating elements of the natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) greater than 95% by weight, most preferably at least 99% by weight, as determined by the Lowry method, and (2) using a spinning cup sequencer. Sufficient to obtain at least 15 residues of the N-terminal or internal amino acid, or (3) one band by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Will be purified. Isolated antibody includes antibodies in recombinant cells since at least one antibody component will not be present in the natural environment. However, isolated antibodies will typically be prepared by one or more purification steps.
[602] As used herein, the term "label" refers to a detectable compound or composition that binds directly or indirectly with an antibody to produce a "labeled" antibody. The label can be detected by itself (eg, a radioisotope label or fluorescent label) or, if it is an enzyme label, can promote chemical modification of the detectable substrate compound or composition.
[603] "Solid phase" means a non-aqueous matrix to which an antibody of the invention may be attached. Examples of solid phases included herein include those formed partially or entirely of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. Included. In some embodiments, depending on the circumstances, the solid phase may comprise the wells of an assay plate, and in other embodiments the solid phase is a purification column (eg, an affinity chromatography column). The term also encompasses discrete particles of a discontinuous solid phase, such as those described in US Pat. No. 4,275,149.
[604] A "liposome" is a small vesicle consisting of various forms of lipids, phospholipids, and / or surfactants useful for delivering a drug (eg, a PRO polypeptide or antibody thereto) to a mammal. Liposomal components are typically arranged in bilayer form, similar to the lipid arrangement of biological membranes.
[605] "Small molecule" is defined herein as having a molecular weight of less than about 500 Daltons.
[606]
[607]
[608]
[609]
[610]
[611]
[612]
[613]
[614]
[615]
[616]
[617]
[618]
[619]
[620]
[621]
[622]
[623]
[624]
[625]
[626]
[627] II. Compositions and Methods of the Invention
[628] A. Full Length PRO Polypeptide
[629] The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as PRO polypeptides. In particular, cDNA encoding various PRO polypeptides were identified and isolated as described in more detail in the Examples below. The PRO number of proteins produced in separate rounds of expression may vary but the UNQ number is specific to any given DNA and protein encoded therefrom and will not change. However, herein, simply, proteins encoded by the full-length natural nucleic acid molecules disclosed herein, and other natural homologs and variants of PRO included in the above definitions, are referred to as "PRO / number" regardless of their origin and manner of preparation. Will be mentioned.
[630] As described in the Examples below, various cDNA clones were deposited with the ATCC. One skilled in the art can readily determine the actual nucleotide sequence of the clone by analyzing the sequence of the deposited clone using conventional methods in the art. General techniques can be used to determine the expected amino acid sequence from the nucleotide sequence. In the case of the PRO polypeptides described herein and the nucleic acids encoding them, the inventors have identified what is considered to be the best identifying frame using sequence information available at the time.
[631] 1.Full Length PRO1484 Polypeptide
[632] The inventors have used WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO1484 (shown in Figure 2 and SEQ ID NO: 2) has some amino acid sequence identity with a portion of the complement related protein (ACR3_MOUSE) of mouse adipocytes. Came out. Therefore, it is presently believed that the PRO1484 disclosed herein is the complement-associated protein homolog of newly identified adipocytes and may have the typical activity of this protein.
[633] 2. Full Length PRO4334 Polypeptide
[634] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4334 (shown in Figures 4 and 9) has some amino acid sequence identity with PC-1. Thus, it is presently believed that the PRO4334 disclosed herein is a newly identified member of the PC-1 family and shares similar mechanisms.
[635] 3. Full Length PRO1122 Polypeptide
[636] The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as PRO1122. In particular, the inventors have identified and isolated cDNA encoding the PRO1122 polypeptide, as described in more detail in the Examples below. We have used the BLAST and FastA sequence alignment computer programs to find that PRO1122 polypeptide has sequence identity with CTLA-8. The amino acid sequence has a region having sequence identity with IL-17. Therefore, it is presently believed that the PRO1122 polypeptide disclosed herein is a novel cytokine and may be involved in an inflammatory response.
[637] 4. Full Length PRO1889 Polypeptide
[638] The inventors have used WU-BLAST2 sequence alignment computer program to reveal that some of the full-length native sequence PRO1889 (shown in FIG. 8 and SEQ ID NO: 16) has some amino acid sequence identity with some of the human E48 antigen protein (HSE48ATGN_1). Came out. Thus, it is presently believed that the PRO1889 disclosed herein is the newly identified E48 homologue and may have the typical activity or properties of the E48 protein.
[639] 5. Full Length PRO1890 Polypeptide
[640] The inventors have used WU-BLAST2 sequence alignment computer program to show that some of the full-length native sequence PRO1890 (shown in FIG. 10 and SEQ ID NO: 18) has some amino acid sequence identity with a portion of layilin protein (AF093673_1). Revealed.
[641] 6. Full Length PRO1887 Polypeptide
[642] We used the WU-BLAST2 sequence alignment computer program to determine which full length native sequence PRO1887 (shown in FIG. 12 and SEQ ID NO: 23) was associated with a carboxyl esterase precursor (identified as "ESTM_MOUSE" in the Dayhof database) between mice. It was found to have some degree of amino acid sequence identity. Thus, it is presently believed that the PRO1887 disclosed herein is a newly identified member of the carboxyl esterase family and may have the typical enzymatic activity of carboxyesterases.
[643] 7. Full Length PRO1785 Polypeptide
[644] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO1785 (shown in FIG. 14 and SEQ ID NO: 29) has some amino acid sequence identity with glutathione peroxidase. Therefore, it is presently believed that PRO1785 disclosed herein is a newly identified member of the peroxidase family and may have antioxidant enzyme activity. Modulating antioxidant activity is of interest in the treatment of cancer and aging.
[645] 8. Full Length PRO4353 Polypeptide
[646] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4353 (shown in FIG. 16 and SEQ ID NO: 35) had some amino acid sequence identity with semaphorin Z. Therefore, it is presently believed that PRO4353 disclosed herein is a newly identified member of the semaphorin Z family and is involved in the inhibition of nerve growth. PRO4353 was used in the analysis to find modulators of semaphorin Z, particularly inhibitors that promote regeneration of the central nervous system.
[647] 9. Full Length PRO4357 Polypeptide
[648] The inventors found that using the WU-BLAST2 sequence alignment computer program, Accession No. P_W48804, consisting of 289 amino acids of the full-length native sequence PRO4357 (shown in FIG. 18 and SEQ ID NO: 40), has some amino acid sequence identity. However, PRO4357 has 213 more amino acids at the N-terminus.
[649] 10. Full Length PRO4405 Polypeptide
[650] As is currently known, the DNA84920-2614 sequence encodes a new factor named PRO4405 herein and using the WU-BLAST2 sequence alignment computer program, it has been found that there is limited sequence identity with known proteins.
[651] 11.Full Length PRO4356 Polypeptide
[652] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4356 (shown in FIG. 22 and SEQ ID NO: 50) has some amino acid sequence identity with the GPI-anchored protein involved in metastasis. Thus, it is presently believed that the PRO4356 disclosed herein is a newly identified member of this family and shares a similar mechanism.
[653] 12. Full Length PRO4352 Polypeptide
[654] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4352 (shown in FIGS. 24 and 52) has some amino acid sequence identity with protocadherin pc3 and protocadherin pc4. Thus, PRO4352 is associated with cell adhesin and is believed to be used in the treatment of differentiating diseases, cell adhesin, neuronal receptors or skin diseases. Screening can also identify agonists and antagonists for the treatment of these diseases.
[655] 13. Full Length PRO4380 Polypeptide
[656] As is currently known, the DNA92234-2602 sequence encodes a new factor named PRO4380 herein and has been found using WU-BLAST2 sequence alignment computer program to have limited sequence identity with proteins of known function.
[657] 14. Full Length PRO4354 Polypeptide
[658] As is now known, the DNA92256-2596 sequence encodes a new factor named PRO4354 herein, and was found to have limited sequence identity with proteins of known function using the WU-BLAST2 sequence alignment computer program.
[659] 15. Full Length PRO4408 Polypeptide
[660] As is currently known, the DNA92274-2617 sequence encodes a new factor named PRO4408 herein and has been found using WU-BLAST2 sequence alignment computer program to have limited sequence identity with proteins of known function.
[661] 16. Full Length PRO5737 Polypeptide
[662] We have used WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO5737 (shown in Figure 32 and SEQ ID NO: 63) has some degree of amino acid sequence identity with IL-1 and / or IL-1Ra. Thus, it is presently believed that the PRO5737 disclosed herein is a newly identified member of this family and shares a similar mechanism.
[663] 17. Full Length PRO4425 Polypeptide
[664] As is currently known, the DNA93011-2637 sequence encodes a new factor named PRO4425 herein, and using the WU-BLAST2 sequence alignment computer program, PRO4425 shows homology with, but not identical to, the protein of Genbank Accession No. HGS_RE295. It turned out that
[665] 18. Full Length PRO5990 Polypeptide
[666] Using the above-mentioned ALIGN-2 sequence alignment computer program, full-length native sequence PRO5990 (shown in FIG. 36 and SEQ ID NO: 67) has some amino acid sequence identity with Secretoranin II (Dayhof No. GEN14673). Revealed. Accordingly, it is presently contemplated that the PRO5990 polypeptides disclosed herein are newly identified members of the secretographin protein family and may have one or more biological and / or immunological activities or properties typical of this protein family.
[667] 19. Full Length PRO6030 Polypeptide
[668] As described in the Examples below, clones of DNA96850-2705 were isolated from human libraries. As is currently known, the DNA96850-2705 nucleotide sequence encodes a new factor named PRO6030 herein, and using the ALIGN-2 sequence alignment computer program, it has been found that there is no significant sequence identity with any known protein.
[669] 20. Full Length PRO4424 Polypeptide
[670] As is currently known, the DNA96857-2636 sequence encodes a new factor, herein designated PRO4424, and is homologous to, but not identical to, the protein of Genbank Accession No. HGS_A135, using a WU-BLAST2 sequence alignment computer program. It turned out that
[671] 21. Full Length PRO4422 Polypeptide
[672] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4422 (shown in Figure 42 and SEQ ID NO: 76) has some amino acid sequence identity with lysozyme g. Thus, it is presently believed that the PRO4422 disclosed herein is a newly identified member of the lysozyme family and may have lysozyme activity.
[673] 22. Full Length PRO4430 Polypeptide
[674] We used the WU-BLAST2 sequence alignment computer program to find that the full length native sequence PRO4430 (shown in Figure 44 and SEQ ID NO: 78) had some amino acid sequence identity with the protein of Genbank Accession No. MMHC213L3_9. Thus, it is presently believed that the PRO4430 disclosed herein is related to the Genbank protein and can share one or more similar mechanisms.
[675] 23. Full Length PRO4499 Polypeptide
[676] As is currently known, the DNA96889-2641 sequence encodes a new factor named PRO4499 herein, and using the WU-BLAST2 sequence alignment computer program, it has been found that there is limited sequence identity with known proteins.
[677] B. PRO Polypeptide Variants
[678] In addition to the full-length native sequence PRO polypeptides described herein, it is contemplated that PRO variants may be prepared. PRO variants can be prepared by introducing suitable nucleotide changes into PRO DNA and / or synthesizing the desired PRO polypeptide. Those skilled in the art will appreciate that amino acid changes, such as altering the number or location of glycosylation sites or changing membrane anchoring properties, can alter the post-translational processing of RPO.
[679] Variations in the various domains of the full-length native sequence PRO or PRO described herein can be prepared using any technique and guidance for conservative and non-conservative mutations, eg, disclosed in US Pat. No. 5,364,934. Modification means that one or more codons encoding PRO can be substituted, deleted or inserted to change the amino acid sequence of PRO relative to native sequence PRO. Optionally, the mutation is produced by replacing one or more amino acids with any other amino acid in one or more domains of PRO. Amino acid residues that can be inserted, substituted or deleted without adversely affecting the desired activity compare the sequence of PRO to the sequence of known homologous protein molecules and minimize the number of amino acid sequence changes generated in regions of high homology. You can decide. Amino acid substitutions may be the result of substitution of one amino acid with another amino acid having similar structural and / or chemical properties, eg, replacement of leucine with serine, ie, conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1-5 amino acids. Acceptable variations can be determined by systematically inserting, deleting, or replacing amino acids in the sequence and testing the activity of the resulting variant represented by the full length or mature native sequence.
[680] The application provides PRO polypeptide fragments. For example, when compared to full-length natural proteins, these fragments may be truncated at the N-terminus or C-terminus or missing internal residues. Some fragments lack amino acid residues that are not essential for the desired biological activity of the PRO polypeptides of the invention.
[681] PRO fragments can be prepared by any of a number of conventional techniques. Desired peptide fragments can be synthesized chemically. Another method is to produce a PRO fragment by enzymatic digestion, for example by treating the protein with an enzyme known to cut a protein at a site defined by a particular amino acid residue, or by enzymatically cutting the DNA with a suitable restriction enzyme. And isolating this desired fragment. Yet another suitable technique includes isolating and amplifying a DNA fragment encoding a desired polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that form the desired end of the DNA fragment are used as 5 'and 3' primers in PCR. Preferably, the PRO polypeptide fragment shares one or more biological and / or immunological activities with the native PRO polypeptide disclosed herein.
[682] In specific embodiments, conservative substitutions of the target are shown in Table 6 under the heading of preferred substitutions. When the biological activity was changed by such substitutions, more substantial changes were introduced and the products were screened as named as substitutions in Table 6 below or as described in more detail below for amino acid species.
[683] Original residuesSubstitution examplePreferred Substitution Ala (A)val, leu, ileval Arg (R)lys, gln, asnlys Asn (N)gln, his, lys, arggln Asp (D)gluglu Cys (C)serser Gln (Q)asnasn Glu (E)aspasp Gly (G)pro, alaala His (H)asn, gln, lys, argarg Ile (I)leu, val, met, ala, phe, norleucineleu Leu (L)norleucine, ile, val, met, ala, pheile Lys (K)arg, gln, asnarg Met (M)leu, phe, ileleu Phe (F)leu, val, ile, ala, tyrleu Pro (P)alaala Ser (S)thrthr Thr (T)serser Trp (W)tyr, phetyr Tyr (Y)trp, phe, thr, serphe Val (V)ile, leu, met, phe, ala, norleucineleu
[684] Substantial modifications of the function or immunological identity of the PRO polypeptides may include (a) maintaining the structure of the polypeptide backbone at the substitution region, for example in sheet or helix form, or (b) maintaining the charge or hydrophobicity of the molecule at the target site. Or (c) selecting a substitution that significantly alters its effect of retaining most of the side chains. Natural residues are divided into the following groups according to common side chain properties:
[685] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[686] (2) neutral hydrophilic: cys, ser, thr;
[687] (3) acidic: asp, glu;
[688] (4) basic: asn, gln, his, lys, arg;
[689] (5) residues affecting chain orientation: gly, pro; And
[690] (6) aromatic; trp, tyr, phe.
[691] Non-conservative substitutions will replace said one type of component with another type. In addition, the residues so substituted may be introduced at conservative substitution sites or more preferably at the remaining (non-conserved) sites.
[692] Variations can be made using methods known in the art such as oligonucleotide-mediated (position-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 : 315 (1985)], restriction selection mutagenesis [Wells et al ., Philos. Trans. R. Soc. London SerA , 317 : 415 (1986)] or other known techniques can be performed on cloned DNA to produce the PRO variant DNA of the present invention.
[693] Scanning amino acid assays can also be used to identify one or more amino acids along adjacent sequences. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Typically, alanine is a preferred scanning amino acid because it is less likely to remove side chains outside the beta-carbon and alter the backbone arrangement of the variants (Cunningham and Wells, Science , 244 : 1081-1085 (1989). )]]. Alanine is also preferred because it is usually the most common amino acid. In addition, alanine is frequently found both in buried and exposed locations (Creighton, The Proteins , (WH Freeman & Co., NY)); And Chothia, J. Mol. Biol. , 150 : 1 (1976)]. If alanine substitutions do not produce adequate amounts of variants, isotopic amino acids can be used.
[694] C. variant of PRO
[695] Covalent modifications of PRO are included within the scope of the present invention. One form of covalent modification involves reacting a target amino acid residue of a PRO polypeptide with an organic derivatizing agent capable of reacting with a selected side chain of PRO, or an N-terminal or C-terminal residue. Derivatization with a bifunctional agent is useful for crosslinking PRO to a water-insoluble support matrix or surface, or vice versa, for example for use in anti-PRO antibody purification methods. Commonly used crosslinkers include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidosalicylic acid, Homo-functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), difunctional maleimides such as bis-N-maleimido-1,8-octane and Substances such as methyl-3 [(p-azidophenyl) dithio] propioimidate.
[696] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, lysine, Methylation of alpha-amino groups of arginine and histidine side chains [TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of N-terminal amines and amidation of C-terminal carboxyl groups.
[697] Other types of covalent modifications of PRO polypeptides within the scope of the present invention include changes in the natural glycosylation pattern of the polypeptide. “A change in natural glycosylation pattern” refers to the deletion of one or more carbohydrate residues found in the native sequence PRO (either by removing potential glycosylation sites or by deleting chemical glycosylation by chemical and / or enzymatic methods) and / or ) Addition of one or more glycosylation sites that are not present in native sequence PRO. The term also encompasses qualitative changes in glycosylation of natural proteins, including changes in the properties and proportions of the various carbohydrate residues present.
[698] The addition of glycosylation sites to the PRO polypeptide can be accomplished by changing the amino acid sequence. The change can be made, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence PRO (for O-linked glycosylation sites). The PRO amino acid sequence can be optionally altered through a change in the DNA level by mutating DNA encoding the PRO polypeptide at a preselected base to produce a codon that is translated into the desired amino acid.
[699] Another means of increasing the number of carbohydrate residues on the PRO polypeptide is to couple the glycoside chemically or enzymatically to the polypeptide. Such methods are described, for example, in WO 87/05330 published September 11, 1987 and in Aplin and Wriston, CRC Crit. Rev. Biochem. , pp. 259-306 (1981).
[700] Removal of carbohydrate residues present in a PRO polypeptide can be accomplished chemically or by substitution by mutation of a codon encoding an amino acid residue that functions by enzyme or as a glycosylation target. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin et al ., Arch. Biochem. Biophys. , 259 : 52 (1987) and by Edge et al ., Anal. Biochem. , 118 : 131 (1981). Enzymatic cleavage of carbohydrate residues on polypeptides can be accomplished using a variety of endoglycosidases and exoglycosidases [Thotakura et al . Meth. Enzymol. , 138 : 350 (1987)].
[701] Other types of covalent modifications of PRO include various nonproteinaceous polymers, eg, in the manner described in US Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337. For example connecting the PRO polypeptide to one of polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.
[702] In addition, the PRO of the present invention may be modified in such a way as to form chimeric molecules comprising PRO fused to other heterologous polypeptide or amino acid sequences.
[703] In one embodiment, such chimeric molecules comprise a fusion of PRO with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Epitope tags are generally located at the amino or carboxyl terminus of PRO. The presence of the PRO in the form with the epitope tag can be detected using an antibody against the tag polypeptide. In addition, the introduction of epitope tags allows for easy purification of PRO by affinity purification using anti-tag antibodies or other types of affinity matrices that bind to epitope tags. Various tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine or poly-histidine-glycine tags, flu HA tag polypeptides and antibodies thereof 12CA5 [Field et al ., Mol. Cell. Biol. , 8 : 2159-2165 (1988)], c-myc tags and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [Evan et al., Molecular and Cellular Biology , 5 : 3610-3616] (1985)], and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990)]. have. Other tag polypeptides include Flag-peptides (Hopp et al., BioTechnology , 6 : 1204-1210 (1988)), KT3 epitope peptides [Martin et al., Science , 255 : 192-194 ( 1992)], alpha-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)].
[704] In other embodiments, the chimeric molecule comprises a fusion of PRO with an immunoglobulin or a specific region of immunoglobulins. For the divalent form of the chimeric molecule (also referred to as "immunoadhesin"), the fusion may be the F _c region of an IgG molecule. This Ig fusion preferably comprises the substitution of a site of one or more variable regions in the Ig molecule with a soluble (membrane or inactivation) form of the PRO polypeptide. In a particularly preferred embodiment, the immunoglobulin fusions comprise the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. For a method of producing immunoglobulin fusions, see US Pat. No. 5,428,130, issued June 27, 1995.
[705] D. Manufacture of PRO
[706] The content described below relates primarily to methods of making PRO by culturing cells transformed or transfected with the vector containing the PRO nucleic acid. Of course, the PRO may be prepared using other methods known in the art. For example, PRO sequences or fragments thereof can be prepared by direct peptide synthesis using solid phase techniques [Stewart et al., Solid-Phase Peptide Synthesis , WH Freeman Co., San Francisco, CA (1969). ) And Merrifield, J. Am. Chem. Soc. , 85 : 2149-2154 (1963). In vitro protein synthesis can be performed by manual or automated methods. Automated synthesis can be performed using, for example, Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. The full length PRO can be prepared by chemically synthesizing the various parts of the PRO separately and combining them using chemical or enzymatic methods.
[707] 1. Isolation of DNA Encoding PRO
[708] DNA encoding PRO may be obtained from a cDNA library prepared from tissues that possess PRO mRNA and are thought to express it at detectable levels. Thus, human PRO DNA can be conveniently obtained from a cDNA library prepared from human tissue as described in the Examples. PRO coding genes can also be obtained from genomic libraries or by known synthetic methods (eg, automated nucleic acid synthesis methods).
[709] Libraries can be screened using probes designed to identify a gene of interest or a protein encoded by the gene (eg, an antibody to PRO or an oligonucleotide consisting of about 20 to 80 or more bases). Screening of cDNAs or genomic libraries using selected probes uses standard methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Haror Laboratory Press, 1989). Can be done. Other means for isolating the gene encoding PRO is to use PCR method [the literature, such as fountain Brook (Sambrook); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Haror Laboratory Press, 1995).
[710] The following examples illustrate techniques for screening cDNA libraries. Oligonucleotide sequences selected as probes should be sufficiently long and sufficiently clear to minimize false positive results. Oligonucleotides are preferably labeled so that they can be detected upon hybridization with DNA in the library being screened. Labeling methods are known in the art and include the use of radiolabels, biotinylation or enzyme labels, such as ³² P-labeled ATP. Hybridization conditions, including moderate stringency and high stringency is provided in the described supra, such as literature Sam Brook, (Sambrook).
[711] The sequences identified in the library screening method can be aligned in comparison to other known sequences available deposited in public databases such as GenBank or other proprietary sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of the molecule or across the full length sequence can be determined using known methods of the prior art and the methods described herein.
[712] Nucleic acid having protein coding sequence is the first use of the estimated amino acid sequence disclosed herein, et al., Sam Brook (Sambrook) to detect the precursor, if necessary, using conventional primer extension method described in [supra selected cDNA or Genomic libraries can be obtained by screening and processing intermediates of mRNA that are not reverse transcribed into cDNA.
[713] 2. Selection and Transformation of Host Cells
[714] Host cells are conventional nutrient media transfected or transformed with the expression or cloning vectors described herein for PRO production and modified to be suitable for induction of a promoter, selection of a transformant or amplification of a gene encoding a sequence of interest. In culture. Those skilled in the art can select culture conditions such as medium, temperature and pH without undue experimentation. In general, principles, protocols, and techniques to maximize productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach , M. Butler, ed. Can be found in (IRL Press, 1991)] and Sam Brook et al., (Sambrook) [supra].
[715] Eukaryotic transfection methods and prokaryotic transformation methods such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Sam Brook (Sambrook) described supra, or electroporation, calcium treatment using calcium chloride method described in, such as is generally used for prokaryotes. Infection with Agrobacterium tumefaciens has been described in particular plants as described in Shaw et al., Gene , 23 : 315 (1983) and WO 89/05859 published June 29, 1989. Used for cell transformation. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. General features of transfection of mammalian cell host systems are described in US Pat. No. 4,399,216. Transformation into yeast is generally described by Van Solingen et al., J. Bact. , 130 : 946 (1977) and Hsiao et al ., Proc. Natl. Acad. Sci. (USA) , 76 : 3829 (1979)]. However, other methods of introducing DNA into cells, such as intranuclear microinjection, electroporation, fusion of circular cells with bacterial protoplasts, or polycations such as polybrene, polyornithine can also be used. . For various techniques for transformation of mammalian cells, see Keown et al., Methods in Enzymology , 185: 527-537 (1990) and Mansour et al . Nature , 336: 348-352 (1988).
[716] Suitable host cells for cloning or expressing DNA in a vector herein include prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells are sedative bacteria, for example Gram negative or Gram positive organisms, for example Enterobacteriaceae, for example E. coli. Coli, including but not limited to. A variety of teeth. E. coli strains, for example E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537). E. coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are readily available. Other suitable prokaryotic host cells are Escherichia, such as E. coli. Coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Sererratia, for example For example Serratia marcescans and Shigella, and Bacillus, for example B. Subtilis and b. Licheniformis (e.g. B. licheniformis 41P described in DD 266,710, published April 12, 1989), Pseudomonas, for example p. Aeruginosa and Streptomyces. This example is for illustrative purposes only and is not intended to be limiting. Strain W3110 is a particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentation. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to result in mutation of the gene encoding the endogenous protein of the host, an example of such a host is E. coli with the complete genotype tonA . E. coli W3110 strain 1A2, E. coli with the complete genotype tonA ptr3 . E. coli W3110 strain 9E4, E. coli with the full genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), E. coli with the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ^r . E. coli W3110 strain 37D6, strain 37D6 having a non-kanamycin resistant degP deletion mutation. E. coli W3110 strain 40B4 and E. coli with the Periplasm protease variant disclosed in US Pat. No. 4,946,783, issued Aug. 7, 1990. E. coli strains. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are suitable.
[717] In addition to prokaryotic cells, eukaryotic microorganisms such as fibrous fungi or yeast are suitable as cloning or expression hosts for PRO-coding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Shichizosaccharomyces pombe (Beach and Nurse, Nature , 290: 140 [1981]); European Patent No. 139,383, published May 2, 1985; Kluyveromyces hosts (US Pat. No. 4,943,529; Fler et al., Bio / Technology , 9: 968-975 (1991), for example K. et al. Lactis [MW98-8C, CBS683, CBS4574; Louvencourt et al ., J. Bacteriol. , 154 (2): 737-742 [1983], k. Fragilis (ATCC 12,424), k. Bulgaricus (ATCC 16,045), K. Wickeramii (ATCC 24,178), K. Waltii (ATCC 56,500), K. Drosophilarum [ATCC 36,906; Van den Berg et al., Bio / Technology , 8: 135 (1990), K. Thermotolerans and K. Marxianus; Yarrowia (European Patent No. 402,226); Pichia pastoris (European Patent 183,070); Sreekrishna et al ., J. Basic Microbiol. , 28: 265-278 [1988]; Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa [Case et al . Proc. Natl. Acad. Sci. USA , 76: 5259-5263 [1979]; Schwanniomyces, such as, for example, occidentalis (European Patent No. 394,538, published October 31, 1990); And fibrous fungi such as neurospora, penicillium, tolypocladium (WO 91/00357 published January 10, 1991) and Aspergillus hosts, for example Listen a. Nidulans (Ballance et al., Biochem. Biophys. Res. Commun. , 112: 284-289 [1983]; Tilburn et al., Gene , 26: 205-221 [1983]; Yelton et al ., Proc. Natl. Acad. Sci. USA , 81: 1470-1474 [1984] and A. Niger (Kelly and Hynes, EMBO J. , 4: 475-479 [1985]). Methyltrophic yeast is suitable and consists of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula Yeast capable of growing on methanol, selected from, but not limited to. A list of specific species that are examples of these types of yeast is described in C. Anthony's The Biochemistry of Methylotrophs , 269 (1982).
[718] Suitable host cells for the expression of glycosylated PRO are derived from multicellular organisms. Examples of invertebrate cells include insect cells and plant cells, such as Drosophila S2 and Spodoptera Sf9. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 cell lines transformed with SV40 (COS-7, ATCC CRL 1651), kidney cell lines of human embryos (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al. J. Gen Virol. , 36:59 (1997)), Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA , 77: 4216 ( 1980)), mouse sertoli cells (TM4, Marter, Biol. Reprod. , 23: 243-251 (1980)), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065) and mice Breast tumors (MMT 060562, ATCC CCL 51). One skilled in the art can readily select suitable host cells.
[719] 3. Selection and use of replicable vectors
[720] Nucleic acid encoding a PRO (eg cDNA or genomic DNA) is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors can be easily obtained. For example, the vector may be in the form of plasmids, cosmids, viral particles or phages. Suitable nucleic acid sequences can be inserted into the vector by various methods. In general, DNA is inserted into a suitable restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, origins of replication, one or more marker genes, enhancer components, promoters and transcription termination sequences. The preparation of suitable vectors comprising one or more such components uses standard ligation techniques known to those skilled in the art.
[721] PRO can be produced not only by direct recombination methods but also as a fusion polypeptide with a heterologous polypeptide that can be a mature protein or other polypeptide or signal sequence having a specific cleavage site at the N terminus of the polypeptide. In general, the signal sequence may be a component of the vector or part of a PRO-encoding DNA inserted into the vector. The signal sequence can be for example a prokaryotic signal sequence selected from the group of alkali phosphatase, penicillinase, lpp or thermostable enterotoxin II leader. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader, an α factor leader (Saccharomyces and Kluyveromyces α-factor leader (described in US Pat. No. 5,010,182)) or an acid phosphatase leader, C. . C. albicans glucoamylase leader (EP 362,179 published April 4, 1990) or a signal described in WO 90/13646 published November 15, 1990. In expression of mammalian cells, mammalian signal sequences, such as signal sequences from secretory polypeptides of the same or related species, and viral secretion leaders can be used to direct the secretion of the protein.
[722] Both expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are known for various bacteria, yeasts and viruses. The origin of replication of plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Do.
[723] Expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Representative selection genes, eg, genes encoding D-alanine racemases for Bacillus, may comprise (a) proteins that confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) Encode proteins that complement nutritional deficiencies or (c) provide important nutrients that are not available from complex media.
[724] Examples of selectable markers suitable for mammalian cells are markers that allow identification of cells capable of receiving PRO-encoding nucleic acids, for example DHFR or thymidine kinase. When wild type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity, and Urlaub et al ., Proc. Natl. Acad. Sci. USA , 77: 4216 (1980)]. Suitable selection genes for use in yeast are the trp1 gene present in yeast plasmid YRp7 (Stinchcomb et al. , Nature , 282: 39 (1979)); Kingsman et al., Gene , 7: 141 (1979); Tschemper et al., Gene , 10: 157 (1980). trp1 gene provides a selection marker for variant strains of yeast that cannot grow using tryptophan (eg, ATCC 44076 or PEP4-1) [Jones, Genetics , 85: 12 (1977)] .
[725] Expression and cloning vectors contain a promoter operably linked to a PRO coding nucleic acid sequence that directs mRNA synthesis. Promoters recognized by various potential host cells are known. Suitable promoters for use in prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al. , Nature , 275: 615 (1978)); Goeddel et al. , Nature , 281: 544 (1979), alkaline phosphatase, tryptophan (trp) promoter system [Goeddel, Nucleic Acid 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)]. In addition, promoters used in bacterial systems will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the PRO.
[726] Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinases [Hitzeman et al . J. Biol. Chem. , 255: 2073 (1980)] or other relevant enzymes (Hess et al ., J. Adv. Enzyme Reg. , 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)]], for example enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- Promoters for 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase and glucokinase.
[727] Other yeast promoters that are inducible promoters with the added benefit of transcription controlled by growth conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes involved in nitrogen metabolism, metallothionein, glyceraldehyde Promoter region for 3-phosphate dehydrogenase, and enzymes that act on maltose and galactose use. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
[728] Transcription of PRO from a vector in a mammalian host cell may include viruses such as polyoma virus, poultry virus (UK No. 2,211,504 published July 5, 1989), adenovirus (eg adenovirus 2), bovine Genomes of viruses such as papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 (SV40), heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, and heat- It is regulated by a promoter that is compatible with the host cell system, obtained from the impact promoter.
[729] Transcription of the DNA encoding PRO by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-functional components of DNA, generally about 10 to 300 bp, that act on the promoter to increase transcription. Currently, many enhancer sequences are known to be derived from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer derived from a virus of a eukaryotic cell. Examples include the SV40 enhancer (bp 100-270) later in the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer later in the origin of replication, and the adenovirus enhancer. The enhancer can be spliced into the vector at the 5 'or 3' position in the PRO coding sequence, but is preferably located at the 5 'site from the promoter.
[730] In addition, expression vectors for use in eukaryotic host cells (multinuclear cells derived from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may comprise sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically obtained from the 5 ', and optionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide fragments that are transcribed into polyadenylation fragments in the untranslated portion of the mRNA encoding PRO.
[731] Other methods, vectors and host cells suitable for application to the synthesis of PRO in the culture of recombinant vertebrate cells are described in Getting et al. , Nature , 293: 620-625 (1981); Mantei et al. , Nature , 281: 40-46 (1979); European Patent No. 117,060 and European Patent No. 117,058.
[732] 4. Detection of Gene Amplification / Expression
[733] Gene amplification and / or expression may be, for example, conventional Southern blotting, Northern blotting to quantify transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA , 77: 5201-5205 (1980)], dot blotting (DNA analysis) or appropriately labeled probes based on the sequences provided herein can be measured directly in the sample by hybridization in situ. . Alternatively, an antibody capable of recognizing a specific double chain can be used, including a DNA double strand, an RNA double strand and a DNA-RNA hybrid double strand or a DNA-protein double strand. In other words, an assay can be performed that can label the antibody, bind the double chain to the surface, and detect the presence of the antibody bound to the double chain when the double chain is formed on the surface.
[734] Alternatively, gene expression can be measured by immunological methods such as immunohistochemical staining of cells or tissue sections and analysis of cell culture or body fluids for direct quantification of expression of gene products. Antibodies useful for immunohistochemical staining and / or analysis of sample fluids can be monoclonal or polyclonal antibodies and can be prepared in any mammal. Conveniently, antibodies to exogenous sequences that are fused to native sequence PRO polypeptides or synthetic peptides based on the DNA sequences provided herein or to PRO DNA and that encode specific antibody epitopes can be prepared.
[735] 5. Purification of Polypeptides
[736] The form of PRO may be recovered from culture medium or from host cell lysate. When bound to the membrane, it can be released from the membrane using a suitable detergent solution (eg Triton-X 100) or by cleavage of the enzyme. Cells used for expression of PRO may be pulverized by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical grinding or cell lysates.
[737] It may be desirable to purify PRO from recombinant cell proteins or polypeptides. Examples of suitable purification methods are fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica or cation exchange resins, for example chromatography on DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, for example Sephadex Gel filtration using G-75, Protein A Sepharose column to remove contaminants such as IgG and metal chelating column to bind epitope tagged forms of PRO. Various protein purification methods can be used and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice , Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the production method used and the properties of the particular PRO produced.
[738] E. Use of PRO
[739] Nucleotide sequences (or complements thereof) that encode PRO have a variety of uses in the field of molecular biology, including their use as hybridization probes, in chromosome and gene mapping, and in the production of antisense RNAs and DNA. PRO nucleic acids will also be useful for the production of PRO polypeptides by the recombinant techniques described herein.
[740] The full length native sequence PRO gene or fragment thereof isolates a full length PRO cDNA or other cDNA (eg, a gene encoding a PRO from a native variant or other species of PRO) having the desired sequence identity with the native PRO sequence disclosed herein. It can be used as a hybridization probe for the cDNA library. Optionally, the probe will be about 20 to about 50 bases in length. Hybridization probes can be derived from at least a portion of a new region of the full-length native nucleotide sequence, where this region can be determined without undue experimentation, or from a genomic sequence comprising a promoter, enhancer component and intron of native sequence PRO. For example, the screening method will include isolating the coding region of the PRO gene using known DNA sequences to synthesize selected probes of about 40 bases. Hybridization probes can be labeled with a variety of labels, including radioactive nucleotides such as ³² P or ³⁵ S, or enzymatic labels such as alkaline phosphatase linked to the probe via an avidin / biotin linkage system. Labeled probes having sequences complementary to those of the PRO genes of the invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine the members of the library that the probe hybridizes to. Hybridization techniques are described in more detail in the Examples below.
[741] Any EST sequence disclosed herein can similarly be used as a probe using the methods described herein.
[742] Other useful fragments of PRO nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (RNA or DNA) capable of binding to a target PRO mRNA (sense) or PRO DNA (antisense) sequence. Antisense or sense oligonucleotides according to the present invention comprise fragments of coding regions of PRO DNA. This fragment generally comprises at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to induce antisense or sense oligonucleotides based on cDNA sequences encoding a given protein is described, for example, in Stein and Cohen ( Cancer Res. 48: 2659, 1988) and van der Krol et al. ( BioTechniques 6: 958, 1988).
[743] Combination of the antisense or sense oligonucleotide with the target nucleic acid sequence forms a double strand that blocks the transcription or translation of the target sequence by one of several or other methods, including enhanced digestion of the double strand, early termination of transcription or translation. . Thus, antisense oligonucleotides can be used to block the expression of the PRO protein. In addition, antisense or sense oligonucleotides include oligonucleotides having modified sugar-phosphodiester backbones (or other sugar chains, eg, sugar chains disclosed in WO 91/06629), wherein such sugar chains are internal Resistance to clease. Such oligonucleotides with resistant sugar chains are stable in vivo (ie, resistant to enzymatic degradation) but retain sequence specificity capable of binding to the target nucleotide sequence.
[744] Other examples of sense or antisense oligonucleotides include organic residues such as those disclosed in WO 90/10048, and other residues that increase the affinity of the oligonucleotide for the target nucleic acid sequence, such as poly- (L-lysine). Oligonucleotides that are covalently linked with In addition, intercalating agents such as ellipsine, and alkylating agents or metal conjugates can be linked to sense or antisense oligonucleotides to change the binding specificity of the antisense or sense oligonucleotides to the target nucleotide sequence.
[745] Cells containing the target nucleic acid sequence, for example, by any gene transfer method including CaPO ₄ -mediated DNA transfection, electroporation, or by using a gene transfer vector such as Epstein-Barr virus. Antisense or sense oligonucleotides can be introduced. In a preferred method, antisense or sense oligonucleotides are inserted into a suitable retroviral vector. The cell comprising the target nucleic acid sequence is contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include vectors derived from double copy vectors designated murine retroviruses M-MuLV, N2 (retrovirus derived from M-MuLV), or DCT5A, DCT5B and DCT5C (see WO 90/13641). Included but not limited to.
[746] In addition, as disclosed in WO 91/04753, it is possible to introduce a sense or antisense oligonucleotide into a cell comprising a target nucleotide sequence by forming a conjugate with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the linkage of the ligand binding molecule does not substantially impede the ability of the ligand binding molecule to bind to the corresponding molecule or receptor, nor does it block the intracellular entry of the sense or antisense oligonucleotides or conjugate forms thereof.
[747] Alternatively, as disclosed in WO 90/10448, oligonucleotide-lipid conjugates can be introduced to introduce a sense or antisense oligonucleotide into a cell comprising a target nucleic acid sequence. Such sense or antisense oligonucleotide-lipid conjugates are preferably separated by internal lipases intracellularly.
[748] Antisense or sense RNA or DNA molecules are generally about 5 bases, about 10 bases, about 15 bases, about 20 bases, about 25 bases, about 30 bases, about 35 bases, about 40 bases in length. Base, about 45 bases, about 50 bases, about 55 bases, about 60 bases, about 65 bases, about 70 bases, about 75 bases, about 80 bases, about 85 bases, about 90 bases Base, about 95 bases, about 100 bases or more.
[749] Probes may also be used in PCR techniques to generate a group of sequences for identification of closely related PRO coding sequences.
[750] In addition, nucleotide sequences encoding PRO can be used to prepare hybridization probes for mapping genes encoding PRO and for genetic analysis of individuals with genetic disorders. Nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using known techniques such as hybridization in situ, linkage analysis for known chromosomal markers, and hybridization screening using libraries.
[751] If the coding sequence of a PRO encodes a protein that binds to another protein (eg where PRO is a receptor), the PRO can be used in an assay to identify other proteins or molecules involved in this binding interaction. By this method, inhibitors of receptor / ligand binding interactions can also be identified. In addition, proteins involved in such binding interactions can be used to screen for peptides or small molecule inhibitors or agonists of binding interactions. Receptor PRO can also be used to isolate similar ligands. Screening assays can be designed to find lead compounds that mimic the biological activity of a receptor for native PRO or PRO. Such screening assays include high throughput screening assays for chemical libraries and would be particularly suitable for identifying small molecule drug candidates. Small molecules include synthetic organic or inorganic compounds. Assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell based assays characterized in the art.
[752] Nucleic acids encoding PRO or variants thereof can also be used to generate transgenic or “knock out” animals useful for the development and screening of useful therapeutics. Transgenic animals (eg, mice or rats) are animals that have cells that contain the transgene, which transgenic genes are present in the animal or its ancestors during the prenatal period, for example the embryonic stage. Is introduced. The transgene is DNA that is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, a cDNA encoding a PRO can be used to clone genomic DNA encoding the PRO according to established techniques and to use the genomic sequence to transgene comprising cells expressing the DNA encoding the PRO. Animals can be produced. In particular, methods for producing transgenic animals such as mice or rats are routine methods in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. Typically, specific cells are targeted in the introduction of a PRO transgene with a tissue specific enhancer. In the embryonic stage, a transgenic animal comprising a copy of a transgene that encodes a PRO introduced into the germ cells of the animal can be used to investigate the effect of increasing the expression of the DNA encoding the PRO. Such animals can be used, for example, as test animals for reagents that are thought to protect against pathological symptoms associated with overexpression of PRO polypeptides. According to this aspect of the invention, treatment of the animal with the reagent results in a lower incidence of pathological symptoms than untreated animals carrying the transgene, indicating a potential therapeutic intervention in the pathological condition.
[753] Or having a defect or modified gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO using the non-human homolog of PRO and the modified genomic DNA encoding PRO introduced into the animal's embryonic cells. You can make PRO "knock out" animals. For example, cDNA encoding PRO can be used to clone genomic DNA encoding PRO according to established techniques. Portions of genomic DNA encoding a PRO may be deleted or substituted with other genes, such as genes encoding selective markers that may be used to monitor integration. In general, thousands of bases of unmodified flanking DNA (at both the 5 'and 3' ends) are included in a vector (e.g., for homologous recombinant vectors, see Thomas and Capecchi, Cell , 51: 503). (1987)). This vector is introduced into a stem cell line (e.g., by electroporation), and cells in which homologous recombination of the introduced DNA and the endogenous DNA are selected (see, eg, Li et al., Cell , 69: 915 (1992). Selected cells are then injected into blastocysts of animals (eg mice or rats) to form aggregated chimeras (see, eg, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). The chimeric embryos were then transplanted into suitable fertility surrogate animals to produce "knock out" animals. Progeny that carry homologous recombinant DNA in germ cells can be identified using standard techniques and used to breed animals in which all cells of the animal contain homologous recombinant DNA. Knockout animals are characterized, for example, by the ability to defend against certain pathological symptoms and the development of pathological symptoms due to the absence of PRO polypeptides.
[754] Nucleic acids encoding PRO polypeptides can also be used in gene therapy. When used in gene therapy, genes are introduced into cells to synthesize therapeutic gene products in vivo, eg to replace defective genes. "Gene therapy" includes both traditional gene therapy where a single effect is achieved and administration of gene therapy, including single or repeated administration of DNA or mRNA effective for treatment. Antisense RNAs and DNAs can be used as therapeutic agents to block the expression of certain genes in vivo. Despite the low intracellular concentration of these oligonucleotides due to the limited uptake of short antisense oligonucleotides through the cell membrane, it has already been found that they can act as inhibitors when introduced into cells (Zamecnik et al., Proc. Natl. Acad). , Sci. USA 83, 4143-4146 (1986)). Such oligonucleotides can enhance the uptake of oligonucleotides by modification, for example by replacing their negatively charged phosphodiester groups with non-charged groups.
[755] There are a variety of techniques that can be used to introduce nucleic acids into living cells. This technique depends on whether the nucleic acid is delivered to cells cultured in vitro or to cells of the desired host in vivo. Suitable techniques for delivering nucleic acids to mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran and calcium phosphate precipitation and the like. Currently preferred for in vivo gene delivery techniques include transfection using viral (usually retroviral) vectors and viral coat protein-liposomal mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 ( 1993). In some cases, it is desirable to provide the nucleic acid source with agents that target the target cell, such as membrane proteins on the cell surface or antibodies specific for the target cell, ligands for receptors on the target cell, and the like. If liposomes are used, proteins that bind to membrane proteins on the cell surface involved in endocytosis can be used for targeting and / or promote uptake, such as, for example, directed to specific cell types. Capsid proteins or fragments thereof, antibodies to proteins that undergo internalization in circulation, and proteins that target intracellular locations and increase intracellular half-life. Receptor mediated endocytosis techniques are described, for example, in Wu et al., J. Biol. Chem. 262, 4429-4432 (1987), and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For a review of gene marking and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).
[756] The PRO polypeptides disclosed herein can also be used as molecular weight markers for protein electrophoresis, and the isolated nucleic acid sequences can be used to recombinantly express these markers.
[757] Nucleic acid molecules or fragments thereof encoding the PRO polypeptides disclosed herein are useful for the identification of chromosomes. In this regard, there is a continuing need to identify new chromosomal markers because there are currently relatively few chromosomal marking reagents available based on actual sequence data. Each of the PRO nucleic acid molecules of the present invention can be used as a chromosome marker.
[758] The PRO polypeptides and nucleic acid molecules of the invention can also be used for tissue typing, wherein the PRO polypeptides of the invention can be differentially expressed in one tissue compared to other tissues. PRO nucleic acid molecules will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis.
[759] The PRO polypeptides disclosed herein may also be used as therapeutic agents. Pharmaceutically useful compositions can be prepared by formulating the PRO polypeptides of the invention according to known methods, wherein the PRO product is combined with a pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient with the desired purity with any physiologically acceptable carrier, excipient or stabilizer ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to the subject of administration at the dosages and concentrations employed, and include antioxidants such as phosphates, citrate and other organic acids, ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, serum Proteins such as 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 dextrins , Chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and / or nonionics such as Tween®, Pluronics® or PEG Surfactants and the like.
[760] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes before or after lyophilization and thawing.
[761] Therapeutic compositions of the present disclosure may generally be contained in a container with a sterile access door, for example, in a stoppered vial that can be pierced by an intravenous solution bag or a hypodermic needle.
[762] Routes of administration include known methods, for example by injection or infusion by intravenous, intraperitoneal, intracranial, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or sustained release.
[763] Dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending upon the particular intended use. Determination of the appropriate dosage and route of administration is well known to those skilled in the art. The results of animal experiments provide reliable guidance in determining dosages effective for human treatment. Scaling effective doses between species is described in Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96).
[764] When a PRO polypeptide or agonist or antagonist thereof is administered in vivo, the general dosage depends on the route of administration and is about 10 ng to 100 mg per kg body weight of the mammal, or preferably about 1 μg / kg per day To 10 mg / kg. Guidance as to specific dosages and methods of delivery is provided in the literature, for example in US Pat. No. 4,657,760, 5,206,344 or 5,225,212. Other agents may be effective for other therapeutic compounds and for other diseases, and administration targeted to one organ or tissue is expected to require delivery in a different manner than, for example, administration to another organ or tissue. do.
[765] If sustained release administration of a PRO polypeptide is required for an agent with release properties suitable for the treatment of a disease or disorder requiring administration of the PRO polypeptide, microencapsulation of the PRO polypeptide is contemplated. Microencapsulation of recombinant proteins for delayed release has been successfully performed using human growth hormone (rhGH), interferon (rhIFN), interleukin-2 and MN rgp120 (Johnson et al., Nat. Med. , 2: 795- 799 (1996); Yasuda, Biomed. Ther. , 27: 1221-1223 (1993); Hora et al., Bio / Technology , 8: 755-758 (1990); Cleland, "Design and Production of Single Immunization Vaccines Using Polyactide Polyglycolide Microsphere Systems, "in Vaccine Design: The Subunit and Adjuvant Approach , Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462: WO 97/03692, WO 96/40072, WO 96 / 07399 and US Pat. No. 5,654,010).
[766] Sustained release preparations of these proteins have been developed using poly-lactic acid-co-glycolic acid (PLGA) polymers due to their biocompatibility and broad biodegradable properties. The degradation products of PLGA, lactic acid and glycolic acid, can be quickly removed from the human body. In addition, the resolution of the polymer can be adjusted from months to years depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents from lactide / glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41).
[767] The invention includes methods for screening compounds to identify (antagonist) compounds that mimic PRO polypeptides or block the effects of (agonist) PRO polypeptides. Screening assays for antagonist drug candidates were designed to identify compounds that bind or conjugate with the PRO polypeptide encoded by the genes identified herein or interfere with the interaction of the encoded polypeptide with other intracellular proteins. Such screening assays include assays capable of high throughput screening for chemical libraries, which would be particularly suitable for identifying small molecule drug candidates.
[768] Such assays can be performed in a variety of formats including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays characterized in the art.
[769] Common to all assays for antagonists is that the PRO polypeptide encoded by the nucleic acids identified herein and the candidate drug must be contacted under conditions and time sufficient to allow them to interact with each other.
[770] In binding assays, the interaction is binding, and the conjugate formed can be isolated or detected in the reaction mixture. In certain embodiments, a PRO polypeptide or drug candidate encoded by a gene identified herein is immobilized to a solid phase, eg, a microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is generally achieved by coating the solid surface with a solution of PRO polypeptide and drying. Alternatively, immobilized antibodies, such as monoclonal antibodies specific for immobilized PRO polypeptides, can be used to anchor them to a solid surface. This analysis is performed by adding an unfixed component that can be labeled by a detectable label to a coded surface comprising a fixed component, for example an anchored component. When the reaction is finished, unreacted components are removed, for example by washing, and the binder anchored to the solid surface is detected. If the component that was not originally immobilized contained a detectable label, detection of the label immobilized on the surface indicates that a complexing reaction occurred. If a component that is not originally immobilized does not include a label, the complexing reaction can be detected, for example, using a labeled antibody that specifically binds to the immobilized conjugate.
[771] If a candidate compound interacts with, but does not bind to, a particular PRO polypeptide encoded by a gene identified herein, the interaction of the candidate compound with that polypeptide can be analyzed by well known methods of detecting protein-protein interactions. . Such assays include conventional methods such as crosslinking, coimmunoprecipitation, and copurification via gradient or chromatography columns. Protein-protein interactions are also described in 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 discussed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA , 89: 5789-5793 (1991), can be used to monitor using the yeast-based gene system described. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other as a transcription-active domain. The yeast expression system described in this publication (generally referred to as "2-hybrid system") takes advantage of this property and uses two hybrid proteins, one of which is that the target protein is associated with the DNA-binding domain of GAL4. The other is a candidate active protein fused with an active domain. The expression of the GAL1-lacZ reporter gene under the control of the GAL4-activation promoter depends on the reconstitution of GAL4 activity through protein-protein interactions. Colonies containing the interacting polypeptide are detected using a pigment producing substrate for β-galactosidase. A complete kit (MATCHMAKER®), which confirms protein-protein interactions between two specific proteins using 2-hybrid technology, can be purchased from Clontech. In addition, the system can be extended to map protein domains involved in specific protein interactions, as well as to pinpoint the location of amino acid residues critical for such interactions.
[772] In the following way, compounds which interfere with the interaction of genes encoding PRO polypeptides identified herein with other intracellular or extracellular components can be tested. A reaction mixture comprising the product of a gene encoding a PRO polypeptide and an intracellular or extracellular component was prepared under conditions and times that allow interaction and binding of the two products. To test the ability of the candidate compound to inhibit binding, the reaction was performed in the presence and absence of the test compound. In addition, placebo may be added to a third reaction mixture that serves as a positive control. The binding (conjugate formation) between the test compound and the intracellular or extracellular components present in the mixture was monitored as described above. The conjugate was formed in the control reactant, but no binder was formed in the reaction mixture comprising the test compound, indicating that the test compound interfered with the interaction of the test compound with its reaction counterpart.
[773] To analyze the antagonist, the PRO polypeptide could be added to the cell along with the compound to screen for specific activity, and the ability of the compound to inhibit the desired activity in the presence of the PRO polypeptide indicated that the compound was an antagonist to the PRO polypeptide. Alternatively, antagonists can be detected by combining the PRO polypeptide and potential antagonist with a membrane-bound PRO polypeptide receptor or recombinant receptor under appropriate conditions for competitive inhibition assays. PRO polypeptides can be labeled with radioactive isotopes, for example, to determine the effect of a potential antagonist using the number of PRO polypeptide molecules that bind to the receptor. Genes encoding receptors could be identified by several methods known to those of skill in the art, for example ligand panning and FACS classification (Coligan et al., Current Protocols in Immun. , 1 (2): Chapter 5 (1991)). . Preferably, expression cloning methods are used, wherein the polyadenylation RNA is prepared from cells reactive to the PRO polypeptide, and the cDNA library generated from this RNA is classified as pool and is non-responsive to the PRO polypeptide or It is used to transfect other cells. Transfected cells growing on glass slides are exposed to labeled PRO polypeptide. PRO polypeptides can be labeled by a variety of methods, including iodide or encapsulation of recognition sites for site-specific protein kinases. After immobilization and incubation, the slides were analyzed by autoradiography. Sub- pools were identified by identifying positive pools and transfected again using interactive sub- pooling and rescreening methods, resulting in a single clone encoding the putative receptor.
[774] As an alternative method of identifying receptors, labeled PRO polypeptides could be photoaffinity-linked with extract samples expressing cell membranes or receptor molecules. The crosslinked material was analyzed by PAGE and exposed to X-ray film. Label conjugates containing receptors can be cut and digested into peptide fragments for protein micro-sequencing. By designing a set of degenerate oligonucleotide probes using amino acid sequences obtained from micro-sequencing, one could screen cDNA libraries and identify genes encoding putative receptors.
[775] In another antagonist assay method, mammalian cell or membrane samples expressing receptors in the presence of candidate compounds were incubated with labeled PRO polypeptide. Thereafter, the ability of the compound to enhance or block the interaction could be measured.
[776] More specific examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins with PRO polypeptides, and in particular poly- and monoclonal antibodies, and antibody fragments, side chain antibodies, anti-genetic antibodies, and such antibodies or fragments thereof. Chimeric or humanized forms of, and antibodies, including human antibodies and antibody fragments. Alternatively, the potential antagonist may be a modified form of a closely related protein, eg, a PRO polypeptide that recognizes but does not affect the receptor but competitively inhibits the action of the PRO polypeptide.
[777] Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where the antisense RNA or DNA molecule acts to directly block the translation of the mRNA by hybridizing with the target mRNA to prevent protein translation. . Antisense techniques can be used to regulate gene expression via triple helix formation or antisense DNA or RNA, all of which are based on the binding of polynucleotides to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature PRO polypeptide herein can be used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of genes involved in transcription (Lee et al., Nucl. Acids Res. , 6: 3073 (1979); Cooney et al, Science , 241). : 456 (1988); Dervan et al., Science , 251: 1360 (1991))) transcription and production of PRO polypeptides. Antisense RNA oligonucleotides hybridized with their mRNAs in vivo to block the translation of mRNA molecules to PRO polypeptides (antisense-Okano, Neurochem. , 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988). In addition, the oligonucleotides described above could be delivered to cells to express antisense RNA or DNA in vivo to inhibit the production of PRO polypeptides. If antisense DNA is used, oligodeoxyribonucleotides derived from transcription-initiation sites, eg, positions between about -10 and +10 of the target gene nucleotide sequence, are preferred.
[778] Potential antagonists include small molecules that bind to the active site, receptor binding site, or small molecules that bind the growth factor or other related binding site of the PRO polypeptide to block the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic bipeptidyl organic or inorganic compounds.
[779] Ribozymes are enzyme RNA molecules that can catalyze specific cleavage of RNA. The ribozyme acts after sequence-specific hybridization with the target RNA complementary thereto, followed by cleavage by endonuclease. Known techniques have been able to identify specific ribozyme cleavage sites within potential RNA targets. See, for example, Rossi, Current Biology , 4: 469-471 (1994) and PCT publication No. WO 97/33551 (published September 18, 1997).
[780] The triple helix form of nucleic acid molecules used to inhibit transcription must be single-stranded and consist of deoxynucleotides. The base composition of such oligonucleotides is designed to facilitate triple helix formation via the Hoogsteen base pair rule, which typically requires significant size purine or pyrimidine stretch in one of the double chains. For further details see, for example, PCT publication No. WO 97/33551, supra.
[781] The small molecules can be identified by one or more screening assays described above and / or by any other screening technique well known to those skilled in the art.
[782] In addition, the use of the molecules disclosed herein may be based on the positive function assays described above and below.
[783] F. Anti-PRO Antibodies
[784] The invention further provides anti-PRO antibodies. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies.
[785] 1. Polyclonal Antibodies
[786] Anti-PRO antibodies constitute polyclonal antibodies. Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in a mammal, for example, by injecting the immunizing agent and, if necessary, an adjuvant one or more times. In general, the immunizing agent and / or adjuvant will be injected into the mammal by several injections subcutaneously or intraperitoneally. Immunizing agents can include PRO polypeptides or fusion proteins 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 tyroglobulin and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freud's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicholinomycolate). Immunotreatment methods can be selected by one skilled in the art without undue experimentation.
[787] 2. Monoclonal Antibodies
[788] The anti-PRO antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods as described in Kohler and Milstein, Nature , 256 : 495 (1975). In hybridoma methods, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes capable of producing or producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.
[789] Immunizing agents generally include PRO polypeptides or fusion proteins thereof. Generally, peripheral blood lymphocytes (“PBLs”) are used when cells of human origin are desired, but spleen cells or lymph node cells are used when a mammalian source other than human is desired. Then, a suitable fusion agent such as polyethylene glycol is used to fuse the lymphocytes with the immortalized cell line to form hybridoma cells. Monoclonal Antibodies: Principle and Practice , Academic Press, (1986) pp. 59-103]. Immortalized cell lines are largely transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. In general, rat or mouse myeloma cell lines are used. Hybridoma cells are preferably cultured in a suitable culture medium containing one or more substances that inhibit the proliferation or survival of unfused immortalized cells. For example, if the parent cell lacks hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzymes, the culture medium for hybridomas ("HAT medium") typically includes hypoxanthine, aminopterin and thymidine. These substances will inhibit the growth of HGPRT-deficient cells.
[790] Preferred immortalized cell lines are those that fuse efficiently, maintain stable levels of antibody production by selected antibody-producing cells, and are susceptible to media such as HAT medium. More preferred immortalized cell lines are, for example, murine myeloma cell lines available from the Salk Institute Cell Distribution Center (San Diego, CA) and the American Type Culture Collection (Manassas, VA). . In addition, human myeloma cell lines and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133 : 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63.
[791] Culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Was measured. Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be found in, for example, Scatchard analysis (Munson and Pollard, Anal. Biochem. , 107 : 220 (1980).
[792] After identifying the desired hybridoma cells, clones were subcloned by restriction dilution procedures and cultured by standard methods (Goding, supra ). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be cultured in vivo as ascites tumors in a mammal.
[793] Monoclonal antibodies secreted by the subclones are for example by conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It can be isolated and purified from culture medium or ascites fluid.
[794] Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be readily obtained using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). It can be isolated and sequenced. The hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector and then transfected with an expression vector to host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that otherwise do not produce immunoglobulin proteins. Monoclonal antibodies can be synthesized in recombinant host cells. In addition, DNA, for example, homologous replacing the murine sequence in the coding sequence for human heavy and light chain constant domain, or (U.S. Patent No. 4,816,567; Morrison (Morrison), etc., supra), or a non-immunoglobulin polypeptide All or part of the coding sequence for may be modified by covalently binding to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of the antibodies of the invention, or the variable domains of one antibody-binding site of the antibodies of the invention can be used to generate chimeric bivalent antibodies.
[795] The antibody may be a monovalent antibody. Methods of making monovalent antibodies are known in the art. For example, one method involves recombinant expression of immunoglobulin light and modified heavy chains. To prevent crosslinking of the heavy chain, the heavy chain is generally truncated at some point in the F _c region. Alternatively, related cysteine residues are substituted or deleted with other amino acid residues to prevent crosslinking.
[796] In vitro methods are also suitable for the preparation of monovalent antibodies. Degradation of the antibody to prepare antibody fragments, particularly Fab fragments, can be carried out using techniques commonly known in the art.
[797] 3. Human and Humanized Antibodies
[798] In addition, the anti-PRO antibodies of the invention may comprise humanized antibodies or human antibodies. Humanized forms of non-human (eg murine) antibodies include immunoglobulin chains or fragments thereof comprising chimeric immunoglobulins, minimal sequences derived from non-human immunoglobulins or fragments thereof (eg, Fv, Fab, Fab ', F (ab') ₂ or other antigen binding small sequence of the antibody). A humanized antibody is a human immunono that has substituted residues from the complementarity determining regions (CDRs) of the receptor with residues from CDRs of species other than the human (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and ability. Globulin (receptive antibody). In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, the humanized antibody may include residues which are not present in the antibody to be received or in the sequence of the CDR or framework to be introduced. In general, humanized antibodies will comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all CDR regions correspond to regions of non-human immunoglobulins, and all or substantially all FRs. The region corresponds to the region of the human immunoglobulin consensus sequence. In addition, humanized antibodies will optimally comprise at least a portion of an immunoglobulin constant region (F _c ), generally a portion of 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)].
[799] Methods of humanizing nonhuman antibodies are well known in the art. In general, humanized antibodies have one or more amino acid residues introduced thereto from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanization consists essentially of methods such as Winter et al. ( Nature , 321 : 522-525 (1986)) by replacing the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. Riechmann et al. , Nature , 332 : 323-327 (1988); It may be carried out according to Verhoeyen et al., Science , 239 : 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially fewer intact human variable domains have been replaced by corresponding sequences from 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 of similar sites in rodent antibodies.
[800] Human antibodies can also be prepared 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. Bio. , 222 : 581 (1991)]. In addition, techniques such as Kohl et al. And Boerner can also be used for the production of human monoclonal antibodies [Cole et al. Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. , 147 (1) : 86-95 (1991)]. Likewise, human antibodies can be prepared by introducing human immunoglobulin loci into a transgenic animal, eg, a mouse, wherein the internal immunoglobulin genes are partially or fully inactivated. After challenge, the production of human antibodies was observed, which was very similar to the antibodies observed in humans in all respects, including gene rearrangement, assembly and antibody listing. This is described, for example, in US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and in scientific publications [Marks et al. Bio / Technology 10 , 779-783 (1992); Lonberg et al. , Nature 368 , 856-859 (1994), Morrison, Nature 368 , 812-13 (1994); Fishwild et al. , Nature Biotechnology 14 , 845-51 (1996); Neuberger, Nature Biotechnology 14 , 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[801] 4. Bispecific Antibodies
[802] Bispecific antibodies are monoclonal, preferably human or humanized antibodies with binding specificities for at least two different antigens. In this case, one of the binding specificities is for PRO and the other is for any other antigen, preferably for cell surface proteins or receptors or receptor subunits.
[803] Methods of making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain / light chain-heavy chain pairs, where the two heavy chains have different specificities [Millstein and Quello Cuello, Nature , 305 : 537-539 (1983). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce possible mixtures of 10 different antibody molecules, only one of which has an accurate bispecific structure. Purification of the correct molecule is generally carried out by affinity chromatography steps. Similar methods are described in WO 93/08829 (published May 13, 1993) and Traunecker et al., EMBO J. , 10: 3655-3659 (1991).
[804] The variable domain (antibody-antigen binding site) of an antibody with the desired binding specificity can be fused to an immunoglobulin constant domain sequence. This fusion is preferably fused with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. It is preferred that a first heavy chain constant region (CH1) comprising a site necessary for light chain binding is present in at least one of the fusions. Immunoglobulin heavy chain fusions, and, if desired, DNAs encoding immunoglobulin light chains are inserted into separate expression vectors and co-transfected into suitable host organisms. For further details for producing bispecific antibodies, see, for example, Suresh et al. , Methods in Enzymmology , 121 : 210 (1986).
[805] According to another method disclosed in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the proportion of heterodimer recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule were replaced with larger side chains (eg tyrosine or tryptophan). Replacement of large amino acid side chains with small amino acid side chains (eg, alanine or threonine) created a supplemental "cavity" of the same or similar size as the large side chain at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers above the yield of other unwanted end-products such as homodimers.
[806] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985), describe a method of cleaving intact antibodies by proteolysis to generate F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol composite material sodium arsenite to stabilize nearby dithiols and prevent the formation of disulfides in the bonsai. The Fab 'fragments generated were then converted to thionitrobenzoate (TNB) derivatives. Thereafter, one of the Fab'-TNB derivatives was reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of other Fab'-TNB derivatives to form bispecific antibodies. The resulting bispecific antibodies can be used as substances for the selective immobilization of enzymes.
[807] this. Bispecific antibodies were formed by directly recovering Fab 'fragments from E. coli and chemically linking them. Shalaby et al ., J. Exp. Med. 175: 217-225 (1992) discloses fully humanized bispecific antibody F (ab ') ₂ molecules. Each Fab 'fragment was secreted separately from this coli and linked by the in vitro chemical method indicated to form a bispecific antibody. The bispecific antibody thus formed was able to bind cells overexpressing the ErbB2 receptor and normal human T cells, as well as initiate the lytic activity of human cytotoxic lymphocytes against human breast cancer targets.
[808] In addition, several techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture are disclosed. For example, bispecific antibodies were prepared using leucine zippers [Kostelny et al ., J. Immunol . 148 (5): 1547-1553 (1992)]. By gene fusion, leucine zipper peptides from Fos and Jun proteins were linked to the Fab 'portion of two different antibodies. The hinge region of the antibody homodimer was reduced to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used as a method for producing antibody homodimers. Hollinger et al ., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides a different mechanism for making bispecific antibody fragments. This fragment comprises a heavy chain variable domain (V _H ) linked by a linker to the light chain variable domain (V _L ), which is too short to pair the two domains with the same chain. Thus, the V _H and V _L domains of one fragment are paired with the complementary V _L and V _H domains of the other fragment and form two antigen-binding sites. Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dimers has been reported (see Gruber et al., J. Immunol. 152: 5368 (1994)). Antibodies having the above valences are also considered, for example, trispecific antibodies can be made (Tutt et al., J. Immunol. 147: 60 (1991)).
[809] Typical bispecific antibodies may bind to two different epitopes of the PRO polypeptides provided herein. Alternatively, in order to focus on intracellular defense mechanisms against cells expressing a particular PRO polypeptide, the arms of the anti-PRO polypeptide may be replaced with an initiating molecule on leukocytes, such as a T-cell receptor molecule (eg, CD2, CD3). , CD28 or B7), or an arm that binds to an F _c receptor (F _c γ R) of IgG, such as F _c γ RI (CD64), F _c γ RII (CD32) and F _c γ RIII (CD16). In addition, bispecific antibodies can be used to locate cytotoxic agents in cells that express particular PRO polypeptides. Such antibodies have a PRO-binding arm and an arm that binds to a cytotoxic substance or radionuclide chelating agent such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to the PRO polypeptide and further binds to tissue factor (TF).
[810] 5. Heteroconjugate Antibodies
[811] Heteroconjugate antibodies are also included within the scope of the present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies can be used, for example, to target immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, US 92/200373, and EP 03089). Ho) suggested. It is contemplated that heterozygous antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving crosslinking agents. For example, immunotoxins can be prepared by forming disulfide exchange reactions or thioether bonds. Suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrimidate and the reagents disclosed in US Pat. No. 4,676,980.
[812] 6. Effector function operation
[813] It may be desirable to modify the antibodies of the invention to effector function to enhance the effect of the antibody, for example in the treatment of cancer. For example, cysteine residues may be introduced into the F _c region to form disulfide bonds between the chains within this region. Homodimeric antibodies thus produced enhance the internalization capacity and / or increase complement-mediated cell death and antibody-dependent intracellular cytotoxicity (ADCC) [Caron et al., J. Exp Med. . , 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148: 2918-2922 (1992). In addition, the heterodifunctional cross-links described in Wolf et al., Cancer Research , 53 : 2560-2565 (1993) can be used to prepare homodimeric antibodies with enhanced anti-tumor activity. Alternatively, antibodies with double F _c regions can be engineered to enhance complement lytic and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design , 3: 219-230 (1989)). do].
[814] 7. Immune Complex
[815] The invention also relates to cytotoxic substances such as chemotherapeutic agents, toxins (eg, enzymatically active bacteria, fungi, plant or animal toxins, or fragments thereof) or radioactive isotopes (ie, radiocomplexes). It relates to an immunocomplex comprising an antibody coupled with.
[816] Chemotherapeutic agents useful in the production of such immunocomplexes are described above. Enzymatically active usable toxins and fragments thereof include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modeine A chain, alpha-sarsine, Aleurites fordii protein, diantine protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), inhibitor of bitter melon (momordica charantia), cumu Leucine, crotin, sapaonaria officinalis inhibitors, gelonin, mitogelin, resrictocin, phenomycin, enomycin, and tricotessenes. Several radionuclides can be used for the preparation of radiocomplex antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.
[817] Difunctional derivatives of various difunctional protein-linking materials, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoesters (e.g. Dimethyl adipimidate HCL), active esters (e.g. disuccinimidyl suverate), aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis (p- Azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg tolyene 2,6-diisocyanate), and Bis-active fluorine compounds (eg, 1,5-difluoro-2,4-dinitrobenzene) can be used to make combinations of antibodies and cytotoxic substances. For example, lysine immunotoxins can be prepared as described in Vitta et al., Science , 238 : 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is a typical chelating agent linking radionucleotides to antibodies (see WO 94/11026). do).
[818] In another embodiment, the antibody may be linked to a "receptor" (eg, streptavidin) used for tumor pretargeting, wherein the antibody-receptor conjugate is administered to the subject and then circulated using a chelating agent. Unbound conjugates are removed from and an "ligand" (eg avidin) that binds to a cytotoxic substance (eg radionucleotide) is administered.
[819] 8. Immunoliposomes
[820] The antibodies disclosed herein may also be formulated with immunoliposomes. Liposomes containing this antibody can be prepared by methods known in the art, such as in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); And US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.
[821] Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes were pushed through filters of defined pore size to yield liposomes with the desired diameter. Martin et al ., J. Biol. Chem. , 257 : 286-288 (1982), can be linked to liposomes via the disulfide-interchange reaction. Chemotherapeutic agents (eg, Doxorubicin) are optionally included in liposomes (Gabizon et al ., J. National Cancer Inst. , 81 (19): 1484 (1989).
[822] 9. Antibody Pharmaceutical Compositions
[823] To treat a variety of diseases, antibodies that specifically bind to the PRO polypeptides identified herein and other molecules identified by the screening assays described above can be administered in the form of pharmaceutical compositions.
[824] If the PRO polypeptide is intracellular and all of the antibody is used as an inhibitor, it is preferred to internalize the antibody. However, lipofection or liposomes can also be used to deliver the antibody or antibody fragment intracellularly. If antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the sequence of the variable-region of an antibody, peptide molecules can be designed that have the ability to bind to the sequence of the target protein. Such peptides can be synthesized chemically and / or prepared by recombinant DNA techniques (eg, Marasco et al ., Proc. Natl. Acad. Sci. USA , 90: 7889-7893 (1993). In addition, the formulations herein may include one or more active compounds, preferably compounds with complementary activities that do not adversely affect each other, as needed for the particular condition to be treated. Alternatively, or in addition, the composition may include substances that enhance their function, such as cytotoxic substances, cytokines, chemotherapeutic agents, or growth-inhibiting agents. Such molecules are suitably formulated in amounts that are effective for the purpose intended.
[825] The active ingredient is for example a coacervation technique or interfacial polymerization, for example in a colloidal drug delivery system (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions respectively, For example, it can be contained in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. This technique is disclosed in Remington's Pharmaceutical Sciences , supra.
[826] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[827] Sustained release formulations may also be prepared. Suitable examples of sustained-release preparations include shaped articles of solid hydrophobic polymers containing the antibody, eg, semipermeable matrices in the form of films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L-glutamic acid Injection consisting of a copolymer of γ ethyl-L-glutamate, a non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer, for example LUPRON DEPOT® (lactic acid-glycolic acid copolymer and leuprolide acetate) Possible microspheres), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for more than 100 days, while some hydrogels release proteins for shorter periods of time. If an encapsulated antibody remains in the body for a long time, it may be denatured or aggregated due to exposure to moisture at 37 ° C., thereby reducing its biological activity and altering immunogenicity. For stabilization, rational strategies can be designed according to the mechanism involved. For example, if the aggregation mechanism is found to form intramolecular SS bonds through thio-disulfide interchange, modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, use of appropriate additives, and Stabilization can be achieved by the development of specific polymer matrix compositions.
[828] G. Uses of Anti-PRO Antibodies
[829] Anti-PRO antibodies of the invention have a variety of utility. For example, anti-PRO antibodies can be used for diagnostic assays for PRO, eg, for detecting expression of PRO in specific cells, tissues or serum. Various diagnostic assay techniques known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays performed on heterologous or homologous phases [Zola's Monoclonal Antibodies: A Manual of Techniques , CRC Press , Inc. (1987) pp. 147-158]. Antibodies used in diagnostic assays can be labeled with detectable residues. Detectable residues must be able to generate a detectable signal either directly or indirectly. For example, the detectable moiety can be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or Luciferin, or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Hunter et al. , Nature , 144 : 945 (1962); David et al., Biochemistry , 13 : 1014 (1974); Pine et al., J. Immunol. Meth. , 40 : 219 (1981); And Nygren, J. Histochem. and Cytochem. , 30 : 407 (1982), including any method known in the art for binding antibodies and detectable moieties.
[830] In addition, anti-PRO antibodies are useful for affinity purification of PRO from recombinant cell culture or natural sources. In this process, the antibody to PRO is immobilized to a suitable support such as Sephadex resin or filter paper using methods known in the art. Thereafter, the immobilized antibody is contacted with a sample containing purified PRO, followed by washing with a suitable solvent that substantially removes all material in the sample except the PRO bound to the immobilized antibody. Finally, the support is washed with other suitable solvent to release the PRO polypeptide from the antibody.
[831] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.
[832] All patents and documents cited in this specification are herein incorporated by reference in their entirety.
[833] EXAMPLE
[834] Commercial reagents mentioned in the examples were used according to the manufacturer's instructions unless otherwise indicated. The source of cells identified by the ATCC accession number throughout the following examples and specification is the American Type Culture Collection (Manassas, VA).
[835] Example 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNAs Encoding It
[836] The EST database was examined using extracellular domain (ECD) sequences (including secretion signal sequences, if present) from about 950 known secreted proteins from the Swiss-Prot co-database. . The EST database includes public databases (e.g. Dayhoff, Jenbank) and proprietary databases (e.g. LIFESEQ ™, Incyte Pharmaceuticals, Palo Alto, Calif.). Included. Investigations were performed using the computer programs BLAST or BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)) as a comparison of the ECD protein sequences to the six frame translations of the EST sequences. Comparatives with BLAST scores of 70 (or in some cases 90) or greater that do not encode known proteins are clustered and consensus DNA sequences using the program "phrap" (Phil Green, University of Washington, Seattle, WA) Assembled.
[837] Using this extracellular domain homology screening, consensus DNA sequences were assembled for other EST sequences identified using phrap. In addition, consensus DNA sequences obtained are often (but not always) stretched using a repeat cycle of BLAST or BLAST-2 and phrap to stretch the consensus sequence as much as possible using the sources of EST sequences discussed above. .
[838] Thereafter, oligonucleotides are synthesized based on the consensus sequence obtained as described above, and used to identify a cDNA library containing the sequence of interest by PCR, and to isolate a clone of the full-length coding sequence for the PRO polypeptide. Used as a probe for. Forward and reverse PCR primers generally range from 20 to 30 nucleotides and are often designed to provide PCR products of about 100 to 1000 bp in length. The length of the probe sequence is typically 40 to 55 bp. In some cases, additional oligonucleotides are synthesized when the consensus sequence is greater than about 1 to 1.5 kbp. To screen several libraries for full-length clones, DNA from these libraries was screened by PCR amplification using PCR primer pairs according to Ausubel et al., Current Protocols in Molecular Biology . Then, clones encoding genes of interest were isolated using one of the probe oligonucleotide and primer pairs by using a positive library.
[839] The cDNA library used to isolate the cDNA clones was constructed by standard methods using commercially available reagents such as reagents from Invitrogen (San Diego, CA, USA). The cDNA is primed with oligo dT with a NotI site, ligated to the hemikinase treated SalI adapter, cleaved with NotI, appropriately sized by gel electrophoresis, and appropriate cloning vector (e.g., pRKB or pRKD; pRK5B is a precursor of pRK5D without the SfiI site; see Holmes et al., Science , 253 : 1278-1280 (1991) and cloned in a defined direction to the only XhoI and NotI sites.
[840] Example 2: Isolation of cDNA Clones by Amylase Screening
[841] 1. Preparation of oligo dT primed cDNA libraries
[842] MRNA was isolated from human tissue of interest using reagents and protocol (Fast Track 2) from Invitrogen (San Diego, Calif.). The RNA was used to generate oligo dT primed cDNA libraries on vector pRK5D using reagents and protocols from Life Technologies, Gettersburg, Maryland, (Super Script Plasmid System). In this process, the double stranded cDNA was made larger than 1000 bp and the SalI / NotI linker attached cDNA was cloned into the XhoI / NotI cleavage vector. pRK5D is a cloning vector comprising a sp6 transcription initiation site, a SfiI restriction enzyme site, and a XhoI / NotI cDNA cloning site in that order.
[843] 2. Preparation of Random Primed cDNA Libraries
[844] Secondary cDNA libraries were generated to preferentially provide the 5 'end of the primary cDNA clone. Sp6 RNA was generated from the primary library (as described above) and random priming to vector pSST-AMY.0 using the RNA using reagents and protocols from Life Technologies (Super Script Plasmid System, as described above) CDNA libraries were generated. In this process, double-stranded cDNAs were made 500-1000 bp in size, ligated to the NotI adapter to the blunt ends, cut into SfiI and cloned into the SfiI / NotI cleavage vector. pSST-AMY.0 is a cloning vector having a yeast alcohol dehydrogenase promoter before the cDNA cloning site and mouse amylase sequence (mature sequence without secretion signal) and a yeast alcohol dehydrogenase terminator after the cloning site. Thus, cDNA cloned into the vector, fused with amylase sequence in frame, will secrete amylase from the appropriately transfected yeast colonies.
[845] 3. Transformation and detection
[846] The DNA from the library described in paragraph 2 above was ice-cooled and 20 ml of electrocompetent DH10B bacteria (Life Technologies) was added. Bacteria and vector mixtures were electroporated as directed by the manufacturer. Then 1 ml of SOC medium (Life Technologies) was added and the mixture was incubated at 37 ° C. for 30 minutes. The transformants were then plated into 20 standard 150 mm LB plates containing ampicillin and incubated for 16 hours (37 ° C.). Positive colonies were removed from the plates and DNA was isolated from bacterial pellets using standard protocols such as CsCl-gradient method. Purified DNA was used to perform the following yeast protocol.
[847] Yeast methods were classified into three categories: 1) transforming yeast using a plasmid / cDNA combination vector, 2) detecting and isolating yeast clones secreting amylase, and 3) inserting from yeast colonies PCR amplification of the sieve directly and purification of DNA for sequencing and further analysis.
[848] The yeast strain used was HD56-5A (ATCC-90785). This strain has a genotype of MAT alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL ⁺ , SUC ⁺ , GAL ⁺ . Preferably, yeast variants lacking post-translational pathways can be used. Such variants may have deficiency alleles due to translocation at sec71, sec72, sec62 , preferably at truncated sec71 . Or an antagonist (antisense nucleotide and (Or) ligands) in combination with amylase expressing yeast may be desirable.
[849] Transformation was performed based on the protocol outlined in Gitz et al., Nucl.Acid.Res ., 20 : 1425 (1992). The transformed cells were then inoculated from agar into 100 ml of YEPD complex media broth and grown overnight at 30 ° C. YEPD broth was prepared as described in Kaiser et al., Methods in Yeast Genetics , Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). Dilute overnight cultures to about 2 x 10 ⁶ cells / ml (about OD ₆₀₀ = 0.1) with 500 ml of fresh YEPD broth and regrow to about 1 x 10 ⁷ cells / ml (about OD ₆₀₀ = 0.4-0.5) I was.
[850] The cells were recovered and then transferred to the GS3 rotor bottle of the Sorval GS3 rotor, centrifuged at 5,000 rpm for 5 minutes, discarded the supernatant and resuspended in sterile water in a 50 ml Falcon tube at 3,500 rpm in a Beckman GS-6KR centrifuge. Transformation was prepared by centrifugation again. The supernatant was discarded and cells were washed with LiAc / TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM Li ₂ OOCCH ₃ ) and resuspended in LiAc / TE (2.5 ml).
[851] In a microcentrifuge tube, 100 μl of the prepared cells were mixed with fresh denatured single stranded salmon testis DNA (Lofstrand Lab, Gaithersburg, Md.) And 1 μg of transformed DNA (vol <10 μl) to perform transformation. After the mixture was briefly mixed by vortexing, 600 μl of 40% PEG / TE (40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li ₂ OOCCH ₃ , pH 7.5) was added. The mixture was gently mixed and incubated at 30 ° C. with stirring for 30 minutes. The cells are then heat shocked at 42 ° C. for 15 minutes, the reaction vessel is centrifuged at 12,000 rpm for 5-10 seconds in a microcentrifuge to drain the supernatant, and 500 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and recentrifuged. Cells were diluted with 1 ml of TE and 200 μl aliquots were plated onto preselected medium in 150 mm growth plates (VWR).
[852] Alternatively, the transformation was performed in one large scale reaction with increasing reagent volume, instead of a small number of multiple reactions.
[853] The selection medium used was a uracil-free synthetic complete dextrose prepared as described by Kaiser et al., Methods in Yeast Genetics , Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 208-210 (1994). It was a baby (SCD-Ura). Transformants were grown at 30 ° C. for 2-3 days.
[854] Detection of amylase secreting colonies was performed by including red starch in the selective growth medium. Starch was coupled with a red dye (reactive Red-120, Sigma) according to the method as described in Biely et al., Anal. Biochem. , 172 : 176-179 (1988). The coupled starch was added to the SCD-Ura agar plate at a final concentration of 0.15% (w / v) and buffered to pH 7.0 with potassium phosphate (final concentration 50-100 mM).
[855] Positive colonies were selected and streaked on fresh selection medium (on 150 mm plates) to obtain well isolated and identifiable single colonies. Red starch was added directly to the buffered SCD-Ura agar to detect well isolated single colonies that were amylase secretion positive. Positive colonies were determined by starch degradability (which produces a clear halo visible to the naked eye directly around the colonies).
[856] 4. Isolation of DNA by PCR Amplification
[857] When the positive colonies were isolated, some of them were removed with a toothpick and diluted in 30 μl of sterile water in a 96 well plate. At this time, positive colonies were frozen and stored for subsequent analysis or immediately amplified. 0.5 μl Klentaq (Clontech, Palo Alto, CA), 4.0 μl 10 mM dNTP (Perkin Elmer-Cetus), 2.5 μl Kentaq buffer (Clonetech), 0.25 μl forward oligonucleotide-1, reverse oligonucleotide 5 μl of cell separation was used as a template for a 25 μl volume PCR reaction containing 0.25 μl of −2 and 12.5 μl of distilled water. The sequence of forward oligonucleotide-1 was as follows:
[858] 5'-TGTAAAACGACGGCCAGT TAAATAGACCTGCAATTATTAATCT -3 '(SEQ ID NO: 3)
[859] The sequence of reverse oligonucleotide 2 was as follows:
[860] 5'-CAGGAAACAGCTATGACC ACCTGCACACCTGCAAATCCATT -3 '(SEQ ID NO: 4)
[861] Thereafter, PCR was performed as follows.
[862] a. Denaturation at 92 ° C. for 5 minutes,
[863] b. Denature for 30 seconds at 92 ° C, anneal for 30 seconds at 59 ° C, stretch for 60 seconds at 72 ° C, three times
[864] * c. Denature for 30 seconds at 92 ° C., annealing for 30 seconds at 57 ° C., stretch for 60 seconds at 72 ° C., three times
[865] d. 25 cycles of denaturation at 92 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds and extension at 72 ° C. for 60 seconds,
[866] e. Maintained at 4 ° C.
[867] The underlined regions of the oligonucleotides were annealed to the ADH promoter region and amylase region, respectively, and amplified the 307 bp region from the vector pSST-AMY.O in the absence of an insert. Typically, the first 18 nucleotides of the 5 'end of the oligonucleotide contained the annealing site of the sequencing primer. Thus, the total product of the PCR reaction from the empty vector was 343 bp. However, cDNA fused signal sequences produced significantly longer nucleotide sequences.
[868] After PCR, the literature the fractions 5 ㎕ of the reaction as described in (Sambrook et al., Supra) using the total Tris-Borate-EDTA (TBE) buffer by agarose gel electrophoresis in a 1% agarose gel Investigated. Clones that produce one powerful PCR product larger than 400 bp were purified on a 96 Qiaquick PCR cleanup column (Qiagen Inc., Chasworth, CA) and further analyzed by DNA sequencing.
[869] Example 3: Isolation of cDNA Clone Using Signal Algorithm Analysis
[870] EST and public (eg, Genbank) and / or proprietary (LIFESEQ (r), Incyte Pharmaceuticals, Inc.) using proprietary signal sequence retrieval algorithms developed by Genentech, South San Francisco, CA. , Palo Alto, Calif.) Various polypeptide-encoding nucleic acid sequences were found in dense and assembled EST fragments from the database. This signal sequence algorithm calculates secretory signal scores based on the properties of the DNA nucleotides around the first and, optionally, the second methionine codon (ATG) at the 5'-end of the subject sequence or sequence fragment. The nucleotide following the first ATG must encode at least 35 distinct amino acids without any stop codons. If the first ATG encodes a given amino acid, the second ATG is not examined. If this requirement is not met, candidate sequences are not scored. To determine if the EST sequence contains the signal sequence, a set of seven sensors (evaluation parameters) known to bind to the secretion signal was used to record the score of the amino acid sequence corresponding to the DNA around the ATG codon. . This algorithm was used to find many polypeptide-encoding nucleic acid sequences.
[871] Example 4: Isolation of cDNA Clone Encoding Human PRO1484
[872] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA39616. Oligonucleotides were synthesized based on the DNA39616 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using a probe to isolate a clone of the full length coding sequence of PRO1484.
[873] PCR primer pairs (forward and reverse) were synthesized:
[874] Forward PCR Primer (39616.f1) 5'-GCAACAATGGAGCCACTGGTCATG-3 '(SEQ ID NO: 5)
[875] Reverse PCR Primer (39616.r1) 5'-GCAAAGGTGGAGAAGCGTTGGTGG-3 '(SEQ ID NO: 6)
[876] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA39616 sequence.
[877] Hybridization Probe (39616.p1)
[878] 5'-CCCACTTCAGCAATCAGAACAGTGGGATTATCTTTCAGCAGTGTTTGAGACC-3 '(SEQ ID NO: 7)
[879] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO1484 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus.
[880] DNA sequence analysis of clones isolated as described above yielded the full length DNA sequence (UNQ753) of PRO1484 (named herein DNA44686-1653 (FIG. 1, SEQ ID NO: 1)) and the protein sequence of PRO1484 derived therefrom.
[881] The full nucleotide sequence of DNA44686-1653 is shown in FIG. 1 (SEQ ID NO: 1). Clone DNA44686-1653 contains a single open reading frame (FIG. 2) that has an apparent translation initiation site at nucleotide positions 77-79 and terminates at the stop codon at nucleotide positions 815-817. The predicted polypeptide precursor is 246 amino acids long (FIG. 2). The estimated molecular weight of the full-length PRO1484 protein shown in FIG. 2 is about 26,994 Daltons and the estimated pI is 6.43. Analysis of the full-length PRO1484 sequence shown in FIG. 2 (SEQ ID NO: 2) revealed a signal peptide of amino acids about 1 to about 22, a C1q domain feature sequence of amino acids about 137 to about 167, and a C1q domain shown in FIG. The presence of various amino acid sequence blocks having homology with the protein was demonstrated. The clone DNA44686-1653 was deposited with the ATCC on January 12, 1999 to be assigned ATCC accession No. 203581.
[882] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 2 (SEQ ID NO: 2). Significant homology between COLE_LEPMA, MMU32107_1, CAS4_EPHMU, A57131, A41207 and CERL_RAT] was demonstrated.
[883] Example 5: Isolation of cDNA Clone Encoding Human PRO4334
[884] Using the signal sequence algorithm described in Example 3 above, the EST cluster sequence could be identified from an Insight database. This EST cluster sequence is then subjected to various expressed sequence tags (EST), including public EST databases (eg, Genbank) and proprietary EST DNA databases (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) ), The homology present was confirmed. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives showing 70 (or 90 in some cases) Blast scores that do not encode known proteins were clustered and consensus using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) Assembled with DNA sequence. The consensus sequence obtained therefrom was named DNA56421 herein.
[885] In view of the observed sequence homology between the DNA56421 sequence and the EST sequences contained in Incyte EST clone no. 3347532, the Incyte clone was purchased and cDNA inserts were obtained and sequenced. The sequence of this cDNA insert is shown in FIG. 3 and is designated herein as DNA59608-2577.
[886] The full length clone shown in FIG. 3 includes a single open reading frame (FIG. 3, SEQ ID NO: 8) which has an apparent translation initiation site at nucleotide positions 83-85 and terminates at the stop codon present at nucleotide positions 1404-1406. The expected polypeptide precursor (FIG. 4, SEQ ID NO: 9) is 440 amino acids long. The estimated molecular weight of PRO4334 is about 50,211 Daltons and the estimated pI is about 8.29.
[887] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using the WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 4 (SEQ ID NO: 9) shows that the PRO4334 amino acid sequence and the Dayhof sequence included herein are [AB020686_1, PC1_HUMAN, Homology between P_R79148, PC1_MOUSE, RNU78788_1, RATPDIB_1, P_W75859, AC005587_1, P_R86595 and PPD1_BOVIN].
[888] The clone DNA59608-2577 was deposited with the ATCC on March 23, 1999 and was assigned ATCC Accession No. 203870.
[889] Example 6: Isolation of cDNA Clone Encoding Human PRO1122
[890] EST was found by examining the expressed sequence tag (EST) database (LIFESEQ ™, Incyte Pharmaceuticals, Palo Alto, Calif.). This EST was Incyte 1347523, also referred to herein as DNA49665. Oligonucleotides were synthesized based on the DNA49665 sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) serving as a probe to isolate a clone of the full-length coding sequence for PRO1122.
[891] PCR primer pairs (forward and reverse) were synthesized:
[892] Forward PCR primer 5′-ATCCACAGAAGCTGGCCTTCGCCG-3 ′ (SEQ ID NO: 12); And
[893] Reverse PCR Primer 5'-GGGACGTGGATGAACTCGGTGTGG-3 '(SEQ ID NO: 13)
[894] In addition, synthetic oligonucleotide hybridization probes were constructed having the following nucleotide sequences:
[895] Hybridization probe
[896] 5'-TATCCACAGAAGCTGGCCTTCGCCGAGTGCCTGTGCAGAG-3 '(SEQ ID NO: 14)
[897] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO1122 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from kidney tissue of human fetus (LIB228).
[898] DNA sequence analysis of clones isolated as described above yielded the full-length DNA sequence of PRO1122 (SEQ ID NO: 10) (named DNA62377-1381 herein) and the protein sequence of PRO1122 derived therefrom.
[899] The full nucleotide sequence of DNA62377-1381 is shown in FIG. 5 (SEQ ID NO: 10). Clones DNA62377-1381 contain a single open reading frame that has a clear translation initiation site at nucleotide positions 50-52 of SEQ ID NO: 10 (FIG. 5) and terminates at the stop codon at nucleotide positions 641-643. The expected polypeptide precursor is 197 amino acids long (FIG. 6). The estimated molecular weight of the full-length PRO1122 protein shown in FIG. 6 is about 21,765 Daltons and the estimated pI is about 8.53. Clones DNA62377-1381 were deposited with the ATCC on December 22, 1998. The deposited clones have actual nucleic acid sequences, and the sequences provided herein are of course based on known sequencing techniques.
[900] Amino acid sequence analysis of the full-length PRO1122 polypeptide suggests that the polypeptide has similarities with CTLA-8 and IL-17, suggesting that PRO1122 may be a novel cytokine. More specifically, analysis of the Dayhof database (version 35.45 Swiss Prot 35) demonstrated significant homology between the PRO1122 amino acid sequence and the Dayhof sequence [P-W13651, VG13_HSVSA and CEF25D1_1].
[901] Example 7: Isolation of cDNA Clone Encoding Human PRO1889
[902] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA49310. Based on the observed homology between the DNA49310 consensus sequence and the EST contained in Incyte EST clone number 2779436, Incyte EST clone number 2779436 was purchased and an insert thereof was sequenced. The sequence of this insert is shown in Figure 7 and is named DNA77623-2524 herein.
[903] The full nucleotide sequence of DNA77623-2524 is shown in FIG. 7 (SEQ ID NO: 15). Clone DNA77623-2524 contains a single open reading frame (FIG. 7) having an apparent translation initiation site at nucleotide positions 39 to 41 and ending at the stop codon at nucleotide positions 330 to 332. The predicted polypeptide precursor is 97 amino acids long (FIG. 8). The estimated molecular weight of the full-length PRO1889 protein shown in FIG. 8 is about 10,160 Daltons and the estimated pI is 6.56. Analysis of the full length PRO1889 sequence shown in FIG. 8 (SEQ ID NO: 16) shows potential N-myristoylation of a signal peptide of amino acids about 1 to about 20, amino acids about 6 to about 11, and amino acids about 33 to about 38 The presence of the prokaryotic membrane lipoprotein lipid attachment site at the site, about 24 to about 34 amino acids, and about 78 to about 88 amino acids, was demonstrated. The clone DNA77623-2524 was deposited with the ATCC on Dec. 22, 1998 to be assigned ATCC Accession No. 203546.
[904] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 8 (SEQ ID NO: 16). Significant homology between P_R70984, CHKSCA2A_1, P_W61628, I48639, BMBUNGKP4_1 and UPAR_HUMAN] was demonstrated.
[905] Example 8: Isolation of cDNA Clone Encoding Human PRO1890
[906] Consensus DNA sequences were assembled for other EST sequences using repeat cycles of BLAST and phrap as described in Example 1 above. This consensus sequence is referred to herein as DNA52162. Oligonucleotides were synthesized based on the DNA52162 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using a probe to isolate a clone of the full length coding sequence of PRO1890.
[907] PCR primer pairs (forward and reverse) were synthesized:
[908] Forward PCR Primer (52162.f1) 5'-CACCAACCAACTGCCAATCCTGGC-3 '(SEQ ID NO: 19)
[909] Reverse PCR primer (52162.r1) 5'-ACCACATTCTGATGGGTGTCTCCTGG-3 '(SEQ ID NO: 20)
[910] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA52162 sequence.
[911] Hybridization Probe (52162.p1)
[912] 5'-GGGTCCCTACCTTTACCAGTGGAATGATGACAGGTGTAACATGAAGCAC-3 '(SEQ ID NO: 21)
[913] RNA for preparing cDNA library was isolated from kidney tissue of human fetus.
[914] DNA sequence analysis of clones isolated as described above gave the full length DNA sequence (UNQ872) of PRO1890 (named DNA79230-2525 (FIG. 9, SEQ ID NO: 17) herein) and the protein sequence of PRO1890 derived therefrom.
[915] The full nucleotide sequence of DNA79230-2525 is shown in FIG. 9 (SEQ ID NO: 17). Clone DNA79230-2525 contains a single open reading frame (FIG. 9) having an apparent translation initiation site at nucleotide positions 378-380 and ending at the stop codon at nucleotide positions 1197-1199. The expected polypeptide precursor is 273 amino acids long (FIG. 10). The estimated molecular weight of the full length PRO1890 protein shown in FIG. 10 is about 30,431 Daltons and the estimated pI is about 6.79. Full length PRO1890 sequencing shown in FIG. 10 (SEQ ID NO: 18) shows a signal peptide of about 1 to about 21 amino acids, the transmembrane domain of about 214 to about 235 amino acids, about 86 to about 89 amino acids, and about 255 to about amino acids Potential N-glycosylation sites of amino acids about 258, cAMP- and cGMP-dependent protein kinase phosphorylation sites of amino acids about 266 to amino acids about 269, and amino acids about 27 to about 32, amino acids about 66 to about 71, amino acids about Potential N of about 91 to about amino acid, about 93 to about amino acid about 98, about amino acid about 102 to about 107, about about 109 to about 114 about amino acid, about 140 to about about amino acid about 145, and about about 212 to about about 217 amino acids The presence of myristoylation sites was demonstrated. The clone DNA79230-2525 was deposited with ATCC on Dec. 22, 1998 and assigned ATCC Accession No. 203549.
[916] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 10 (SEQ ID NO: 18). AF026547_1, LEC2_MEGRO, PGCV_HUMAN, GEN12667, P_R06331 and CELF52E1_9] have demonstrated significant homology.
[917] Example 9: Isolation of cDNA Clone Encoding Human PRO1887
[918] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA43041. Oligonucleotides were synthesized based on this consensus sequence, 1) identifying the cDNA library containing the sequence of interest by PCR, and 2) using the probe for isolation of a clone of the full-length coding sequence of PRO1887.
[919] PCR primer pairs (forward and reverse) were synthesized:
[920] Forward PCR primers : 5′-GCAAAGCTCTGCCTCCTTGGCC-3 ′ (SEQ ID NO: 24); And
[921] Reverse PCR primers : 5'-GGGTGGACTGTGCTCTAATGGACGC-3 '(SEQ ID NO: 25), and
[922] 5'-CGTGGCACTGGGTTGATC-3 '(SEQ ID NO: 26).
[923] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from the consensus DNA43041 sequence.
[924] Hybridization probe : 5'-GATGCAGTTCTGGTCAGAGACGCTCCCCAGCAAGATACAACAGTG-3 '(SEQ ID NO: 27).
[925] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO1887 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from human bone marrow.
[926] DNA sequence analysis of clones isolated as described above yielded the full-length DNA sequence of PRO1887 (hereafter designated DNA79862-2522 (FIG. 11, SEQ ID NO: 11)) and the protein sequence of PRO1887 derived therefrom.
[927] DNA79862-2522 is shown in FIG. 11 (SEQ ID NO: 22). Clone DNA79862-2522 contains a single open reading frame with an explicit translation initiation site at nucleotide positions 6-8 and an apparent stop codon at nucleotide positions 1719-1721. The expected polypeptide precursor is 571 amino acids long. The estimated molecular weight of the full-length PRO1887 protein shown in FIG. 12 is about 62,282 Daltons and the estimated pI is about 5.56. Additional features of the PRO1887 protein include a signal peptide of amino acid about 1 to about 27, a transmembrane domain of amino acid about 226 to amino acid about 245, a potential N-glycosylation site of amino acid about 105 to amino acid about 108, amino acid about 10 to about Amino acid about 15, amino acid about 49 to amino acid about 54, amino acid about 62 to amino acid about 67, amino acid about 86 to amino acid about 91, amino acid about 150 to amino acid about 155, amino acid about 155 to amino acid about 160, amino acid about 162 to amino acid about 167, amino acid about 217 to amino acid about 222, amino acid about 227 to amino acid about 232, amino acid about 228 to amino acid about 233, amino acid about 232 to amino acid about 237, amino acid about 262 to amino acid about 267, amino acid about 257 to amino acid about 362, And an N-myristoylation site of amino acids about 461 to about 466, Amino acids from about 12 to about 22 amino acid prokaryotic membrane lipoprotein lipid attachment sites of, and about amino acid 216 to amino acid type carboxyl esterase of about 231 and a -B serine active site.
[928] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 12 (SEQ ID NO: 23). Homology between P_W39078, GEN13248, P_R58980, A31800_1 and P_R45189].
[929] The clone DNA79862-2522 was deposited with the ATCC on December 22, 1998 and assigned ATCC Accession No. 203550.
[930] Example 10 Isolation of cDNA Clone Encoding Human PRO1785
[931] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is named DNA35718 herein. Oligonucleotides were synthesized based on the DNA35718 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using as a probe to isolate a clone of the full-length coding sequence of PRO1785.
[932] PCR primer pairs (forward and reverse) were synthesized:
[933] Forward PCR primers : 5′-ATCCTCCAACATGGAGCCTCTTGC-3 ′ (SEQ ID NO: 30);
[934] Forward PCR primers : 5′-GTATCTTGTCAACCCTGAGG-3 ′ (SEQ ID NO: 31); And
[935] Reverse PCR primer : 5'-TAACCAGAGCTGCTATGTCAGGCC-3 '(SEQ ID NO: 32);
[936] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from the consensus DNA35718 sequence.
[937] Hybridization probe : 5'-AGGCAAAGTTTCACTAGTTGTAAACGTGGCCAGTGACTGCCAACTCACAG-3 '(SEQ ID NO: 33).
[938] In order to screen several libraries for the purpose of finding a source of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO1785 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from endothelial cells of human aorta.
[939] DNA sequence analysis of clones isolated as described above yielded the full-length DNA sequence of PRO1785 (named herein DNA80136-2503 (FIG. 13, SEQ ID NO: 28)) and the protein sequence of PRO1785 derived therefrom.
[940] The entire coding sequence of PRO1785 is shown in FIG. 13 (SEQ ID NO: 28). Clone DNA80136-2503 comprises a single open reading frame with a clear initiation site at nucleotide positions 2-4 of SEQ ID NO: 28 and a clear stop codon at nucleotide positions 629-631. The expected polypeptide precursor is 209 amino acids long. The signal peptide is present at amino acid about 1 to about 31 amino acid of SEQ ID NO: 29, the transmembrane domain at about 18 to about 37 amino acid, and the glutathione peroxidase feature is at about 104 to about amino acid amino acid. The clone DNA80136-2503 was deposited with ATCC and assigned to ATCC Accession No. 203541. The estimated molecular weight of the full-length PRO1785 protein shown in FIG. 14 is about 23,909 Daltons, and the estimated pI is about 9.68.
[941] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 14 (SEQ ID NO: 29). AC004151_3, BTUE_ECOLI, GSHC_HUMAN, P_R89910, PWU88907_1 and D37916_1] have been demonstrated.
[942] Example 11: Isolation of cDNA Clone Encoding Human PRO4353
[943] Consensus DNA sequences were assembled for other EST sequences using repeat cycles of BLAST and phrap as described in Example 1 above. This consensus sequence is named DNA39482 herein. Oligonucleotides were synthesized based on the DNA39482 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using a probe to isolate a clone of the full-length coding sequence of PRO4353.
[944] PCR primer pairs (forward and reverse) were synthesized:
[945] Forward PCR primers : 5'-GAGGACCTACCGGCCGGACAG-3 '(SEQ ID NO: 36) and
[946] Reverse PCR primers : 5'-ATACACCCCGAGTACTGCTGGCAG-3 '(SEQ ID NO: 37)
[947] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from the consensus DNA39482 sequence.
[948] Hybridization Probe : 5'-AGACAGGGCAGCGGCTGCTGAGCTTGGAGCTGGACGCAGCTT-3 '(SEQ ID NO: 38)
[949] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO4353 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from endothelial cells of human aorta.
[950] DNA sequence analysis of the clones isolated as described above gave the full-length DNA sequence of PRO4353 (named herein DNA80145-2594 (FIG. 15, SEQ ID NO: 34)) and the protein sequence of PRO4353 derived therefrom.
[951] The entire coding sequence of PRO4353 is shown in FIG. 15 (SEQ ID NO: 34). Clone DNA80145-2594 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 19 to 21 and an apparent stop codon at nucleotide positions 2683 to 2685. The expected polypeptide precursor is 888 amino acids long. The clone DNA80145-2594 was deposited with the ATCC to be assigned ATCC Accession No. 204-PTA. The estimated molecular weight of the full-length PRO4353 protein shown in FIG. 16 is about 95,285 Daltons, and the estimated pI is about 8.89.
[952] As a result of analysis of the Dayhof database (version 35.45 Swiss Prot 35) using the WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 16 (SEQ ID NO: 34), the PRO4353 amino acid sequence and the Dayhof sequence (sequence and related text are included herein) Homology between [P_W19857, AB000776_1, P_W57260, JH0798, P_R71382, CEY54E5B_1, I48747, MUSC1_1, P_R71383 and P_W63748] was demonstrated.
[953] Example 12 Isolation of cDNA Clone Encoding Human PRO4357
[954] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA80155. Oligonucleotides were synthesized based on the DNA80155 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR and 2) using a probe to isolate a clone of the full-length coding sequence of PRO4357.
[955] PCR primer pairs (forward and reverse) were synthesized:
[956] Forward PCR primers : 5'-GAAGGTGGAAATTAAATTCCAAGGGC-3 '(SEQ ID NO: 41) and
[957] Reverse PCR primers : 5'-CGATAAGCTGCTACAGTGCCATCG-3 '(SEQ ID NO: 42)
[958] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from the consensus DNA80155 sequence.
[959] Hybridization Probe : 5'-GTGACTGTCCTCTGCAAGATAGTGCAGCCTGGCTACGGGA-3 '(SEQ ID NO: 43)
[960] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO4357 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from endothelial cells of human aorta.
[961] DNA sequence analysis of clones isolated as described above yielded the full-length DNA sequence of PRO4357 and the protein sequence of PRO4357 derived therefrom.
[962] The entire coding sequence of PRO4357 is shown in FIG. 17 (SEQ ID NO: 39). Clone DNA84917-2597 comprises a single open reading frame with an apparent translation initiation site at nucleotide positions 286-288 and an apparent stop codon at nucleotide positions 1792-1794. The expected polypeptide precursor is 502 amino acids long. The clone DNA84917-2597 was deposited with ATCC and assigned to ATCC Accession No. 203863. The estimated molecular weight of the full-length PRO4357 protein shown in FIG. 18 is about 58,043 Daltons and the estimated pI is about 7.94.
[963] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 18 (SEQ ID NO: 40). Homology between GELA_DICDI, EHU70560_1, AF089841_1, ABP2_HMAN, P_W19349 and A49551.
[964] Example 13: Isolation of cDNA Clone Encoding Human PRO4405
[965] Consensus DNA sequences were assembled for other EST sequences using repeated cycles of BLAST and phrap. This consensus sequence is referred to herein as DNA80170. Oligonucleotides were synthesized based on the DNA80170 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using a probe to isolate a clone of the full-length coding sequence of PRO4405.
[966] PCR primer pairs (forward and reverse) were synthesized:
[967] Forward PCR primers : 5'-CGGGACTTTCGCTACCTGTTGC-3 '(SEQ ID NO: 46) and
[968] Reverse PCR primers : 5'-CATCATATTCCACAAAATGCTTTGGG-3 '(SEQ ID NO: 47)
[969] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from consensus sequences.
[970] Hybridization probe : 5'-CCTTCGGGGATTCTTCCCGGCTCCCGTTCGTTCCTCTG-3 '(SEQ ID NO: 48)
[971] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO4405 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA libraries was isolated from the kidneys of human fetuses.
[972] DNA sequence analysis of clones isolated as described above yielded the full-length DNA sequence of PRO4405 (named herein DNA84920-2614 (FIG. 19, SEQ ID NO: 44)) and the protein sequence of PRO4405 derived therefrom.
[973] The entire coding sequence of PRO4405 is shown in FIG. 19 (SEQ ID NO: 44). Clone DNA84920-2614 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 79-81 and an apparent stop codon at nucleotide positions 1009-1011. The expected polypeptide precursor is 310 amino acids long. The clone DNA84920-2614 was deposited with ATCC and assigned to ATCC Accession No. 203966. The estimated molecular weight of the full-length PRO4405 protein shown in FIG. 20 is about 33,875 Daltons and the estimated pI is about 7.08.
[974] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 20 (SEQ ID NO: 45), wherein the PRO4405 amino acid sequence and the Dayhof sequence (sequences and related texts are included herein) Homology between [YA93_SCHPO, S62432, YJG2_YEAST, AC004472_3, AB004539_7, S64782, CELC27A12_8, AF109219_1, AF086791_10 and P_W75859] was demonstrated.
[975] Example 14 Isolation of cDNA Clone Encoding Human PRO4356
[976] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is designated herein as DNA80200. Based on the observed homology between the DNA80200 consensus sequence and the EST sequence contained in Merck EST clone 248287, Merck EST clone 248287 was purchased and its insert obtained and sequenced to provide DNA86576-2595.
[977] The entire coding sequence of PRO4356 is shown in FIG. 21 (SEQ ID NO: 49). Clone DNA86576-2595 contains a single open reading frame with an apparent translation initiation site at nucleotide positions 55-57 and an apparent stop codon at nucleotide positions 808-810. The expected polypeptide precursor is 251 amino acids long. The clone DNA86576-2595 was deposited with ATCC and assigned to ATCC Accession No. 203868. The estimated molecular weight of the full-length PRO4356 protein shown in FIG. 22 is about 26,935 Daltons and the estimated pI is about 7.42.
[978] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 22 (SEQ ID NO: 50), showing the PRO4356 amino acid sequence and the Dayhof sequence included herein , S42152, AF007789_1, UPAR_RAT, UPAR_MOUSE, P_W31165, P_W31168, P_R44423 and P_W26359.
[979] Example 15 Isolation of cDNA Clone Encoding Human PRO4352
[980] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA83397. Oligonucleotides were synthesized based on the DNA83397 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using a probe to isolate a clone of the full-length coding sequence of PRO4352.
[981] PCR primer pairs (forward and reverse) were synthesized:
[982] Forward PCR primers : 5'-CTGGGGAGTGTCCTTGGCAGGTTC-3 '(SEQ ID NO: 53) and
[983] Reverse PCR primers : 5'-CAGCATACAGGGCTCTTTAGGGCACAC-3 '(SEQ ID NO: 54)
[984] In addition, synthetic oligonucleotide hybridization probes having the following nucleotide sequences were constructed from the consensus DNA83397 sequence.
[985] Hybridization Probe : 5'-CGGTGACTGAGGAAACAGAGAAAGGATCCTTTGTGGTCAATCTGGC-3 '(SEQ ID NO: 55)
[986] To screen several libraries for the purpose of finding sources of full length clones, the DNA of the libraries was screened by PCR amplification using the PCR primer pairs identified above. Then, clones encoding the PRO4352 gene were isolated using one of the probe oligonucleotides and PCR primers in the positive library. RNA for preparing cDNA library was isolated from the brain of a human fetus.
[987] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence of PRO4352 (designated herein as DNA87976-2593 (FIG. 23, SEQ ID NO: 51)) and the protein sequence of PRO4352 derived therefrom.
[988] The entire coding sequence of PRO4352 is shown in FIG. 23 (SEQ ID NO: 51). Clone DNA87976-2593 comprises a single open reading frame with an obvious translation initiation site at nucleotide positions 179 to 181 of SEQ ID NO: 51 and an apparent stop codon at nucleotide positions 2579 to 2581. The expected polypeptide precursor is 800 amino acids long. The clone DNA87976-2593 was deposited with ATCC and assigned to ATCC Accession No. 203888. The estimated molecular weight of the full-length PRO4352 protein shown in FIG. 24 is about 87,621 Daltons and the estimated pI is about 4.77.
[989] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 24 (SEQ ID NO: 52). Homology between MMU88549_1, D86917_1, AB008179_1, P_R58907, HSHFATPRO_1 and AF031572_1].
[990] Example 16: Isolation of cDNA Clone Encoding Human PRO4380
[991] The signal sequence algorithm described in Example 3 can be used to confirm the EST cluster sequence from the Incyte database. The EST cluster sequence is then subjected to various expressed sequence tags, including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) (EST) The homology present was confirmed as compared to the database. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives with BLAST scores of 70 (in some cases 90) or higher, which do not encode known proteins, are clustered and organized into program "phrap" (Phil Green, University of Washington, Seattle, Washington) to obtain consensus DNA sequences It was. The consensus sequence thus obtained was named DNA79132 herein. Considering DNA79132, DNA92234-2602 was found.
[992] The full length clones shown in FIG. 25 include a single open reading frame (FIG. 25, SEQ ID NO: 56) having nucleotide positions 201 to 203 apparent translation initiation sites and ending at the apparent stop codon at nucleotide positions 1722 to 1724. The expected polypeptide precursor (FIG. 26, SEQ ID NO: 57) is 507 amino acids long. The estimated molecular weight of PRO4380 is about 56,692 Daltons and the estimated pI is about 5.22.
[993] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 26 (SEQ ID NO: 57), wherein the PRO4380 amino acid sequence and the Dayhof sequence (sequences and related texts are included herein) Homology between [CER11H6_1, S56299, D89150_1, G70870, S43914, LMO34616_5, LLU78036_1, AF055904_2, P_W79066 and ARGE_ECOLI] was demonstrated.
[994] The clone DNA92234-2602 was deposited with ATCC and assigned to ATCC Accession No. 203948.
[995] Example 17 Isolation of cDNA Clone Encoding Human PRO4354
[996] The signal sequence algorithm described in Example 3 can be used to identify the EST cluster (92909) sequence, designated DNA10195 herein. The EST cluster sequence is then subjected to various expressed sequence tags, including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) (EST) The homology present was confirmed as compared to the database. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives with BLAST scores of 70 (in some cases 90) or greater, which do not encode known proteins, are clustered and assembled into the program "phrap" (Phil Green, University of Washington, Seattle, Washington) and assembled into consensus DNA sequences It was. The consensus sequence thus obtained was named DNA56063 herein. Considering DNA56063, DNA92256-2596 was found.
[997] The full-length clone shown in FIG. 27 includes a single open reading frame (FIG. 27, SEQ ID NO: 58) with nucleotide positions 108-110 apparent translation initiation site and ending at the stop codon present at nucleotide positions 852-854. The expected polypeptide precursor (FIG. 28, SEQ ID NO: 59) is 248 amino acids long. The estimated molecular weight of PRO4354 is about 28,310 Daltons and the estimated pI is about 4.63.
[998] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using the WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 28 (SEQ ID NO: 59) shows the PRO4354 amino acid sequence and the Dayhof sequence included herein [HGS_RF300, Homology between CEVK04G11_2, CEC11H1_7, HSU80744_1, CEF09E8_2, RNAJ2967_1, DDICOI_1, AB020648_1, P_W33887 and A64319].
[999] The clone DNA92256-2596 was deposited with the ATCC on March 30, 1999 to be assigned ATCC Accession No. 2030551.
[1000] Example 18 Isolation of cDNA Clones Encoding Human PRO4408
[1001] The signal sequence algorithm described in Example 3 can be used to confirm the EST cluster sequence from the Incyte database. The EST cluster sequence is then subjected to various expressed sequence tags, including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) (EST) The homology present was confirmed as compared to the database. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives with BLAST scores of 70 (in some cases 90) or greater, which do not encode known proteins, are clustered and assembled into the program "phrap" (Phil Green, University of Washington, Seattle, Washington) and assembled into consensus DNA sequences It was. The consensus sequence thus obtained was named DNA79298 herein. Considering DNA79298, DNA92274-2617 was found and fully sequenced.
[1002] The full length clone shown in FIG. 29 includes a single open reading frame (FIG. 29, SEQ ID NO: 60) which has an apparent translation initiation site at nucleotide positions 89-91 and terminates at a stop codon present at nucleotide positions 758-760. The predicted polypeptide precursor (FIG. 30, SEQ ID NO: 61) is 223 amino acids long. The estimated molecular weight of PRO4408 is about 25,402 daltons and the estimated pI is about 8.14.
[1003] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 30 (SEQ ID NO: 61). Homology between D1ONCVO, PC4214, OV16_ONCVO, P_R27718, GEN10789 and OBA5_DROME.
[1004] The clone DNA92274-2617 was deposited with the ATCC to be assigned ATCC Accession No. 203971.
[1005] Example 19 Isolation of cDNA Clone Encoding Human PRO5737
[1006] The sequence tag (EST) DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) Expressed using human interleukin-1 receptor antagonist (hIL-1Ra) sequences was examined to identify hIL-1Ra known proteins. An EST sequence named 1433156 was found herein showing homology. EST clone 1433156 was purchased from Incyte Pharmaceuticals (Palo Alto, Calif.) And the cDNA insert was obtained and sequenced throughout to provide the DNA92929-2534 sequence.
[1007] The full nucleotide sequence of DNA92929-2534 is shown in FIG. 31 (SEQ ID NO: 62). Clone DNA92929-2534 comprises a single open reading frame (FIG. 31, SEQ ID NO: 62) with an apparent translation initiation site at nucleotide positions 96-98 and a stop codon at nucleotide positions 498-500. The expected polypeptide precursor (hIL-1Ra2) is 134 amino acids long. The putative signal sequence spans amino acid positions 1 to 17. The clone DNA92929-2534 was deposited with ATCC and assigned to ATCC Accession No. 203586. The estimated molecular weight of the full-length hIL-1ra2 protein shown in FIG. 32 is about 14,927 Daltons and the estimated pI is about 4.8.
[1008] Based on BLAST and FastA sequence alignment analysis of the full length sequence (using the ALIGN-2 computer program), hIL-1Ra2 (FIG. 32, SEQ ID NO: 63) shows significant amino acid sequence identity with the hIL-1Rαβ protein. hIL-1Ra2 is thought to be a splice variant of hIL-1Rαβ.
[1009] Example 20 Isolation of cDNA Clones Encoding Human PRO4425
[1010] The signal sequence algorithm described in Example 3 can be used to confirm the EST cluster sequence from the Incyte database. The EST cluster sequence is then subjected to various expressed sequence tags, including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) (EST) The homology present was confirmed as compared to the database. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives with BLAST scores of 70 (in some cases 90) or greater, which do not encode known proteins, are clustered and assembled into the program "phrap" (Phil Green, University of Washington, Seattle, Washington) and assembled into consensus DNA sequences It was. The consensus sequence thus obtained was named DNA81099 herein.
[1011] In view of the observed sequence homology between the DNA81099 sequence and the EST sequence contained in EST clone number AA448744, EST clone AA448744 was purchased from Merck, and cDNA inserts were obtained and sequenced. The sequence of this cDNA insert is shown in FIG. 33 and is designated herein as DNA93011-2637.
[1012] The full length clone shown in FIG. 33 includes a single open reading frame (FIG. 33, SEQ ID NO: 64) which has an apparent translation initiation site at nucleotide positions 27-29 and terminates at a stop codon present at nucleotide positions 435-437. The expected polypeptide precursor (FIG. 34, SEQ ID NO: 65) is 136 amino acids long. The estimated molecular weight of PRO4425 is about 15,577 Daltons and the estimated pI is about 8.88.
[1013] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 34 (SEQ ID NO: 65). Homology between P_R39520, P_R65332, P_R39388, TGL4_HUMAN, YKAB_CAEEL and S71105] was demonstrated.
[1014] The clone DNA93011-2637 was deposited with the ATCC to be assigned ATCC Accession No. 20-PTA.
[1015] Example 21 Isolation of cDNA Clone Encoding Human PRO5990
[1016] Consensus DNA sequences were assembled for other EST sequences using phraps as described in Example 1 above. This consensus sequence is referred to herein as DNA86602. Oligonucleotides were synthesized based on the DNA86602 consensus sequence, 1) identifying a cDNA library containing the sequence of interest by PCR, and 2) using as a probe to isolate a clone of the full-length coding sequence of PRO5990.
[1017] PCR primer pairs (forward and reverse) were synthesized:
[1018] Forward PCR primers : 5'-CGTCACAGGAACTTCAGCACCC-3 '(SEQ ID NO: 68)
[1019] Reverse PCR primers : 5'-GTCTTGGCTTCCTCCAGGTTTGG-3 '(SEQ ID NO: 69)
[1020] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA86602 sequence.
[1021] Hybridization probes :
[1022] 5'-GGACAGCGCTCCCCTCTACCTGGAGACTTGACTCCCGC-3 '(SEQ ID NO: 70)
[1023] RNA for preparing cDNA library was isolated from brain tissue of a human fetus.
[1024] DNA sequence analysis of the clones isolated as described above yielded the full length DNA sequence of full length PRO5990 (herein named DNA96042-2682 (FIG. 35, SEQ ID NO: 35)) and the protein sequence of the PRO5990 polypeptide derived therefrom.
[1025] The full length clones identified above comprise a single open reading frame (FIG. 35, SEQ ID NO: 35) having an apparent translation initiation site at nucleotide positions 265-267 and a stop codon at nucleotide positions 1669-1671. The expected polypeptide precursor is 468 amino acids long, the estimated molecular weight is about 53,005 Daltons and the estimated pI is about 4.98. Analysis of the full length PRO5990 sequence shown in FIG. 36 (SEQ ID NO: 67) demonstrated the presence of several important polypeptide domains as shown in FIG. 36, wherein the positions indicated in these important polypeptide domains are approximate as described above. The clone DNA96042-2682 was deposited with the ATCC on July 20, 1999 to be assigned ATCC Accession No. 382-PTA.
[1026] Analysis of the Dayhof database (Version 35.45 Swiss Prot 35) using the ALIGN-2 sequence alignment analysis of the full length sequence shown in FIG. 36 (SEQ ID NO: 67). The PRO5990 amino acid sequence and the Dayhof sequence [SG3_MOUSE; SG3_RAT; GEN14673; ENHMHCAX_1; MYS2_DICDI; NFU43192_1; US01_YEAST; A56577; PFLSA13_1; Sequence identity between CELF12F3_3].
[1027] Example 22 Isolation of cDNA Clone Encoding Human PRO6030
[1028] The EST cluster sequence, designated CLU20900 herein, can be identified from the LIFESEQ® (Incyte Pharmaceuticals, Palo Alto, Calif.) Database using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then subjected to various expressed sequence tags (including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) Compared with EST), the homology present was confirmed. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Consensus DNA using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) by clustering a comparison showing a blast score of 70 (or 90 in some cases) or higher that does not encode known proteins Assembled in sequence. The consensus sequence obtained therefrom was named DNA81229 herein.
[1029] In view of the observed sequence homology between the DNA81229 sequence and the EST sequence contained in clone 4040130H1 from the Incyte (Incyte Pharmaceuticals, Palo Alto, Calif.) Database, clone 4040130H1 was purchased and a cDNA insert was obtained. Sequencing was performed. It was found herein that the cDNA insert encodes a storage protein. The sequence of this cDNA insert is shown in FIG. 37 and is designated herein as DNA96850-2705.
[1030] Clone DNA96850-2705 contains a single open reading frame (FIG. 37) with an apparent translation initiation site at nucleotide positions 60-62 and terminating at a stop codon at nucleotide positions 1026-1028. The expected polypeptide precursor is 322 amino acids long (FIG. 38). The estimated molecular weight of the full-length PRO6030 protein shown in FIG. 38 is about 34,793 Daltons and the estimated pI is about 6.34. Analysis of the full length PRO6030 sequence shown in FIG. 38 (SEQ ID NO: 72) demonstrated the presence of several important polypeptide domains as shown in FIG. 38, wherein the positions indicated in these important polypeptide domains are approximate as described above. The clone DNA96850-2705 was deposited with ATCC on August 3, 1999 to be assigned ATCC Accession No. 479-PTA.
[1031] Analysis of the Deihof database (version 35.45 Swiss Prot 35) using the ALIGN-2 sequence alignment analysis of the full length sequence shown in FIG. 38 (SEQ ID NO: 72) shows that the PRO6030 amino acid sequence and the Deihof sequence [AF059571_1; I38346; AF035835_1; P_W83138; P_R54714; P_R65166; P_P93995; BGP1_HUMAN; P_W06873; A43165_1] demonstrated sequence identity.
[1032] Example 23 Isolation of cDNA Clone Encoding Human PRO4424
[1033] The EST cluster sequence could be identified from the Incyte database using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then subjected to various expressed sequence tags (including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) EST) compared to the database to confirm the homology that exists. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives showing 70 (or 90 in some cases) Blast scores that do not encode known proteins were clustered and consensus using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) Assembled with DNA sequence. The extended consensus sequence obtained therefrom is named DNA80820 herein. Considering DNA80820, DNA96857-2636 was found and sequenced.
[1034] The full length clone shown in FIG. 39 includes a single open reading frame (FIG. 39, SEQ ID NO: 73) which has an apparent translation initiation site at nucleotide positions 52-54 and terminates at the stop codon present at nucleotide positions 715-717. The expected polypeptide precursor (FIG. 40, SEQ ID NO: 74) is 221 amino acids long. The estimated molecular weight of PRO4424 is about 23,598 Daltons and the estimated pI is about 6.96.
[1035] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 40 (SEQ ID NO: 74). Homology between P_R88556, CELR12E2_13, DMC34F3_8, ATG13D4_7, HGS_A204, S58331.
[1036] The clone DNA96857-2636 was deposited with ATCC to be assigned ATCC Accession No. 17-PTA.
[1037] Example 24 Isolation of cDNA Clone Encoding Human PRO4422
[1038] The EST cluster sequence could be identified from the Incyte database using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then subjected to various expressed sequence tags (including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) EST) compared to the database to confirm the homology that exists. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives showing 70 (or 90 in some cases) Blast scores that do not encode known proteins were clustered and consensus using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) Assembled with DNA sequence. The consensus sequence obtained therefrom was named DNA80134 herein. Considering DNA80134, DNA96867-2620 was found and fully sequenced.
[1039] The full length clone shown in FIG. 41 includes a single open reading frame (FIG. 41, SEQ ID NO: 75) which has an apparent translation initiation site at nucleotide positions 318-320 and terminates at a stop codon present at nucleotide positions 900-902. The expected polypeptide precursor (FIG. 42, SEQ ID NO: 76) is 194 amino acids long. The estimated molecular weight of PRO4422 is about 21,431 Daltons and the estimated pI is about 8.57.
[1040] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 42 (SEQ ID NO: 76). Homology between P_W69515, ATAC003680_7, ACCA_HAEIN, I64065, A70853 and AF074611_71.
[1041] Clones DNA96867-2620 were deposited with ATCC to be assigned ATCC Accession No. 203972.
[1042] Example 25 Isolation of cDNA Clone Encoding Human PRO4430
[1043] The EST cluster sequence could be identified from the Incyte database using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then subjected to various expressed sequence tags (including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) EST) compared to the database to confirm the homology that exists. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives showing 70 (or 90 in some cases) Blast scores that do not encode known proteins were clustered and consensus using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) Assembled with DNA sequence. The extended consensus sequence obtained therefrom was named DNA82380 herein. Considering DNA82380, DNA96878-2626 was found and sequenced.
[1044] The full length clone shown in FIG. 43 includes a single open reading frame (FIG. 43, SEQ ID NO: 77) which has an apparent translation initiation site at nucleotide positions 56-58 and terminates at the stop codon present at nucleotide positions 431-433. The expected polypeptide precursor (FIG. 44, SEQ ID NO: 78) is 125 amino acids long. The estimated molecular weight of PRO4430 is about 13,821 Daltons and the estimated pI is about 8.6.
[1045] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 44 (SEQ ID NO: 78). Homology between AF043498_1, P_W62066, LY6C_MOUSE, LY6A_MOUSE, P_R58710 and P_R86315].
[1046] Clone DNA96878-2626 was deposited with ATCC to be assigned ATCC Accession No. 23-PTA.
[1047] Example 26 Isolation of cDNA Clone Encoding Human PRO4499
[1048] The EST cluster sequence could be identified from the Incyte database using the signal sequence algorithm described in Example 3 above. This EST cluster sequence is then subjected to various expressed sequence tags (including a common EST database (eg, Genbank) and a proprietary EST DNA database (LIFESEQ (r), Incyte Pharmaceuticals, Palo Alto, Calif.) EST) compared to the database to confirm the homology that exists. Homology investigations were performed using the computer programs BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266: 460-480 (1996)). Comparatives showing 70 (or 90 in some cases) Blast scores that do not encode known proteins were clustered and consensus using the program "phrap" (Phil Green, Seattle, University of Washington, Seattle) Assembled with DNA sequence. The consensus sequence obtained therefrom was named DNA81155 herein. Considering DNA81155, DNA96889-2641 was found and sequenced.
[1049] The full length clone shown in FIG. 45 includes a single open reading frame (FIG. 45, SEQ ID NO: 79) which has an apparent translation initiation site at nucleotide positions 185-187 and terminates at the stop codon present at nucleotide positions 1202-1204. The expected polypeptide precursor (FIG. 46, SEQ ID NO: 80) is 339 amino acids long. The estimated molecular weight of PRO4499 is about 36,975 Daltons and the estimated pI is about 7.85.
[1050] Analysis of the Dayhof database (version 35.45 Swiss Prot 35) using WU-BLAST2 sequence alignment analysis of the full length sequence shown in FIG. 46 (SEQ ID NO: 80). Homology between AF098967_1, AF007140_1, ROA3_HUMAN, E70969, CEY3C12B_5].
[1051] The clone DNA96889-2641 was deposited with ATCC to be assigned ATCC Accession No. 119-PTA.
[1052] Example 27: Use of PRO as Hybridization Probe
[1053] The following method describes the use of hybridization probes of nucleotide sequences encoding PRO.
[1054] Homologous DNA (eg, encoding native variants of PRO) in a human tissue cDNA library or human tissue genome library using DNA comprising the coding sequence of full length or mature PRO as described herein as a probe Screened.
[1055] Hybridization and washing of the filter comprising each library DNA was performed under the following high stringency conditions. Hybridization of the radiolabeled PRO-derived probe and filter was performed using 50% formamide, 5 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 x Denhardt's solution and 10% The solution was performed at 42 ° C. for 20 hours in a solution of dextran sulfate. The filter was washed in an aqueous solution of 0.1 x SSC and 0.1% SDS at 42 ° C.
[1056] The DNA encoding the full length native sequence PRO and the DNA having the desired sequence identity were then identified by standard methods known in the art.
[1057] Example 28: E. Expression of PRO in E. coli
[1058] In this embodiment, A method for producing a non-glycosylated form of PRO by recombinant expression in E. coli is described.
[1059] First, DNA sequences encoding PRO were amplified using the selected PCR primers. The primer should include a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. Various expression vectors can be used. Examples of suitable vectors include pBR322, which comprises ampicillin and tetracycline resistance genes. Derived from E. coli, Bolivar et al., (Bolivar) [Gene, 2: 95 (1977)]] a. This vector was digested with restriction enzymes and dephosphorylated. PCR amplified sequences were then ligated to the vector. The vector preferably encodes an antibiotic resistance gene, a trp promoter, a poly-his leader (including the first six STII codons, a poly-his sequence and an enterokinase cleavage site), a PRO coding region, a lambda transcription terminator and an argU gene. Will comprise the sequence.
[1060] Subsequently, the ligation mixture was used to select E. coli by the method described in Sambrook et al., Supra. E. coli strains were transformed. After confirming that the transformants can grow on LB plates, colonies with antibiotic resistance were selected. Plasmid DNA was isolated and confirmed by restriction analysis and DNA sequencing.
[1061] Selected clones were incubated overnight in liquid culture medium such as LB broth supplemented with antibiotics. This culture was used to inoculate larger scale cultures. The cells were then incubated to the desired optical density, during which the expression promoter was activated.
[1062] After culturing the cells for several hours or more, the cells could be recovered by centrifugation. Cell pellets obtained by centrifugation can be lysed using various reagents known in the art, and the solubilized PRO protein can be purified under conditions that bind proteins tightly using metal chelating columns.
[1063] Here's how to do this. It was expressed in E. coli in poly-His tagged form. First, the DNA encoding the PRO was amplified using the selected PCR primers. The primers included restriction enzyme sites corresponding to the restriction enzyme sites on the selected expression vector, and other useful sequences that provide efficient and reliable initiation of translation, rapid purification on metal chelate columns, and proteolytic removal with enterokinase. PCR-amplified poly-His tagged sequences were then ligated to the expression vectors, which were used to determine E. coli based on strain 52. E. coli host (W3110fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq) was transformed firstly, transformants were transformed until LOD600 reached 3-5 at 50 mg / ml carbenicillin containing LB. Shake culture was performed at 30 ° C. The culture medium was then cultured with CRAP medium (3.57 g of (NH ₄ ) ₂ SO _{4 in} 500 ml of water, 0.71 g of sodium citrate.2H ₂ O, 1.07 g of KCl, 5.36 g of Difco yeast extract, Sheffield Hicaze. (Sheffield hycase) SF 5.36 g, prepared by mixing 110 mM MPOS (pH 7.3), 0.55% (w / v) glucose and 7 mM MgSO ₄ ) at 50 to 100-fold and about 20 to 30 at 30 ° C. Shake incubation for hours Samples were taken to confirm expression by SDS-PAGE analysis, the cells were pelleted by centrifugation of the bulk culture, and the cell pellet was frozen until purification and refolding.
[1064] E. obtained from 0.5-1 L fermentation broth. E. coli paste (pellets 6-10 g) was resuspended in 10-fold volume (w / v) of 7 M guanidine, 20 mM Tris buffer (pH 8). Solid sodium sulfite and sodium sationate were added to bring final concentrations to 0.1 M and 0.02 M, respectively, and the solution was stirred at 4 ° C. overnight. At this stage, desulphation produced a denatured protein that blocked all cysteine residues. This solution was centrifuged for 30 minutes at 40,000 rpm with a Beckman ultracentrifuge. The supernatant was diluted with 3 to 5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and clarified by filtration with a 0.22 micron filter. The clarified extracts were loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in metal chelate column buffer. The column was washed with another buffer (Calbiochem, Utrol grade) containing 50 mM imidazole (pH 7.4). The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were collected and stored at 4 ° C. Protein concentration was measured by absorbance at 280 nm using the extinction coefficient calculated on the basis of the amino acid sequence.
[1065] The protein was refolded by slowly diluting the sample in freshly prepared refolding buffer consisting of 20 mM Tris (pH 8.6), 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. The refolding volume was chosen such that the final protein concentration was between 50 and 100 μg / ml. The refolding solution was stirred slowly at 4 ° C. for 12-36 hours. The refolding reaction was stopped by adding TFA to a final concentration of 0.4% (about pH 3). Prior to further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to bring the final concentration to 2-10%. The refolded protein was chromatographed by eluting with acetonitrile concentration gradient from 10% to 80% in a Poros R1 / H reversed phase column using 0.1% TFA transfer buffer. Aliquots of fractions showing A ₂₈₀ absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were collected. In general, most protein species that are properly refolded are eluted at the lowest acetonitrile concentration because they contain the hydrophobic interior that is protected from interaction with the reversed phase resin and are the most dense. Aggregated proteins are usually eluted at higher acetonitrile concentrations. In the reverse phase, the desired type of protein is not only separated from the misfolded protein, but also endotoxin is removed from the sample.
[1066] Fractions containing the desired folded PRO polypeptide were pooled and nitrogen stream was slowly added to the solution to remove acetonitrile. Proteins were combined and sterile filtered in 20 mM Hepes (pH 6.8) containing 0.14 M sodium chloride and 4% mannitol by dialysis or gel filtration using G25 Superfine, Pharmacia resin equilibrated with formulation buffer. .
[1067] Many of the PRO polypeptides described herein have been successfully expressed as described above.
[1068] Example 29 Expression of PRO in Mammalian Cells
[1069] This example describes a method for producing a potentially glycosylated form of PRO by recombinant expression in mammalian cells.
[1070] Vector pRK5 (see European Patent No. 307,247 published March 15, 1989) was used as the expression vector. Optionally, using the ligation method described by Sambrook et al., Supra, PRO DNA was ligated to pRK5 using a selected restriction enzyme to insert PRO DNA. The generated vector is called pRK5-PRO.
[1071] In one embodiment, 293 cells can be selected as the host cell. Human 293 cells (ATCC CCL 1573) were incubated in tissue culture plates in medium such as fetal calf serum and optionally DMEM supplemented with nutrients and / or antibiotics. About 10 μg of pRK5-PRO DNA was mixed with about 1 μg of DNA encoding a VA RNA gene (Timmmappaya et al., Cell , 31 : 543 (1982)), and 500 μl of 1 mM Tris- It was dissolved in HCl, 0.1 mM EDTA and 0.227 M CaCl ₂ . 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise to this mixture to form a precipitate at 25 ° C. for 10 minutes. The precipitate was suspended and added to 293 cells and left at 37 ° C. for about 4 hours. The culture medium was aspirated off and 2 ml of 20% glycerol in PBS was added for 30 seconds. The 293 cells were then washed with serum free medium, fresh medium was added and the cells incubated for about 5 days.
[1072] Transfection approximately 24 hours to remove the culture media and replaced with culture medium containing a culture medium (alone) or 200 μCi / ㎖ ³⁵ S- cysteine and 200 μCi / ㎖ ³⁵ S- methionine. After 12 hours of incubation, the conditioned medium was recovered, concentrated in a rotary filter and loaded onto a 15% SDS gel. The treated gel was dried and exposed to the film for a period of time to confirm the presence of the PRO polypeptide. Cultures containing the transfected cells were further incubated (in serum free medium) and the medium was tested by the chosen bioassay.
[1073] Other techniques include Somparyrac et al ., Proc. Natl. Acad. Sci. , 12 : 7575 (1981), can be used to temporarily introduce PRO into 293 cells using the dextran sulfate method. 293 cells were cultured to a maximum density in a spinner flask and 700 μg of pRK5-PRO DNA was added. Cells were first concentrated from spinner flasks by centrifugation and washed with PBS. DNA-dextran precipitate was incubated for 4 hours in cell pellet. Cells were treated with 20% glycerol for 90 seconds and washed with tissue culture medium and then placed back into the spinner flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days the conditioned media was centrifuged and filtered to remove cells and debris. Samples containing the expressed PRO were concentrated and purified by any selected method such as dialysis and / or column chromatography.
[1074] In another embodiment, PRO can be expressed in CHO cells. pRK5-PRO can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE-dextran. As mentioned above, the cell cultures were incubated and the medium was replaced with culture medium (alone) or with a medium containing radiolabels such as ³⁵ S-methionine. After confirming the presence of the PRO polypeptide, the culture medium can be replaced with a serum free medium. Preferably, the cultures were incubated for about 6 days and then the conditioned medium was recovered. The medium containing the expressed PRO can then be concentrated and purified by any chosen method.
[1075] Epitope-tagged PRO could also be expressed in host CHO cells. PRO subclones from the pRK5 vector. The subclonal insert is fused in-frame to the baculovirus expression vector with a selected epitope tag such as a poly-his tag by PCR. Poly-his tagged PRO inserts are subcloned into SV40 induction vectors containing selection markers such as DHFR to select stable clones. Finally, CHO cells can be transfected with SV40 induction vectors (as above). Labeling may be performed in the same manner as above to confirm expression. The culture medium containing the expressed poly-His tagged PRO is then concentrated and purified by any selected method such as Ni ²⁺ -chelate affinity chromatography.
[1076] PRO may be expressed in CHO and / or COS cells by transient expression methods or in CHO cells by other stable expression methods.
[1077] The following method was used to stably express in CHO cells. A protein is an IgG construct (immunoadhesine) in which the coding sequence of the soluble form of each protein (eg, extracellular domain) is fused to the sequence of an IgG1 constant region comprising a hinge, CH2 and CH2 domain, and (Or) expressed as a poly-His tagged form.
[1078] Following PCR amplification, each DNA was subcloned into a CHO expression vector using standard methods 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 restriction sites suitable for 5 'and 3' of the DNA so that cDNA can be conveniently shuttled. Vectors used for expression in CHO cells are described by Lucas et al. Nucl. Acids Res. 24 : 9, 1774-1779 (1996), using the SV40 early promoter / enhancer to express the cDNA and dihydrofolate reductase (DHFR). DHFR expression allowed selection of plasmids that remained stable after transfection.
[1079] 12 μg of the target plasmid DNA was prepared using a commercially available transfection reagent Superfect® (Qiagen), Dosper® or Fugene® (Boehringer Mannheim). It was introduced into 10,000 CHO cells. Cells were cultured according to the method described by Lucas et al., Supra. About 3 × 10 ⁻⁷ cells were frozen in ampoules according to the following method for culturing and producing cells later.
[1080] Ampoules containing plasmid DNA were dissolved in a water bath and vortexed and mixed. The contents were pipetted into a centrifuge tube containing 10 ml of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells resuspended in 10 ml of selection medium (0.2 μm filtered PS20 containing 5% fetal bovine serum with 0.2 μm diafiltration). Cells were dispensed into 100 ml spinners containing 90 ml of selection medium. After 1-2 days, cells were transferred to 250 ml spinner containing 150 ml of selective culture medium and incubated at 37 ° C. After 2-3 days 3 × 10 ⁵ cells / ml were seeded in 250 ml, 500 ml and 2000 ml spinners. Cell medium was centrifuged, exchanged with fresh medium and resuspended in production medium. Any suitable CHO medium may be used and in practice the production medium described in US Pat. No. 5,122,469 (June 16, 1992) was used. Three liters of production spinners were seeded at 1.2 × 10 ⁶ cells / ml. On day 0, cell number and pH were measured. On day 1, samples were taken from the spinner and sprayed with filtered air. On day 2, sample was taken from the spinner and the temperature was changed to 33 ° C. and 30 ml of 500 g / l glucose and 0.6 ml of 10% antifoam (e.g. 35% polydimethyl siloxane emulsion, Dow Corning 365 pharmaceutical grade emulsion) ) Was added. Throughout the production process, the pH should be maintained at about 7.2. After 10 days or when viability dropped below 70%, the cell culture was recovered by centrifugation and filtered through a 0.22 μm filter. The filtrate was either stored at 4 ° C. or immediately loaded into the column for purification.
[1081] For poly-his tagged constructs, proteins were purified using a Ni-NTA column (Qiagen). Imidazole was added to the conditioned medium at a concentration of 5 mM before purification. The conditioned medium was pumped to a 6 ml Ni-NTA column equilibrated in 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml / min at 4 ° C. After loading, the column was further washed with equilibration buffer and protein was eluted with equilibration buffer containing 0.25 M imidazole. This highly purified protein was then desalted in storage buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol using a 25 ml G25 Superfine (Pharmacia) column and at -80 ° C. Stored.
[1082] The immunoadhesin (Fc-containing) constructs were purified from the conditioned media as follows. The conditioned medium was pumped to a 5 ml Protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After loading, the column was washed thoroughly with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was collected in 1 ml fractions and immediately neutralized in a tube containing 275 μl of 1 M Tris buffer (pH 9). The highly purified protein was then removed from the storage buffer by the method described above for the poly-His tagged protein. Uniformity was evaluated by N-terminal amino acid sequencing by SDS-polyacrylamide gel and Edman digestion.
[1083] Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.
[1084] Example 30 Expression of PRO in Yeast
[1085] The following method is a description of recombinant expression of PRO in yeast.
[1086] First, yeast expression vectors were constructed for intracellular production or secretion of PRO from the ADH2 / GAPDH promoter. The DNA encoding the PRO and the promoter were inserted into a suitable restriction enzyme site of the plasmid selected for intracellular expression of PRO. In the case of secretion, the DNA encoding PRO is used for expression of the ADH2 / GAPDH promoter, the native PRO signal peptide or other mammalian signal sequence, or for example the yeast alpha-factor or invertase secretion signal / leader sequence, and PRO. The linker sequence (if needed) can be cloned into a selected plasmid with DNA encoding it.
[1087] Yeast cells, for example yeast strain AB110, can be transformed with the above expression plasmids and cultured in selected fermentation medium. Transformed yeast supernatants can be precipitated with 10% trichloroacetic acid, separated by SDS-PAGE and analyzed by staining the gel with Coomassie Blue.
[1088] Thereafter, the yeast cells can be removed from the fermentation medium by centrifugation, and the medium can be concentrated using the cartridge filter of choice to isolate and purify the recombinant PRO. The concentrate containing PRO can be further purified using selected column chromatography resins.
[1089] Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.
[1090] Example 31 Expression of PRO in Insect Cells Infected with Baculovirus
[1091] The following method is a description of recombinant expression of PRO in baculovirus-infected insect cells.
[1092] The sequence encoding the PRO was fused upstream of the epitope tag included in the baculovirus expression vector. The epitope tag includes a poly-his tag and an immunoglobulin tag (similar to the Fc region of IgG). Several plasmids can be used, including plasmids commercially available, for example plasmids derived from pVL1393 (Novagen). In short, a sequence encoding a PRO or a desired portion of a sequence encoding a PRO (eg, a sequence encoding an extracellular domain of a transmembrane protein, or a sequence encoding a mature protein if the protein is an extracellular protein) is 5 PCR was amplified using primers complementary to the 'and 3' regions. The 5 'primer may comprise a (selected) restriction enzyme site adjacent to both sides. The product was then cleaved with the selected restriction enzymes and subcloned into the expression vector.
[1093] Recombinant baculoviruses use the lipofectin (purchased from GIBCO-BRL) to convert the plasmid and BaculoGold® virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9"). Produced by co-transfection into cells (ATCC CRL 1711). After 4-5 days incubation at 28 ° C., the released virus was recovered and used for subsequent amplification. Viral infection and protein expression were performed as described in O'Reilley et al., Baculovirus Expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994).
[1094] The expressed poly-his tagged PRO can then be purified, for example by Ni ²⁺ -chelate affinity chromatography. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362: 175-179 (1993). In sum, Sf9 cells were washed and resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl) and placed on ice for 20 seconds. Sonication was in turn. The sonicate was removed by centrifugation and the supernatant diluted 50-fold in loading buffer (50 mM phosphoric acid, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni ²⁺ -NTA agarose column (commercially available from Quiiagen) was prepared with a 5 ml fill volume, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extracts were loaded into the column at 0.5 ml per minute. The column was washed with loading buffer to A ₂₈₀ baseline, at which point fraction recovery was started. Next, the column was washed with secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) to elute nonspecifically bound proteins. Again, the A ₂₈₀ baseline was reached and the column developed using an imidazole gradient from 0 to 500 mM in secondary wash buffer. 1 ml fractions were recovered and analyzed by SDS-PAGE and silver staining or by Western blotting with Ni ²⁺ -NTA (Qiagen) combined with alkaline phosphatase. Fractions containing eluted His ₁₀ -tagged PRO were pooled and dialyzed with loading buffer.
[1095] Alternatively, purification of IgG tagged (or Fc tagged) PRO can be performed using known chromatography techniques such as Protein A or Protein G column chromatography.
[1096] Many of the PRO polypeptides disclosed herein have been successfully expressed as described above.
[1097] Example 32 Preparation of Antibodies Binding to PRO
[1098] This example describes a method of making a monoclonal antibody that can specifically bind a PRO polypeptide.
[1099] Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Godding, supra. Immunogens that can be used include purified PRO, fusion proteins comprising PRO and cells expressing recombinant PRO on the cell surface. Those skilled in the art can select immunogens without undue experimentation.
[1100] Mice, eg Balb / c, were immunized by subcutaneous or intraperitoneal injection of emulsified PRO immunogen in an Freund's complete adjuvant in an amount of 1-100 μg. Alternatively, immunogens were emulsified in MPL-TDM supplements (Ribi Immunochemical Research, Hamilton, Montana, USA) and injected into the hind paws of animals. Immunized mice were then further antigen-promoted with additional immunogens emulsified in selected adjuvants after 10-12 days. Thereafter, mice can be further antigen-promoted for several weeks with additional immunoinjection. Blood samples were collected from the rear of the orbit and serum samples were taken periodically from the mice, and anti-PRO antibodies were detected by ELISA assay.
[1101] Once a suitable antibody titer was detected, PRO was injected intravenously to animals that were "positive" for the antibody. After 3-4 days, the mice were sacrificed to recover spleen cells. The spleen cells were then fused with P3X63AgU.1 available from selected murine myeloma cell lines, eg ATCC No. CRL 1597 (using 35% polyethylene glycol). With this fusion, hybridoma cells that can be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin and thymidine) media that inhibit the proliferation of non-fusion cells, myeloma hybrids and splenocyte hybrids. Was created.
[1102] The reactivity of these hybridoma cells to PRO can be screened by ELISA. Methods of determining "positive" hybridoma cells that secrete the desired monoclonal antibodies to PRO are well known to those skilled in the art.
[1103] Positive hybridoma cells can be injected intraperitoneally so that syngeneic Balb / c mice produce ascites containing anti-PRO monoclonal antibodies. Alternatively, hybridoma cells can be cultured in tissue culture flasks or roller bottles. Monoclonal antibodies produced in the ascites can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of the antibody to protein A or protein G may be used.
[1104] Example 33 Purification of PRO Polypeptides Using Specific Antibodies
[1105] Natural or recombinant PRO polypeptides can be purified by various standard techniques in the field of protein purification. For example, pro-PRO polypeptides, mature PRO polypeptides, or pre-PRO polypeptides are purified by immunoaffinity chromatography using antibodies specific for the desired PRO polypeptide. In general, immunoaffinity columns are constructed by coupling anti-PRO polypeptide antibodies to the activated chromatography resin by covalent bonds.
[1106] Polyclonal immunoglobulins were prepared by precipitating with ammonium sulfate from immune serum or by purification on immobilized protein A (Pharmacia LKB Biotechnology, Fitzkataway, NJ). Similarly, monoclonal antibodies were prepared by ammonium sulfate precipitation or chromatography on immobilized Protein A from mouse ascites. Partially purified immunoglobulin was covalently attached to a chromatography resin, such as CnBr-activated SEPHAROSE® (Pharmacia LKB Biotechnology). The antibody was coupled to the resin, the resin was blocked, and the derived resin was washed according to the manufacturer's instructions.
[1107] Such immunoaffinity columns are used for purification of PRO polypeptide by preparing fractions containing the PRO polypeptide in soluble form from the cells. Prepared by the addition of detergent or by the solubilization of the cell fraction obtained through solubilization or differential centrifugation of whole cells by other methods well known in the art. Alternatively, soluble PRO polypeptides comprising signal sequences may be secreted in useful amounts into the medium in which the cells are cultured.
[1108] Soluble PRO polypeptide-containing preparations were passed through an immunoaffinity column and the columns were washed under conditions that allow preferential absorption of the PRO polypeptide (eg, high ion concentration buffer in the presence of detergent). The column is then eluted under conditions that disrupt antibody / PRO polypeptide binding (e.g., low pH buffers such as about pH 2-3 or high concentrations of urea or chaotrope such as thiocyanate ions), PRO polypeptide was recovered.
[1109] Example 34 Drug Screening
[1110] The present invention is particularly useful for screening compounds with any of various drug screening techniques using PRO polypeptides or binding fragments thereof. The PRO polypeptide or fragment thereof used in this test may be free in solution, fixed to a solid support, retained on the cell surface, or located within the cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells stably transformed with recombinant nucleic acids expressing a PRO polypeptide or fragment thereof. Drugs were screened for the transformed cells in a competitive binding assay. The cells, whether viable or fixed, can be used for standard binding assays. For example, the formation of a complex between a PRO polypeptide or fragment thereof and a test substance can be measured. Alternatively, a decrease in complex formation between the PRO polypeptide and its target cells or target receptors caused by the test substance may be examined.
[1111] As such, the present invention provides a method for screening a drug or any other substance that may affect a PRO polypeptide-related disease or disorder. These methods include contacting such a substance with a PRO polypeptide or fragment thereof, and methods well known in the art, such as (i) the presence of a complex between the substance and the PRO polypeptide or fragment thereof or (ii) with the PRO polypeptide or fragment thereof. Analysis for the presence of complexes between cells. In this competitive binding assay, the PRO polypeptide or fragment thereof is usually labeled. After suitable incubation, the free PRO polypeptide or fragment thereof is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular substance to bind to or inhibit the PRO polypeptide / cell complex. do.
[1112] Another drug screening technique is a high-volume screening method for compounds having a suitable binding affinity with a polypeptide, which is described in detail in WO84 / 03564 (published Sep. 13, 1984). In sum, many different small peptide test compounds are synthesized on solid substrates such as plastic pins or some other surface. As with the PRO polypeptide, the peptide test compound is reacted with and washed with the PRO polypeptide. Bound PRO polypeptides are detected by methods well known in the art. Purified PRO polypeptides may also be coated directly onto the plates used in the aforementioned drug screening techniques. Non-neutralizing antibodies can also be used to capture peptides and immobilize them on a solid support.
[1113] The present invention also encompasses the use of screening assays of competing drugs for which neutralizing antibodies capable of binding a PRO polypeptide specifically compete with the test compound for binding to the PRO polypeptide or fragment thereof. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with the PRO polypeptide.
[1114] Example 35 Rational Drug Design
[1115] The goal of rational drug design is to produce structural polypeptides of biological activity target polypeptides (ie PRO polypeptides) or small molecules that interact with them, such as agonists, antagonists or inhibitors. Any of these examples can be used to make drugs that are more active or more stable forms of PRO polypeptides, or drugs that enhance or inhibit the function of PRO polypeptides in vivo (Hodgson, Bio ). / Technology , 9 : 19-21 (1991)].
[1116] In one method, the three-dimensional structure of a PRO polypeptide or PRO polypeptide-inhibitor complex is determined by x-ray crystallography, computer modeling or most commonly a combination of the two methods. In order to elucidate the structure of the molecule and determine the active site, both the form and the charge of the PRO polypeptide must be identified. Although less frequent, useful information about the structure of a PRO polypeptide can also be obtained by modeling based on the structure of the homologous protein. In both cases, relevant structural information is used to design cognate PRO polypeptide-like molecules or to identify effective inhibitors. Useful examples of rational drug design include molecules that enhance activity or stability, as shown by Braxton and Wells ( Biochemistry, 31 : 7796-7801 (1992)), or Athauda et al. In J. Biochem. , 113 : 742-746 (1993), molecules that act as inhibitors, agonists or antagonists of natural peptides.
[1117] In addition, a target-specific antibody selected by functional analysis may be isolated as described above and then its crystal structure analyzed. In principle, this method provides a pharmacore that can be the basis of subsequent drug design. Protein determination can also be omitted by producing anti-genetic antibodies (anti-ids) against functional pharmacologically active antibodies. In the mirror image, the binding site of the anti-id is thought to be the homologue of the original receptor. Anti-ids can then be used to identify and isolate peptides from chemically or biologically produced peptide banks. The isolated peptide is then used as Pharmacore.
[1118] With the present invention, a sufficient amount of PRO polypeptide has been made available to perform analytical studies such as x-ray crystallography. In addition, the amino acid sequence information of the PRO polypeptides provided herein will provide guidance in using computer modeling techniques in place of or in addition to x-ray determination.
[1119] Example 36 Chondrocyte Regeneration Assay (Analysis 110)
[1120] This analysis indicates that certain polypeptides of the present invention are thought to be useful in the treatment of various bone and / or cartilage diseases such as, for example, sports injury and arthritis because they act to induce differentiation of chondrocytes. The analysis is performed as follows. Porcine chondrocytes were isolated by digesting the articular cartilage of the metacarpal joints of sows 4-6 months of age with collagenase overnight. Isolated cells were then seeded at 25,000 cells / cm ² in Ham F-12 containing 10 μg FBS and 4 μg / ml of gentamycin. The culture medium was exchanged every 3 days and cells were seeded at 5,000 cells per well in 100 μl of the same medium without serum in 96 well plates, and the test PRO polypeptide, staurosporin (positive control) or medium alone (negative). 100 μl of control) was added to make a total volume of 200 μl per well. After incubation at 37 ° C. for 5 days, photographs of each well were taken to determine the differentiation state of chondrocytes. In this assay, positive regeneration of chondrocytes was determined to be more similar in the positive control than in the negative control.
[1121] In this assay, the PRO1484, PRO1890, PRO1887, PRO4353, PRO4357, PRO4405, PRO5737 and PRO5990 polypeptides were tested as positive.
[1122] Example 37: Detection of Polypeptides Affecting Glucose or FFA Uptake in Skeletal Muscle (Analysis 106)
[1123] This assay was designed to determine if the PRO polypeptide exhibited the ability to affect glucose or FFA uptake by skeletal muscle. PRO polypeptides tested positive in this assay are expected to be useful in the treatment of diseases in which stimulation or inhibition of glucose uptake by skeletal muscle is beneficial, including diabetes, hyperinsulinemia or hypoinsulinemia.
[1124] In a 96 well format, the PRO polypeptide to be analyzed was added to the differentiated skeletal muscle of the primary rat and incubated overnight. Next, fresh medium containing PRO polypeptide and +/- insulin was added to the wells. This sample medium was then monitored to measure glucose and FFA uptake by skeletal muscle. Insulin will stimulate glucose and FFA uptake by skeletal muscle, and insulin in medium without PRO polypeptide was used as a limit for score recording as a positive control. Since the PRO polypeptide to be tested can stimulate or inhibit glucose and FFA uptake, the results were recorded as positive if at least 1.5 times or less than 0.5 times the insulin control.
[1125] In this assay, the PRO1484, PRO1122, PRO1889, PRO4357 and PRO4380 polypeptides were tested as positive as stimulants or inhibitors of glucose and / or FFA uptake.
[1126] Example 38: Detection of PRO Polypeptides Influencing Glucose or FFA Uptake by Primary Rat Adipocytes (Analysis 94)
[1127] This assay was designed to determine whether the PRO polypeptide exhibited the ability to affect glucose or FFA uptake by adipocytes. PRO polypeptides tested positive in this assay are expected to be useful for the treatment of diseases where stimulation or inhibition of glucose uptake by adipocytes is beneficial, including obesity, diabetes, hyperinsulinemia or hypoinsulinemia.
[1128] In 96 well format, the PRO polypeptide to be analyzed was added to primary rat adipocytes and incubated overnight. Samples were injected at 4 and 16 hours and evaluated for glycerol, glucose and FFA uptake. After incubation for 16 hours, insulin was added to the medium and incubated for 4 hours. At this time, a sample was injected and glycerol, glucose and FFA uptake were measured. Media containing insulin without PRO polypeptide was used as a positive reference control. The PRO polypeptide to be tested was able to stimulate or inhibit glucose and FFA uptake, and the results were recorded as positive if at least 1.5 times or less than 0.5 times the insulin control.
[1129] In this assay, the PRO1890, PRO1785 and PRO4422 polypeptides were tested to be positive as stimulators of glucose and / or FFA absorption.
[1130] In this assay, the PRO4334, PRO4425, PRO4424 and PRO4430 polypeptides were tested as inhibitors of glucose and / or FFA uptake.
[1131] Example 39: Induction of differentiation of pancreatic β-progenitor cells (analysis 89)
[1132] This analysis indicates that certain polypeptides of the present invention are useful for the treatment of various insulin deficiency symptoms, including diabetes mellitus, in mammals because they act to induce differentiation of pancreatic β-cells into progenitor pancreatic β-cells. . The analysis is performed as follows. The analysis utilizes primary cultures of pancreatic cells in mouse fetuses and the primary readings show that the expression of markers representing β-progenitor cells or mature β-cells is altered. Expression of the marker is measured by real time quantitative PCR (RTQ-PCR), where the marker measured is insulin.
[1133] The pancreas was excised from E14 embryos (CD1 mice). The pancreas was then digested by treatment with collagenase / dispase in F12 / DMEM for 40-60 minutes at 37 ° C. (collagenase / dispase, 1.37 mg / ml, Boehringer Mannheim (# 1097113). Volume 5% BSA was used to neutralize digests and cells washed once with RPMI1640. On day 1, cells were pre-coated with 12-well tissue culture plates (20 μg / ml laminin in PBS, Boehringer Mannheim, # Seed from the pancreas of 1 to 2 embryos was distributed per well The culture medium for this primary culture was 14 F / 1640. On day 2, the medium was removed and attached using RPMI / 1640. In addition to the protein to be tested, at least 2 ml of medium was added, on day 4, the medium was removed and RNA was prepared from the cells to analyze the expression of the markers by real-time quantitative RT-PCR. This untreated stand Case of increasing the expression of a cell marker associated β- than the sphere, the protein was considered to be active.
[1134] 14F / 1640 added the following component to RPMI1640 (Gibco).
[1135] Group A 1: 1000
[1136] Group B 1: 1000
[1137] 10 μg / ml recombinant human insulin
[1138] * Aprotinin (50 μg / ml) 1: 2000 (Boehringer Mannheim # 981532)
[1139] 60 ㎍ / ml bovine pituitary extract (BPE)
[1140] Gentamicin 100 ng / ml
[1141] Group A: (in 10 ml PBS)
[1142] Transferrin, 100 mg (Sigma T2252)
[1143] Epidermal growth factor, 100 μg (BRL 100004)
[1144] 10 μl of 5 × ^10-6 M triiodothyronine (Sigma T5516)
[1145] 100 μl 10 ⁻¹ M ethanolamine (Sigma E0135)
[1146] 100 μl 10 ⁻¹ M phosphoethanolamine (Sigma P0503)
[1147] 4 μl of 10 ^-1 M selenium (Aesar # 12574)
[1148] Group C: (in 10 ml of 100% ethanol)
[1149] 2 μl of 5 × 10 ⁻³ M hydrocortisone (Sigma # H0135)
[1150] 100 μl of 1 × ^10-3 M progesterone (Sigma # P6149)
[1151] 20 500 μl of mM forskolin (calbiochem # 344270)
[1152] Minimal medium: RPMI 1640, and transferrin (10 μg / ml), insulin (1 μg / ml), gentamycin (100 ng / ml), aprotinin (50 μg / ml) and BPE (15 μg / ml).
[1153] Restriction medium: RPMI 1640, and transferrin (10 μg / ml), insulin (1 μg / ml), gentamycin (100 ng / ml) and aprotinin (50 μg / ml).
[1154] In this assay, the PRO4356 polypeptide was positive.
[1155] Example 40 Fetal Hemoglobin Induction in Erythrocyte Cell Line (Analysis 107)
[1156] This assay is useful for screening whether PRO polypeptides can induce the transition from adult hemoglobin to fetal hemoglobin in erythroid cell lines. If the molecule tested in this assay is positive, it is expected to be useful for the therapeutic treatment of various mammalian hemoglobin-related diseases such as thalassemia. This analysis is performed as follows. Erythrocyte cells are plated in standard growth medium at 1000 cells per well in 96 well format. PRO polypeptide is added to the growth medium at a concentration of 0.2% or 2% and the cells are incubated at 37 ° C. for 5 days. Cells are treated with 100 μM hemin as a positive control and cells are not treated as a negative control. After 5 days, cell lysates are prepared and analyzed for expression of gamma globin (fetal marker). Positive in this assay means an amount of gamma globin that is at least two times higher than that of the negative control.
[1157] In this assay, the PRO4352, PRO4354, PRO4408, PRO6030 and PRO4499 polypeptides were tested as positive.
[1158] Example 41: Analysis of proliferation of hemangioblasts of mouse kidney (assay 92)
[1159] This assay suggests that Berger's disease, Schöleinlein-Henoch purpura, Chronic Digestive Disorder, and Herpes, because certain polypeptides of the invention act to induce proliferation of hepatic stem cells in mammalian kidneys. It has been shown to be useful for treating renal disease associated with impaired function of hepatic stem cells, such as sexual dermatitis or other neuropathy associated with Crohn's disease. The analysis is performed as follows. On day 1, hepatoblasts of mouse kidneys were grown in 96-well plates in a 3: 1 mixture of growth medium [Dulbecco's modified Eagle's medium and Ham F12 medium, 95% fetal bovine serum, 5% 14 mM HEPES Supplemented] and incubated overnight. On day 2, PRO polypeptide was diluted to two concentrations (1% and 0.1%) in serum-free medium and added to cells. The control sample is serum-free medium alone. On day 4, Cell Titer 96 Aqueous One-Component Solution Reagent (Promega) was added to each well and the color reaction was allowed to proceed for 2 hours. Then, absorbance (OD) was measured at 490 nm. Positive in these assays are those with absorbance values that are at least 15% higher than control readings.
[1160] In this assay, the PRO4380, PRO4408, and PRO4425 polypeptides were tested positive.
[1161] <Material deposit>
[1162] The following materials have been deposited with the American Type Culture Collection (ATCC), Rockville Parklow Drive 12301, Maryland, USA.
[1163] TABLE 7
[1164] Substance ATCC Deposit Number Deposit Date
[1165] * DNA44686-1653 203581 January 12, 1999
[1166] DNA59608-2577 203870 March 23, 1999
[1167] DNA62377-1381 203552 Dec 22, 1998
[1168] DNA77623-2524 203546 December 22, 1998
[1169] DNA79230-2525 203549 Dec 22, 1998
[1170] DNA79862-2522 203550 Dec 22, 1998
[1171] DNA80136-2503 203541 Dec 15, 1998
[1172] DNA80145-2594 204-PTA June 8, 1999
[1173] DNA84917-2597 203863 March 23, 1999
[1174] DNA84920-2614 203966 April 27, 1999
[1175] DNA86576-2595 203868 March 23, 1999
[1176] DNA87976-2593 203888 March 30, 1999
[1177] DNA92234-2602 203948 April 20, 1999
[1178] DNA92256-2596 203891 March 30, 1999
[1179] DNA92274-2617 203971 April 27, 1999
[1180] DNA92929-2534 203586 January 12, 1999
[1181] DNA93011-2637 20-PTA May 4, 1999
[1182] DNA96042-2682 382-PTA July 20, 1999
[1183] DNA96850-2705 479-PTA August 3, 1999
[1184] DNA96857-2636 17-PTA May 4, 1999
[1185] DNA96867-2620 203972 April 27, 1999
[1186] DNA96878-2626 23-PTA May 4, 1999
[1187] DNA96899-2641 119-PTA May 25, 1999
[1188] These deposits were made under the provisions of the Budapest Treaty and its rules (Budapest Treaty) on the international approval of microbial deposits under the patent procedure. This ensures maintenance of the viable culture of the deposit for 30 years from the date of deposit. The deposits will be distributed from ATCC under the Convention of the Budapest Treaty in accordance with the agreement between Genentech Inc and ATCC, which shall be deposited with the public upon the granting of the relevant US patent or upon publication of the US or foreign patent application, whichever comes first. To ensure the permanent and non-limiting distribution of water culture progeny, and to the US Patent and Trademark Commissioner in 35 USC §122 and the rules of the US Patent and Trademark Commissioner (including 37 CFR §1.14, in particular see 886 OG 638). Progenie's sale is assured to those who have the right to do so.
[1189] The assignee of the present application agrees that upon incubation under appropriate conditions, if the culture of the deposited material is killed, lost or damaged, the material will be replaced immediately with another identical material upon notification. The sale of deposited materials should not be construed as an authorization of the governments to carry out the invention in violation of the rights granted under the patent law.
[1190] The foregoing description is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not limited to the scope of the deposited structure as the deposited embodiment is intended as an illustration of certain aspects of the present invention, and any structure that is functionally equivalent is within the scope of the present invention. The deposit of a substance herein does not mean that the disclosure contained herein is inappropriate for carrying out any aspect, including the best mode of the invention, and is intended to limit the scope of the claims to the specific description set forth in the specification. It should not be interpreted. Indeed, various modifications of the invention in addition to those shown and described herein above are apparent to those skilled in the art and will be within the scope of the appended claims.
[1191] The present invention provides novel secretion and transmembrane polypeptides and nucleic acids encoding them. Polypeptides of the invention have the ability to induce cell differentiation and, in particular, act to induce differentiation of chondrocytes and are therefore useful for the treatment of various bone and / or cartilage diseases such as, for example, sports injury and arthritis. It is thought to be.
<110> Genentech, Inc.      Desnoyers, Luc      Eaton, Dan L.      Goddard, Audrey      Godowski, Paul J.      Gurney, Austin L.      Pan, James      Stewart, Timothy A.      Watanabe, Colin K.      Wood, William I.      Zhang, Zemin <120> SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC      ACIDS ENCODING THE SAME <130> P3030R1PCT <140> PCT / US00 / 05601 <141> 2000-03-01 <150> US 60 / 125,774 <151> 1999-03-23 <150> US 60 / 125,778 <151> 1999-03-23 <150> US 60 / 125,826 <151> 1999-03-24 <150> US 60 / 127,035 <151> 1999-03-31 <150> US 60 / 127,706 <151> 1999-04-05 <150> US 60 / 130,359 <151> 1999-04-21 <150> US 60 / 131,270 <151> 1999-04-27 <150> US 60 / 131,272 <151> 1999-04-27 <150> US 60 / 131,291 <151> 1999-04-27 <150> US 60 / 132,371 <151> 1999-05-04 <150> US 60 / 132,379 <151> 1999-05-04 <150> US 60 / 132,383 <151> 1999-05-04 <150> US 60 / 135,750 <151> 1999-05-25 <150> US 60 / 138,166 <151> 1999-06-08 <150> US 60 / 144,791 <151> 1999-07-20 <150> US 60 / 146,970 <151> 1999-08-03 <150> US 60 / 170,262 <151> 1999-12-09 <160> 80 <210> 1 <211> 1712 <212> DNA <213> Homo Sapien <400> 1 ggcatctgcc cgaggagacc acgctcctgg agctctgctg tcttctcagg 50 gagactctga ggctctgttg agaatcatgc tttggaggca gctcatctat 100 tggcaactgc tggctttgtt tttcctccct ttttgcctgt gtcaagatga 150 atacatggag tctccacaaa ccggaggact acccccagac tgcagtaagt 200 gttgtcatgg agactacagc tttcgaggct accaaggccc ccctgggcca 250 ccgggccctc ctggcattcc aggaaaccat ggaaacaatg gcaacaatgg 300 agccactggt catgaaggag ccaaaggtga gaagggcgac aaaggtgacc 350 tggggcctcg aggggagcgg gggcagcatg gccccaaagg agagaagggc 400 tacccgggga ttccaccaga acttcagatt gcattcatgg cttctctggc 450 aacccacttc agcaatcaga acagtgggat tatcttcagc agtgttgaga 500 ccaacattgg aaacttcttt gatgtcatga ctggtagatt tggggcccca 550 gtatcaggtg tgtatttctt caccttcagc atgatgaagc atgaggatgt 600 tgaggaagtg tatgtgtacc ttatgcacaa tggcaacaca gtcttcagca 650 tgtacagcta tgaaatgaag ggcaaatcag atacatccag caatcatgct 700 gtgctgaagc tagccaaagg ggatgaggtt tggctgcgaa tgggcaatgg 750 cgctctccat ggggaccacc aacgcttctc cacctttgca ggattcctgc 800 tctttgaaac taagtaaata tatgactaga atagctccac tttggggaag 850 acttgtagct gagctgattt gttacgatct gaggaacatt aaagttgagg 900 gttttacatt gctgtattca aaaaattatt ggttgcaatg ttgttcacgc 950 tacaggtaca ccaataatgt tggacaattc aggggctcag aagaatcaac 1000 cacaaaatag tcttctcaga tgaccttgac taatatactc agcatcttta 1050 tcactctttc cttggcacct aaaagataat tctcctctga cgcaggttgg 1100 aaatattttt ttctatcaca gaagtcattt gcaaagaatt ttgactactc 1150 tgcttttaat ttaataccag ttttcaggaa cccctgaagt tttaagttca 1200 ttattcttta taacatttga gagaatcgga tgtagtgata tgacagggct 1250 ggggcaagaa caggggcact agctgcctta ttagctaatt tagtgccctc 1300 cgtgttcagc ttagcctttg accctttcct tttgatccac aaaatacatt 1350 aaaactctga attcacatac aatgctattt taaagtcaat agattttagc 1400 tataaagtgc ttgaccagta atgtggttgt aattttgtgt atgttccccc 1450 acatcgcccc caacttcgga tgtggggtca ggaggttgag gttcactatt 1500 aacaaatgtc ataaatatct catagaggta cagtgccaat agatattcaa 1550 atgttgcatg ttgaccagag ggattttata tctgaagaac atacactatt 1600 aataaatacc ttagagaaag attttgacct ggctttagat aaaactgtgg 1650 caagaaaaat gtaatgagca atatatggaa ataaacacac ctttgttaaa 1700 gataaaaaaa aa 1712 <210> 2 <211> 246 <212> PRT <213> Homo Sapien <400> 2 Met Leu Trp Arg Gln Leu Ile Tyr Trp Gln Leu Leu Ala Leu Phe   1 5 10 15 Phe Leu Pro Phe Cys Leu Cys Gln Asp Glu Tyr Met Glu Ser Pro                  20 25 30 Gln Thr Gly Gly Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly                  35 40 45 Asp Tyr Ser Phe Arg Gly Tyr Gln Gly Pro Pro Gly Pro Pro Gly                  50 55 60 Pro Pro Gly Ile Pro Gly Asn His Gly Asn Asn Gly Asn Asn Gly                  65 70 75 Ala Thr Gly His Glu Gly Ala Lys Gly Glu Lys Gly Asp Lys Gly                  80 85 90 Asp Leu Gly Pro Arg Gly Glu Arg Gly Gln His Gly Pro Lys Gly                  95 100 105 Glu Lys Gly Tyr Pro Gly Ile Pro Pro Glu Leu Gln Ile Ala Phe                 110 115 120 Met Ala Ser Leu Ala Thr His Phe Ser Asn Gln Asn Ser Gly Ile                 125 130 135 Ile Phe Ser Ser Val Glu Thr Asn Ile Gly Asn Phe Phe Asp Val                 140 145 150 Met Thr Gly Arg Phe Gly Ala Pro Val Ser Gly Val Tyr Phe Phe                 155 160 165 Thr Phe Ser Met Met Lys His Glu Asp Val Glu Glu Val Tyr Val                 170 175 180 Tyr Leu Met His Asn Gly Asn Thr Val Phe Ser Met Tyr Ser Tyr                 185 190 195 Glu Met Lys Gly Lys Ser Asp Thr Ser Ser Asn His Ala Val Leu                 200 205 210 Lys Leu Ala Lys Gly Asp Glu Val Trp Leu Arg Met Gly Asn Gly                 215 220 225 Ala Leu His Gly Asp His Gln Arg Phe Ser Thr Phe Ala Gly Phe                 230 235 240 Leu Leu Phe Glu Thr Lys                 245 <210> 3 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 3 tgtaaaacga cggccagtta aatagacctg caattattaa tct 43 <210> 4 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 4 caggaaacag ctatgaccac ctgcacacct gcaaatccat t 41 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 5 gcaacaatgg agccactggt catg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 6 gcaaaggtgg agaagcgttg gtgg 24 <210> 7 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 7 cccacttcag caatcagaac agtgggatta tctttcagca gtgtttgaga 50 cc 52 <210> 8 <211> 1579 <212> DNA <213> Homo Sapien <400> 8 gagagaatag ctacagattc tccatcctca gtctttgcaa ggcgacagct 50 gtgccagccg ggctctggca ggctcctggc agcatggcag tgaagcttgg 100 gaccctcctg ctggcccttg ccctgggcct ggcccagcca gcctctgccc 150 gccggaagct gctggtgttt ctgctggatg gttttcgctc agactacatc 200 agtgatgagg cgctggagtc attgcctggt ttcaaagaga ttgtgagcag 250 gggagtaaaa gtggattact tgactccaga cttccctagt ctctcgtatc 300 ccaattatta taccctaatg actggccgcc attgtgaagt ccatcagatg 350 atcgggaact acatgtggga ccccaccacc aacaagtcct ttgacattgg 400 cgtcaacaaa gacagcctaa tgcctctctg gtggaatgga tcagaacctc 450 tgtgggtcac tctgaccaag gccaaaagga aggtctacat gtactactgg 500 ccaggctgtg aggttgagat tctgggtgtc agacccacct actgcctaga 550 atataaaaat gtcccaacgg atatcaattt tgccaatgca gtcagcgatg 600 ctcttgactc cttcaagagt ggccgggccg acctggcagc catataccat 650 gagcgcattg acgtggaagg ccaccactac gggcctgcat ctccgcagag 700 gaaagatgcc ctcaaggctg tagacactgt cctgaagtac atgaccaagt 750 ggatccagga gcggggcctg caggaccgcc tgaacgtcat tattttctcg 800 gatcacggaa tgaccgacat tttctggatg gacaaagtga ttgagctgaa 850 taagtacatc agcctgaatg acctgcagca agtgaaggac cgcgggcctg 900 ttgtgagcct ttggccggcc cctgggaaac actctgagat atataacaaa 950 ctgagcacag tggaacacat gactgtctac gagaaagaag ccatcccaag 1000 caggttctat tacaagaaag gaaagtttgt ctctcctttg actttagtgg 1050 ctgatgaagg ctggttcata actgagaatc gagagatgct tccgttttgg 1100 atgaacagca ccggcaggcg ggaaggttgg cagcgtggat ggcacggcta 1150 cgacaacgag ctcatggaca tgcggggcat cttcctggcc ttcggacctg 1200 atttcaaatc caacttcaga gctgctccta tcaggtcggt ggacgtctac 1250 aatgtcatgt gcaatgtggt gggcatcacc ccgctgccca acaacggatc 1300 ctggtccagg gtgatgtgca tgctgaaggg ccgcgccggc actgccccgc 1350 ctgtctggcc cagccactgt gccctggcac tgattcttct cttcctgctt 1400 gcataactga tcatattgct tgtctcagaa aaaaacacca tcagcaaagt 1450 gggcctccaa agccagatga ttttcatttt atgtgtgaat aatagcttca 1500 ttaacacaat caagaccatg cacattgtaa atacattatt cttggataat 1550 tctatacata aaagttccta cttgttaaa 1579 <210> 9 <211> 440 <212> PRT <213> Homo Sapien <400> 9 Met Ala Val Lys Leu Gly Thr Leu Leu Leu Ala Leu Ala Leu Gly   1 5 10 15 Leu Ala Gln Pro Ala Ser Ala Arg Arg Lys Leu Leu Val Phe Leu                  20 25 30 Leu Asp Gly Phe Arg Ser Asp Tyr Ile Ser Asp Glu Ala Leu Glu                  35 40 45 Ser Leu Pro Gly Phe Lys Glu Ile Val Ser Arg Gly Val Lys Val                  50 55 60 Asp Tyr Leu Thr Pro Asp Phe Pro Ser Leu Ser Tyr Pro Asn Tyr                  65 70 75 Tyr Thr Leu Met Thr Gly Arg His Cys Glu Val His Gln Met Ile                  80 85 90 Gly Asn Tyr Met Trp Asp Pro Thr Thr Asn Lys Ser Phe Asp Ile                  95 100 105 Gly Val Asn Lys Asp Ser Leu Met Pro Leu Trp Trp Asn Gly Ser                 110 115 120 Glu Pro Leu Trp Val Thr Leu Thr Lys Ala Lys Arg Lys Val Tyr                 125 130 135 Met Tyr Tyr Trp Pro Gly Cys Glu Val Glu Ile Leu Gly Val Arg                 140 145 150 Pro Thr Tyr Cys Leu Glu Tyr Lys Asn Val Pro Thr Asp Ile Asn                 155 160 165 Phe Ala Asn Ala Val Ser Asp Ala Leu Asp Ser Phe Lys Ser Gly                 170 175 180 Arg Ala Asp Leu Ala Ala Ile Tyr His Glu Arg Ile Asp Val Glu                 185 190 195 Gly His His Tyr Gly Pro Ala Ser Pro Gln Arg Lys Asp Ala Leu                 200 205 210 Lys Ala Val Asp Thr Val Leu Lys Tyr Met Thr Lys Trp Ile Gln                 215 220 225 Glu Arg Gly Leu Gln Asp Arg Leu Asn Val Ile Ile Phe Ser Asp                 230 235 240 His Gly Met Thr Asp Ile Phe Trp Met Asp Lys Val Ile Glu Leu                 245 250 255 Asn Lys Tyr Ile Ser Leu Asn Asp Leu Gln Gln Val Lys Asp Arg                 260 265 270 Gly Pro Val Val Ser Leu Trp Pro Ala Pro Gly Lys His Ser Glu                 275 280 285 Ile Tyr Asn Lys Leu Ser Thr Val Glu His Met Thr Val Tyr Glu                 290 295 300 Lys Glu Ala Ile Pro Ser Arg Phe Tyr Tyr Lys Lys Gly Lys Phe                 305 310 315 Val Ser Pro Leu Thr Leu Val Ala Asp Glu Gly Trp Phe Ile Thr                 320 325 330 Glu Asn Arg Glu Met Leu Pro Phe Trp Met Asn Ser Thr Gly Arg                 335 340 345 Arg Glu Gly Trp Gln Arg Gly Trp His Gly Tyr Asp Asn Glu Leu                 350 355 360 Met Asp Met Arg Gly Ile Phe Leu Ala Phe Gly Pro Asp Phe Lys                 365 370 375 Ser Asn Phe Arg Ala Ala Pro Ile Arg Ser Val Asp Val Tyr Asn                 380 385 390 Val Met Cys Asn Val Val Gly Ile Thr Pro Leu Pro Asn Asn Gly                 395 400 405 Ser Trp Ser Arg Val Met Cys Met Leu Lys Gly Arg Ala Gly Thr                 410 415 420 Ala Pro Pro Val Trp Pro Ser His Cys Ala Leu Ala Leu Ile Leu                 425 430 435 Leu Phe Leu Leu Ala                 440 <210> 10 <211> 1047 <212> DNA <213> Homo Sapien <400> 10 gccaggtgtg caggccgctc caagcccagc ctgccccgct gccgccacca 50 tgacgctcct ccccggcctc ctgtttctga cctggctgca cacatgcctg 100 gcccaccatg acccctccct cagggggcac ccccacagtc acggtacccc 150 acactgctac tcggctgagg aactgcccct cggccaggcc cccccacacc 200 tgctggctcg aggtgccaag tgggggcagg ctttgcctgt agccctggtg 250 tccagcctgg aggcagcaag ccacaggggg aggcacgaga ggccctcagc 300 tacgacccag tgcccggtgc tgcggccgga ggaggtgttg gaggcagaca 350 cccaccagcg ctccatctca ccctggagat accgtgtgga cacggatgag 400 gaccgctatc cacagaagct ggccttcgcc gagtgcctgt gcagaggctg 450 tatcgatgca cggacgggcc gcgagacagc tgcgctcaac tccgtgcggc 500 tgctccagag cctgctggtg ctgcgccgcc ggccctgctc ccgcgacggc 550 tcggggctcc ccacacctgg ggcctttgcc ttccacaccg agttcatcca 600 cgtccccgtc ggctgcacct gcgtgctgcc ccgttcagtg tgaccgccga 650 ggccgtgggg cccctagact ggacacgtgt gctccccaga gggcaccccc 700 tatttatgtg tatttattgt tatttatatg cctcccccaa cactaccctt 750 ggggtctggg cattccccgt gtctggagga cagcccccca ctgttctcct 800 catctccagc ctcagtagtt gggggtagaa ggagctcagc acctcttcca 850 gcccttaaag ctgcagaaaa ggtgtcacac ggctgcctgt accttggctc 900 cctgtcctgc tcccggcttc ccttacccta tcactggcct caggccccgc 950 aggctgcctc ttcccaacct ccttggaagt acccctgttt cttaaacaat 1000 tatttaagtg tacgtgtatt attaaactga tgaacacatc cccaaaa 1047 <210> 11 <211> 197 <212> PRT <213> Homo Sapien <400> 11 Met Thr Leu Leu Pro Gly Leu Leu Phe Leu Thr Trp Leu His Thr   1 5 10 15 Cys Leu Ala His His Asp Pro Ser Leu Arg Gly His Pro His Ser                  20 25 30 His Gly Thr Pro His Cys Tyr Ser Ala Glu Glu Leu Pro Leu Gly                  35 40 45 Gln Ala Pro Pro His Leu Leu Ala Arg Gly Ala Lys Trp Gly Gln                  50 55 60 Ala Leu Pro Val Ala Leu Val Ser Ser Leu Glu Ala Ala Ser His                  65 70 75 Arg Gly Arg His Glu Arg Pro Ser Ala Thr Thr Gln Cys Pro Val                  80 85 90 Leu Arg Pro Glu Glu Val Leu Glu Ala Asp Thr His Gln Arg Ser                  95 100 105 Ile Ser Pro Trp Arg Tyr Arg Val Asp Thr Asp Glu Asp Arg Tyr                 110 115 120 Pro Gln Lys Leu Ala Phe Ala Glu Cys Leu Cys Arg Gly Cys Ile                 125 130 135 Asp Ala Arg Thr Gly Arg Glu Thr Ala Ala Leu Asn Ser Val Arg                 140 145 150 Leu Leu Gln Ser Leu Leu Val Leu Arg Arg Arg Pro Cys Ser Arg                 155 160 165 Asp Gly Ser Gly Leu Pro Thr Pro Gly Ala Phe Ala Phe His Thr                 170 175 180 Glu Phe Ile His Val Pro Val Gly Cys Thr Cys Val Leu Pro Arg                 185 190 195 Ser val <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 12 atccacagaa gctggccttc gccg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 13 gggacgtgga tgaactcggt gtgg 24 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 14 tatccacaga agctggcctt cgccgagtgc ctgtgcagag 40 <210> 15 <211> 660 <212> DNA <213> Homo Sapien <400> 15 cggccagggc gccgacagcc cgacctcacc aggagaacat gcagctcggc 50 actgggctcc tgctggccgc cgtcctgagc ctgcagctgg ctgcagccga 100 agccatatgg tgtcaccagt gcacgggctt cggagggtgc tcccatggat 150 ccagatgcct gagggactcc acccactgtg tcaccactgc cacccgggtc 200 ctcagcaaca ccgaggattt gcctctggtc accaagatgt gccacatagg 250 ctgccccgat atccccagcc tgggcctggg cccctacgta tccatcgctt 300 gctgccagac cagcctctgc aaccatgact gacggctgcc ctcctccagg 350 cccccggacg ctcagccccc acagccccca cagcctggcg ccagggctca 400 cggccgcccc tccctcgaga ctggccagcc cacctctccc ggcctctgca 450 gccaccgtcc agcaccgctt gtcctaggga agtcctgcgt ggagtcttgc 500 ctcaatctgc tgccgtccaa gcctggggcc catcgtgcct gccgcccctt 550 caggtcccga cctccccaca ataaaatgtg attggatcgt gtggtacaaa 600 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 650 aaaaaaaaaa 660 <210> 16 <211> 97 <212> PRT <213> Homo Sapien <400> 16 Met Gln Leu Gly Thr Gly Leu Leu Lela Ala Ala Val Leu Ser Leu   1 5 10 15 Gln Leu Ala Ala Ala Glu Ala Ile Trp Cys His Gln Cys Thr Gly                  20 25 30 Phe Gly Gly Cys Ser His Gly Ser Arg Cys Leu Arg Asp Ser Thr                  35 40 45 His Cys Val Thr Thr Ala Thr Arg Val Leu Ser Asn Thr Glu Asp                  50 55 60 Leu Pro Leu Val Thr Lys Met Cys His Ile Gly Cys Pro Asp Ile                  65 70 75 Pro Ser Leu Gly Leu Gly Pro Tyr Val Ser Ile Ala Cys Cys Gln                  80 85 90 Thr Ser Leu Cys Asn His Asp                  95 <210> 17 <211> 2570 <212> DNA <213> Homo Sapien <400> 17 ccaggaccag ggcgcaccgg ctcagcctct cacttgtcag aggccgggga 50 agagaagcaa agcgcaacgg tgtggtccaa gccggggctt ctgcttcgcc 100 tctaggacat acacgggacc ccctaacttc agtcccccaa acgcgcaccc 150 tcgaagtctt gaactccagc cccgcacatc cacgcgcggc acaggcgcgg 200 caggcggcag gtcccggccg aaggcgatgc gcgcaggggg tcgggcagct 250 gggctcgggc ggcgggagta gggcccggca gggaggcagg gaggctgcat 300 attcagagtc gcgggctgcg ccctgggcag aggccgccct cgctccacgc 350 aacacctgct gctgccaccg cgccgcgatg agccgcgtgg tctcgctgct 400 gctgggcgcc gcgctgctct gcggccacgg agccttctgc cgccgcgtgg 450 tcagcggcca aaaggtgtgt tttgctgact tcaagcatcc ctgctacaaa 500 atggcctact tccatgaact gtccagccga gtgagctttc aggaggcacg 550 cctggcttgt gagagtgagg gaggagtcct cctcagcctt gagaatgaag 600 cagaacagaa gttaatagag agcatgttgc aaaacctgac aaaacccggg 650 acagggattt ctgatggtga tttctggata gggctttgga ggaatggaga 700 tgggcaaaca tctggtgcct gcccagatct ctaccagtgg tctgatggaa 750 gcaattccca gtaccgaaac tggtacacag atgaaccttc ctgcggaagt 800 gaaaagtgtg ttgtgatgta tcaccaacca actgccaatc ctggccttgg 850 gggtccctac ctttaccagt ggaatgatga caggtgtaac atgaagcaca 900 attatatttg caagtatgaa ccagagatta atccaacagc ccctgtagaa 950 aagccttatc ttacaaatca accaggagac acccatcaga atgtggttgt 1000 tactgaagca ggtataattc ccaatctaat ttatgttgtt ataccaacaa 1050 tacccctgct cttactgata ctggttgctt ttggaacctg ttgtttccag 1100 atgctgcata aaagtaaagg aagaacaaaa actagtccaa accagtctac 1150 actgtggatt tcaaagagta ccagaaaaga aagtggcatg gaagtataat 1200 aactcattga cttggttcca gaattttgta attctggatc tgtataagga 1250 atggcatcag aacaatagct tggaatggct tgaaatcaca aaggatctgc 1300 aagatgaact gtaagctccc ccttgaggca aatattaaag taatttttat 1350 atgtctatta tttcatttaa agaatatgct gtgctaataa tggagtgaga 1400 catgcttatt ttgctaaagg atgcacccaa acttcaaact tcaagcaaat 1450 gaaatggaca atgcagataa agttgttatc aacacgtcgg gagtatgtgt 1500 gttagaagca attcctttta tttctttcac ctttcataag ttgttatcta 1550 gtcaatgtaa tgtatattgt attgaaattt acagtgtgca aaagtatttt 1600 acctttgcat aagtgtttga taaaaatgaa ctgttctaat atttattttt 1650 atggcatctc atttttcaat acatgctctt ttgattaaag aaacttatta 1700 ctgttgtcaa ctgaattcac acacacacaa atatagtacc atagaaaaag 1750 tttgttttct cgaaataatt catctttcag cttctctgct tttggtcaat 1800 gtctaggaaa tctcttcaga aataagaagc tatttcatta agtgtgatat 1850 aaacctcctc aaacatttta cttagaggca aggattgtct aatttcaatt 1900 gtgcaagaca tgtgccttat aattattttt agcttaaaat taaacagatt 1950 ttgtaataat gtaactttgt taataggtgc ataaacacta atgcagtcaa 2000 tttgaacaaa agaagtgaca tacacaatat aaatcatatg tcttcacacg 2050 ttgcctatat aatgagaagc agctctctga gggttctgaa atcaatgtgg 2100 tccctctctt gcccactaaa caaagatggt tgttcggggt ttgggattga 2150 cactggaggc agatagttgc aaagttagtc taaggtttcc ctagctgtat 2200 ttagcctctg actatattag tatacaaaga ggtcatgtgg ttgagaccag 2250 gtgaatagtc actatcagtg tggagacaag cacagcacac agacatttta 2300 ggaaggaaag gaactacgaa atcgtgtgaa aatgggttgg aacccatcag 2350 tgatcgcata ttcattgatg agggtttgct tgagatagaa aatggtggct 2400 cctttctgtc ttatctccta gtttcttcaa tgcttacgcc ttgttcttct 2450 caagagaaag ttgtaactct ctggtcttca tatgtccctg tgctcctttt 2500 aaccaaataa agagttcttg tttctggggg aaaaaaaaaa aaaaaaaaaa 2550 aaaaaaaaaa aaaaaaaaaa 2570 <210> 18 <211> 273 <212> PRT <213> Homo Sapien <400> 18 Met Ser Arg Val Val Ser Leu Leu Leu Gly Ala Ala Leu Leu Cys   1 5 10 15 Gly His Gly Ala Phe Cys Arg Arg Val Val Ser Gly Gln Lys Val                  20 25 30 Cys Phe Ala Asp Phe Lys His Pro Cys Tyr Lys Met Ala Tyr Phe                  35 40 45 His Glu Leu Ser Ser Arg Val Ser Phe Gln Glu Ala Arg Leu Ala                  50 55 60 Cys Glu Ser Glu Gly Gly Val Leu Leu Ser Leu Glu Asn Glu Ala                  65 70 75 Glu Gln Lys Leu Ile Glu Ser Met Leu Gln Asn Leu Thr Lys Pro                  80 85 90 Gly Thr Gly Ile Ser Asp Gly Asp Phe Trp Ile Gly Leu Trp Arg                  95 100 105 Asn Gly Asp Gly Gln Thr Ser Gly Ala Cys Pro Asp Leu Tyr Gln                 110 115 120 Trp Ser Asp Gly Ser Asn Ser Gln Tyr Arg Asn Trp Tyr Thr Asp                 125 130 135 Glu Pro Ser Cys Gly Ser Glu Lys Cys Val Val Met Tyr His Gln                 140 145 150 Pro Thr Ala Asn Pro Gly Leu Gly Gly Pro Tyr Leu Tyr Gln Trp                 155 160 165 Asn Asp Asp Arg Cys Asn Met Lys His Asn Tyr Ile Cys Lys Tyr                 170 175 180 Glu Pro Glu Ile Asn Pro Thr Ala Pro Val Glu Lys Pro Tyr Leu                 185 190 195 Thr Asn Gln Pro Gly Asp Thr His Gln Asn Val Val Val Thr Glu                 200 205 210 Ala Gly Ile Ile Pro Asn Leu Ile Tyr Val Val Ile Pro Thr Ile                 215 220 225 Pro Leu Leu Leu Leu Ile Leu Val Ala Phe Gly Thr Cys Cys Phe                 230 235 240 Gln Met Leu His Lys Ser Lys Gly Arg Thr Lys Thr Ser Pro Asn                 245 250 255 Gln Ser Thr Leu Trp Ile Ser Lys Ser Thr Arg Lys Glu Ser Gly                 260 265 270 Met glu val <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 19 caccaaccaa ctgccaatcc tggc 24 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 20 accacattct gatgggtgtc tcctgg 26 <210> 21 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 21 gggtccctac ctttaccagt ggaatgatga caggtgtaac atgaagcac 49 <210> 22 <211> 3824 <212> DNA <213> Homo Sapien <400> 22 ggagaatgga gagagcagtg agagtggagt ccggggtcct ggtcggggtg 50 gtctgtctgc tcctggcatg ccctgccaca gccactgggc ccgaagttgc 100 tcagcctgaa gtagacacca ccctgggtcg tgtgcgaggc cggcaggtgg 150 gcgtgaaggg cacagaccgc cttgtgaatg tctttctggg cattccattt 200 gcccagccgc cactgggccc tgaccggttc tcagccccac acccagcaca 250 gccctgggag ggtgtgcggg atgccagcac tgcgccccca atgtgcctac 300 aagacgtgga gagcatgaac agcagcagat ttgtcctcaa cggaaaacag 350 cagatcttct ccgtttcaga ggactgcctg gtcctcaacg tctatagccc 400 agctgaggtc cccgcagggt ccggtaggcc ggtcatggta tgggtccatg 450 gaggcgctct gataactggc gctgccacct cctacgatgg atcagctctg 500 gctgcctatg gggatgtggt cgtggttaca gtccagtacc gccttggggt 550 ccttggcttc ttcagcactg gagatgagca tgcacctggc aaccagggct 600 tcctagatgt ggtagctgct ttgcgctggg tgcaagaaaa catcgccccc 650 ttcgggggtg acctcaactg tgtcactgtc tttggtggat ctgccggtgg 700 gagcatcatc tctggcctgg tcctgtcccc agtggctgca gggctgttcc 750 acagagccat cacacagagt ggggtcatca ccaccccagg gatcatcgac 800 tctcaccctt ggcccctagc tcagaaaatc gcaaacacct tggcctgcag 850 ctccagctcc ccggctgaga tggtgcagtg ccttcagcag aaagaaggag 900 aagagctggt ccttagcaag aagctgaaaa atactatcta tcctctcacc 950 gttgatggca ctgtcttccc caaaagcccc aaggaactcc tgaaggagaa 1000 gcccttccac tctgtgccct tcctcatggg tgtcaacaac catgagttca 1050 gctggctcat ccccaggggc tggggtctcc tggatacaat ggagcagatg 1100 agccgggagg acatgctggc catctcaaca cccgtcttga ccagtctgga 1150 tgtgccccct gagatgatgc ccaccgtcat agatgaatac ctaggaagca 1200 actcggacgc acaagccaaa tgccaggcgt tccaggaatt catgggtgac 1250 gtattcatca atgttcccac cgtcagtttt tcaagatacc ttcgagattc 1300 tggaagccct gtctttttct atgagttcca gcatcgaccc agttcttttg 1350 cgaagatcaa acctgcctgg gtgaaggctg atcatggggc cgagggtgct 1400 tttgtgttcg gaggtccctt cctcatggac gagagctccc gcctggcctt 1450 tccagaggcc acagaggagg agaagcagct aagcctcacc atgatggccc 1500 agtggaccca ctttgcccgg acaggggacc ccaatagcaa ggctctgcct 1550 ccttggcccc aattcaacca ggcggaacaa tatctggaga tcaacccagt 1600 gccacgggcc ggacagaagt tcagggaggc ctggatgcag ttctggtcag 1650 agacgctccc cagcaagata caacagtggc accagaagca gaagaacagg 1700 aaggcccagg aggacctctg aggccaggcc tgaaccttct tggctggggc 1750 aaaccactct tcaagtggtg gcagagtccc agcacggcag cccgcctctc 1800 cccctgctga gactttaatc tccaccagcc cttaaagtgt cggccgctct 1850 gtgactggag ttatgctctt ttgaaatgtc acaaggccgc ctcccacctc 1900 tggggcattg tacaagttct tccctctccc tgaagtgcct ttcctgcttt 1950 cttcgtggta ggttctagca cattcctcta gcttcctgga ggactcactc 2000 cccaggaagc cttccctgcc ttctctgggc tgtgcggccc cgagtctgcg 2050 tccattagag cacagtccac ccgaggctag caccgtgtct gtgtctgtct 2100 ccccctcaga ggagctctct caaaatgggg attagcctaa ccccactctg 2150 tcacccacac caggatcggg tgggacctgg agctaggggg tgtttgctga 2200 gtgagtgagt gaaacacaga atatgggaat ggcagctgct gaacttgaac 2250 ccagagcctt caggtgccaa agccatactc aggcccccac cgacattgtc 2300 caccctggcc agaagggtgc atgccaatgg cagagacctg ggatgggaga 2350 agtcctgggg cgccagggga tccagcctag agcagacctt agcccctgac 2400 taaggcctca gactagggcg ggaggggtct cctcctctct gctgcccagt 2450 cctggcccct gcacaagaca acagaatcca tcagggccat gagtgtcacc 2500 cagacctgac cctcaccaat tccagcccct gaccctcagg acgctggatg 2550 ccagctccca gccccagtgc cgggtcctcc ctcccttcct ggcttgggga 2600 gaccagtttc tggggagctt ccaagagcac ccaccaagac acagcaggac 2650 aggccagggg agggcatctg gaccagggca tccgtcgggc tattgtcaca 2700 gagaaaagaa gagacccacc cactcgggct gcaaaaggtg aaaagcacca 2750 agaggttttc agatggaagt gagaggtgac agtgtgctgg cagccctcac 2800 agccctcgct tgctctccct gccgcctctg cctgggctcc cactttggca 2850 gcacttgagg agcccttcaa cccgccgctg cactgtagga gcccctttct 2900 gggctggcca aggccggagc cagctccctc agcttgcggg gaggtgcgga 2950 gggagagggg cgggcaggaa ccggggctgc gcgcagcgct tgcgggccag 3000 agtgagttcc gggtgggcgt gggctcggcg gggccccact cagagcagct 3050 ggccggcccc aggcagtgag ggccttagca cctgggccag cagctgctgt 3100 gctcgatttc tcgctgggcc ttagctgcct ccccgcgggg cagggctcgg 3150 gacctgcagc cctccatgcc tgaccctccc cccacccccc gtgggctcct 3200 gtgcggccgg agcctcccca aggagcgccg ccccctgctc cacagcgccc 3250 agtcccatcg accacccaag ggctgaggag tgcgggtgca cagcgcggga 3300 ctggcaggca gctccacctg ctgccccagt gctggatcca ctgggtgaag 3350 ccagctgggc tcctgagtct ggtggggact tggagaacct ttatgtctag 3400 ctaagggatt gtaaatacac cgatgggcac tctgtatcta gctcaaggtt 3450 tgtaaacaca ccaatcagca ccctgtgtct agctcagtgt ttgtgaatgc 3500 accaatccac actctgtatc tggctactct ggtggggact tggagaacct 3550 ttgtgtccac actctgtatc tagctaatct agtggggatg tggagaacct 3600 ttgtgtctag ctcagggatc gtaaacgcac caatcagcac cctgtcaaaa 3650 cagaccactt gactctctgt aaaatggacc aatcagcagg atgtgggtgg 3700 ggcgagacaa gagaataaaa gcaggctgcc tgagccagca gtgacaaccc 3750 ccctcgggtc ccctcccacg ccgtggaagc tttgttcttt cgctctttgc 3800 aataaatctt gctactgccc aaaa 3824 <210> 23 <211> 571 <212> PRT <213> Homo Sapien <400> 23 Met Glu Arg Ala Val Arg Val Glu Ser Gly Val Leu Val Gly Val   1 5 10 15 Val Cys Leu Leu Leu Ala Cys Pro Ala Thr Ala Thr Gly Pro Glu                  20 25 30 Val Ala Gln Pro Glu Val Asp Thr Thr Leu Gly Arg Val Arg Gly                  35 40 45 Arg Gln Val Gly Val Lys Gly Thr Asp Arg Leu Val Asn Val Phe                  50 55 60 Leu Gly Ile Pro Phe Ala Gln Pro Pro Leu Gly Pro Asp Arg Phe                  65 70 75 Ser Ala Pro His Pro Ala Gln Pro Trp Glu Gly Val Arg Asp Ala                  80 85 90 Ser Thr Ala Pro Pro Met Cys Leu Gln Asp Val Glu Ser Met Asn                  95 100 105 Ser Ser Arg Phe Val Leu Asn Gly Lys Gln Gln Ile Phe Ser Val                 110 115 120 Ser Glu Asp Cys Leu Val Leu Asn Val Tyr Ser Pro Ala Glu Val                 125 130 135 Pro Ala Gly Ser Gly Arg Pro Val Met Val Trp Val His Gly Gly                 140 145 150 Ala Leu Ile Thr Gly Ala Ala Thr Ser Tyr Asp Gly Ser Ala Leu                 155 160 165 Ala Ala Tyr Gly Asp Val Val Val Val Thr Val Gln Tyr Arg Leu                 170 175 180 Gly Val Leu Gly Phe Phe Ser Thr Gly Asp Glu His Ala Pro Gly                 185 190 195 Asn Gln Gly Phe Leu Asp Val Val Ala Ala Leu Arg Trp Val Gln                 200 205 210 Glu Asn Ile Ala Pro Phe Gly Gly Asp Leu Asn Cys Val Thr Val                 215 220 225 Phe Gly Gly Ser Ala Gly Gly Ser Ile Ile Ser Gly Leu Val Leu                 230 235 240 Ser Pro Val Ala Ala Gly Leu Phe His Arg Ala Ile Thr Gln Ser                 245 250 255 Gly Val Ile Thr Thr Pro Gly Ile Ile Asp Ser His Pro Trp Pro                 260 265 270 Leu Ala Gln Lys Ile Ala Asn Thr Leu Ala Cys Ser Ser Ser Ser                 275 280 285 Pro Ala Glu Met Val Gln Cys Leu Gln Gln Lys Glu Gly Glu Glu                 290 295 300 Leu Val Leu Ser Lys Lys Leu Lys Asn Thr Ile Tyr Pro Leu Thr                 305 310 315 Val Asp Gly Thr Val Phe Pro Lys Ser Pro Lys Glu Leu Leu Lys                 320 325 330 Glu Lys Pro Phe His Ser Val Pro Phe Leu Met Gly Val Asn Asn                 335 340 345 His Glu Phe Ser Trp Leu Ile Pro Arg Gly Trp Gly Leu Leu Asp                 350 355 360 Thr Met Glu Gln Met Ser Arg Glu Asp Met Leu Ala Ile Ser Thr                 365 370 375 Pro Val Leu Thr Ser Leu Asp Val Pro Pro Glu Met Met Pro Thr                 380 385 390 Val Ile Asp Glu Tyr Leu Gly Ser Asn Ser Asp Ala Gln Ala Lys                 395 400 405 Cys Gln Ala Phe Gln Glu Phe Met Gly Asp Val Phe Ile Asn Val                 410 415 420 Pro Thr Val Ser Phe Ser Arg Tyr Leu Arg Asp Ser Gly Ser Pro                 425 430 435 Val Phe Phe Tyr Glu Phe Gln His Arg Pro Ser Ser Phe Ala Lys                 440 445 450 Ile Lys Pro Ala Trp Val Lys Ala Asp His Gly Ala Glu Gly Ala                 455 460 465 Phe Val Phe Gly Gly Pro Phe Leu Met Asp Glu Ser Ser Arg Leu                 470 475 480 Ala Phe Pro Glu Ala Thr Glu Glu Glu Lys Gln Leu Ser Leu Thr                 485 490 495 Met Met Ala Gln Trp Thr His Phe Ala Arg Thr Gly Asp Pro Asn                 500 505 510 Ser Lys Ala Leu Pro Pro Trp Pro Gln Phe Asn Gln Ala Glu Gln                 515 520 525 Tyr Leu Glu Ile Asn Pro Val Pro Arg Ala Gly Gln Lys Phe Arg                 530 535 540 Glu Ala Trp Met Gln Phe Trp Ser Glu Thr Leu Pro Ser Lys Ile                 545 550 555 Gln Gln Trp His Gln Lys Gln Lys Asn Arg Lys Ala Gln Glu Asp                 560 565 570 Leu <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 24 gcaaagctct gcctccttgg cc 22 <210> 25 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 25 gggtggactg tgctctaatg gacgc 25 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 26 cgtggcactg ggttgatc 18 <210> 27 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 27 gatgcagttc tggtcagaga cgctccccag caagatacaa cagtg 45 <210> 28 <211> 1342 <212> DNA <213> Homo Sapien <400> 28 catggagcct cttgcagctt acccgctaaa atgttccggg cccagagcaa 50 aggtatttgc agttttgctg tctatagttc tatgcacagt aacgctattt 100 cttctacaac taaaattcct caaacctaaa atcaacagct tttatgcctt 150 tgaagtgaag gatgcaaaag gaagaactgt ttctctggaa aagtataaag 200 gcaaagtttc actagttgta aacgtggcca gtgactgcca actcacagac 250 agaaattact tagggctgaa ggaactgcac aaagagtttg gaccatccca 300 cttcagcgtg ttggcttttc cctgcaatca gtttggagaa tcggagcccc 350 gcccaagcaa ggaagtagaa tcttttgcaa gaaaaaacta cggagtaact 400 ttccccatct tccacaagat taagattcta ggatctgaag gagaacctgc 450 atttagattt cttgttgatt cttcaaagaa ggaaccaagg tggaattttt 500 ggaagtatct tgtcaaccct gagggtcaag ttgtgaagtt ctggaggcca 550 gaggagccca ttgaagtcat caggcctgac atagcagctc tggttagaca 600 agtgatcata aaaaagaaag aggatctatg agaatgccat tgcgtttcta 650 atagaacaga gaaatgtctc catgagggtt tggtctcatt ttaaacattt 700 tttttttgga gacagtgtct cactctgtca cccaggctgg agtgcagtag 750 tgcgttctca gctcattgca acctctgcct ttttaaacat gctattaaat 800 gtggcaatga aggatttttt tttaatgtta tcttgctatt aagtggtaat 850 gaatgttccc aggatgagga tgttacccaa agcaaaaatc aagagtagcc 900 aaagaatcaa catgaaatat attaactact tcctctgacc atactaaaga 950 attcagaata cacagtgacc aatgtgcctc aatatcttat tgttcaactt 1000 gacattttct aggactgtac ttgatgaaaa tgccaacaca ctagaccact 1050 ctttggattc aagagcactg tgtatgactg aaatttctgg aataactgta 1100 aatggttatg ttaatggaat aaaacacaaa tgttgaaaaa tgtaaaatat 1150 atatacatag attcaaatcc ttatatatgt atgcttgttt tgtgtacagg 1200 attttgtttt ttctttttaa gtacaggttc ctagtgtttt actataactg 1250 tcactatgta tgtaactgac atatataaat agtcatttat aaatgaccgt 1300 attataacat ttgaaaaagt cttcatcaaa aaaaaaaaaa aa 1342 <210> 29 <211> 209 <212> PRT <213> Homo Sapien <400> 29 Met Glu Pro Leu Ala Ala Tyr Pro Leu Lys Cys Ser Gly Pro Arg   1 5 10 15 Ala Lys Val Phe Ala Val Leu Leu Ser Ile Val Leu Cys Thr Val                  20 25 30 Thr Leu Phe Leu Leu Gln Leu Lys Phe Leu Lys Pro Lys Ile Asn                  35 40 45 Ser Phe Tyr Ala Phe Glu Val Lys Asp Ala Lys Gly Arg Thr Val                  50 55 60 Ser Leu Glu Lys Tyr Lys Gly Lys Val Ser Leu Val Val Asn Val                  65 70 75 Ala Ser Asp Cys Gln Leu Thr Asp Arg Asn Tyr Leu Gly Leu Lys                  80 85 90 Glu Leu His Lys Glu Phe Gly Pro Ser His Phe Ser Val Leu Ala                  95 100 105 Phe Pro Cys Asn Gln Phe Gly Glu Ser Glu Pro Arg Pro Ser Lys                 110 115 120 Glu Val Glu Ser Phe Ala Arg Lys Asn Tyr Gly Val Thr Phe Pro                 125 130 135 Ile Phe His Lys Ile Lys Ile Leu Gly Ser Glu Gly Glu Pro Ala                 140 145 150 Phe Arg Phe Leu Val Asp Ser Ser Lys Lys Glu Pro Arg Trp Asn                 155 160 165 Phe Trp Lys Tyr Leu Val Asn Pro Glu Gly Gln Val Val Lys Phe                 170 175 180 Trp Arg Pro Glu Glu Pro Ile Glu Val Ile Arg Pro Asp Ile Ala                 185 190 195 Ala Leu Val Arg Gln Val Ile Ile Lys Lys Lys Glu Asp Leu                 200 205 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 30 atcctccaac atggagcctc ttgc 24 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 31 gtatcttgtc aaccctgagg 20 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 32 taaccagagc tgctatgtca ggcc 24 <210> 33 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 33 aggcaaagtt tcactagttg taaacgtggc cagtgactgc caactcacag 50 <210> 34 <211> 3721 <212> DNA <213> Homo Sapien <400> 34 tgtcgcctgg ccctcgccat gcagaccccg cgagcgtccc ctccccgccc 50 ggccctcctg cttctgctgc tgctactggg gggcgcccac ggcctctttc 100 ctgaggagcc gccgccgctt agcgtggccc ccagggacta cctgaaccac 150 tatcccgtgt ttgtgggcag cgggcccgga cgcctgaccc ccgcagaagg 200 tgctgacgac ctcaacatcc agcgagtcct gcgggtcaac aggacgctgt 250 tcattgggga cagggacaac ctctaccgcg tagagctgga gccccccacg 300 tccacggagc tgcggtacca gaggaagctg acctggagat ctaaccccag 350 cgacataaac gtgtgtcgga tgaagggcaa acaggagggc gagtgtcgaa 400 acttcgtaaa ggtgctgctc cttcgggacg agtccacgct ctttgtgtgc 450 ggttccaacg ccttcaaccc ggtgtgcgcc aactacagca tagacaccct 500 gcagcccgtc ggagacaaca tcagcggtat ggcccgctgc ccgtacgacc 550 ccaagcacgc caatgttgcc ctcttctctg acgggatgct cttcacagct 600 actgttaccg acttcctagc cattgatgct gtcatctacc gcagcctcgg 650 ggacaggccc accctgcgca ccgtgaaaca tgactccaag tggttcaaag 700 agccttactt tgtccatgcg gtggagtggg gcagccatgt ctacttcttc 750 ttccgggaga ttgcgatgga gtttaactac ctggagaagg tggtggtgtc 800 ccgcgtggcc cgagtgtgca agaacgacgt gggaggctcc ccccgcgtgc 850 tggagaagca gtggacgtcc ttcctgaagg cgcggctcaa ctgctctgta 900 cccggagact cccatttcta cttcaacgtg ctgcaggctg tcacgggcgt 950 ggtcagcctc gggggccggc ccgtggtcct ggccgttttt tccacgccca 1000 gcaacagcat ccctggctcg gctgtctgcg cctttgacct gacacaggtg 1050 gcagctgtgt ttgaaggccg cttccgagag cagaagtccc ccgagtccat 1100 ctggacgccg gtgccggagg atcaggtgcc tcgaccccgg cccgggtgct 1150 gcgcagcccc cgggatgcag tacaatgcct ccagcgcctt gccggatgac 1200 atcctcaact ttgtcaagac ccaccctctg atggacgagg cggtgccctc 1250 gctgggccat gcgccctgga tcctgcggac cctgatgagg caccagctga 1300 ctcgagtggc tgtggacgtg ggagccggcc cctggggcaa ccagaccgtt 1350 gtcttcctgg gttctgaggc ggggacggtc ctcaagttcc tcgtccggcc 1400 caatgccagc acctcaggga cgtctgggct cagtgtcttc ctggaggagt 1450 ttgagaccta ccggccggac aggtgtggac ggcccggcgg tggcgagaca 1500 gggcagcggc tgctgagctt ggagctggac gcagcttcgg ggggcctgct 1550 ggctgccttc ccccgctgcg tggtccgagt gcctgtggct cgctgccagc 1600 agtactcggg gtgtatgaag aactgtatcg gcagtcagga cccctactgc 1650 gggtgggccc ccgacggctc ctgcatcttc ctcagcccgg gcaccagagc 1700 cgcctttgag caggacgtgt ccggggccag cacctcaggc ttaggggact 1750 gcacaggact cctgcgggcc agcctctccg aggaccgcgc ggggctggtg 1800 tcggtgaacc tgctggtaac gtcgtcggtg gcggccttcg tggtgggagc 1850 cgtggtgtcc ggcttcagcg tgggctggtt cgtgggcctc cgtgagcggc 1900 gggagctggc ccggcgcaag gacaaggagg ccatcctggc gcacggggcg 1950 ggcgaggcgg tgctgagcgt cagccgcctg ggcgagcgca gggcgcaggg 2000 tcccgggggc cggggcggag gcggtggcgg tggcgccggg gttcccccgg 2050 aggccctgct ggcgcccctg atgcagaacg gctgggccaa ggccacgctg 2100 ctgcagggcg ggccccacga cctggactcg gggctgctgc ccacgcccga 2150 gcagacgccg ctgccgcaga agcgcctgcc cactccgcac ccgcaccccc 2200 acgccctggg cccccgcgcc tgggaccacg gccaccccct gctcccggcc 2250 tccgcttcat cctccctcct gctgctggcg cccgcccggg cccccgagca 2300 gccccccgcg cctggggagc cgacccccga cggccgcctc tatgctgccc 2350 ggcccggccg cgcctcccac ggcgacttcc cgctcacccc ccacgccagc 2400 ccggaccgcc ggcgggtggt gtccgcgccc acgggcccct tggacccagc 2450 ctcagccgcc gatggcctcc cgcggccctg gagcccgccc ccgacgggca 2500 gcctgaggag gccactgggc ccccacgccc ctccggccgc caccctgcgc 2550 cgcacccaca cgttcaacag cggcgaggcc cggcctgggg accgccaccg 2600 cggctgccac gcccggccgg gcacagactt ggcccacctc ctcccctatg 2650 ggggggcgga caggactgcg ccccccgtgc cctaggccgg gggccccccg 2700 atgccttggc agtgccagcc acgggaacca ggagcgagag acggtgccag 2750 aacgccgggg cccggggcaa ctccgagtgg gtgctcaagt cccccccgcg 2800 acccacccgc ggagtggggg gccccctccg ccacaaggaa gcacaaccag 2850 ctcgccctcc ccctacccgg ggccgcagga cgctgagacg gtttgggggt 2900 gggtgggcgg gaggactttg ctatggattt gaggttgacc ttatgcgcgt 2950 aggttttggt ttttttttgc agttttggtt tcttttgcgg ttttctaacc 3000 aattgcacaa ctccgttctc ggggtggcgg caggcagggg aggcttggac 3050 gccggtgggg aatggggggc cacagctgca gacctaagcc ctcccccacc 3100 cctggaaagg tccctcccca acccaggccc ctggcgtgtg tgggtgtgcg 3150 tgcgtgtgcg tgccgtgttc gtgtgcaagg ggccggggag gtgggcgtgt 3200 gtgtgcgtgc cagcgaaggc tgctgtgggc gtgtgtgtca agtgggccac 3250 gcgtgcaggg tgtgtgtcca cgagcgacga tcgtggtggc cccagcggcc 3300 tgggcgttgg ctgagccgac gctggggctt ccagaaggcc cgggggtctc 3350 cgaggtgccg gttaggagtt tgaacccccc ccactctgca gagggaagcg 3400 gggacaatgc cggggtttca ggcaggagac acgaggaggg cctgcccgga 3450 agtcacatcg gcagcagctg tctaaagggc ttgggggcct ggggggcggc 3500 gaaggtgggt ggggcccctc tgtaaatacg gccccagggt ggtgagagag 3550 tcccatgcca cccgtcccct tgtgacctcc cccctatgac ctccagctga 3600 ccatgcatgc cacgtggctg gctgggtcct ctgccctctt tggagtttgc 3650 ctcccccagc cccctcccca tcaataaaac tctgtttaca accaaaaaaa 3700 aaaaaaaaaa aaaaaaaaaa a 3721 <210> 35 <211> 888 <212> PRT <213> Homo Sapien <400> 35 Met Gln Thr Pro Arg Ala Ser Pro Pro Arg Pro Ala Leu Leu Leu   1 5 10 15 Leu Leu Leu Leu Leu Gly Gly Ala His Gly Leu Phe Pro Glu Glu                  20 25 30 Pro Pro Pro Leu Ser Val Ala Pro Arg Asp Tyr Leu Asn His Tyr                  35 40 45 Pro Val Phe Val Gly Ser Gly Pro Gly Arg Leu Thr Pro Ala Glu                  50 55 60 Gly Ala Asp Asp Leu Asn Ile Gln Arg Val Leu Arg Val Asn Arg                  65 70 75 Thr Leu Phe Ile Gly Asp Arg Asp Asn Leu Tyr Arg Val Glu Leu                  80 85 90 Glu Pro Pro Thr Ser Thr Glu Leu Arg Tyr Gln Arg Lys Leu Thr                  95 100 105 Trp Arg Ser Asn Pro Ser Asp Ile Asn Val Cys Arg Met Lys Gly                 110 115 120 Lys Gln Glu Gly Glu Cys Arg Asn Phe Val Lys Val Leu Leu Leu                 125 130 135 Arg Asp Glu Ser Thr Leu Phe Val Cys Gly Ser Asn Ala Phe Asn                 140 145 150 Pro Val Cys Ala Asn Tyr Ser Ile Asp Thr Leu Gln Pro Val Gly                 155 160 165 Asp Asn Ile Ser Gly Met Ala Arg Cys Pro Tyr Asp Pro Lys His                 170 175 180 Ala Asn Val Ala Leu Phe Ser Asp Gly Met Leu Phe Thr Ala Thr                 185 190 195 Val Thr Asp Phe Leu Ala Ile Asp Ala Val Ile Tyr Arg Ser Leu                 200 205 210 Gly Asp Arg Pro Thr Leu Arg Thr Val Lys His Asp Ser Lys Trp                 215 220 225 Phe Lys Glu Pro Tyr Phe Val His Ala Val Glu Trp Gly Ser His                 230 235 240 Val Tyr Phe Phe Phe Arg Glu Ile Ala Met Glu Phe Asn Tyr Leu                 245 250 255 Glu Lys Val Val Val Ser Arg Val Ala Arg Val Cys Lys Asn Asp                 260 265 270 Val Gly Gly Ser Pro Arg Val Leu Glu Lys Gln Trp Thr Ser Phe                 275 280 285 Leu Lys Ala Arg Leu Asn Cys Ser Val Pro Gly Asp Ser His Phe                 290 295 300 Tyr Phe Asn Val Leu Gln Ala Val Thr Gly Val Val Ser Leu Gly                 305 310 315 Gly Arg Pro Val Val Leu Ala Val Phe Ser Thr Pro Ser Asn Ser                 320 325 330 Ile Pro Gly Ser Ala Val Cys Ala Phe Asp Leu Thr Gln Val Ala                 335 340 345 Ala Val Phe Glu Gly Arg Phe Arg Glu Gln Lys Ser Pro Glu Ser                 350 355 360 Ile Trp Thr Pro Val Pro Glu Asp Gln Val Pro Arg Pro Arg Pro                 365 370 375 Gly Cys Cys Ala Ala Pro Gly Met Gln Tyr Asn Ala Ser Ser Ala                 380 385 390 Leu Pro Asp Asp Ile Leu Asn Phe Val Lys Thr His Pro Leu Met                 395 400 405 Asp Glu Ala Val Pro Ser Leu Gly His Ala Pro Trp Ile Leu Arg                 410 415 420 Thr Leu Met Arg His Gln Leu Thr Arg Val Ala Val Asp Val Gly                 425 430 435 Ala Gly Pro Trp Gly Asn Gln Thr Val Val Phe Leu Gly Ser Glu                 440 445 450 Ala Gly Thr Val Leu Lys Phe Leu Val Arg Pro Asn Ala Ser Thr                 455 460 465 Ser Gly Thr Ser Gly Leu Ser Val Phe Leu Glu Glu Phe Glu Thr                 470 475 480 Tyr Arg Pro Asp Arg Cys Gly Arg Pro Gly Gly Gly Glu Thr Gly                 485 490 495 Gln Arg Leu Leu Ser Leu Glu Leu Asp Ala Ala Ser Gly Gly Leu                 500 505 510 Leu Ala Ala Phe Pro Arg Cys Val Val Arg Val Pro Val Ala Arg                 515 520 525 Cys Gln Gln Tyr Ser Gly Cys Met Lys Asn Cys Ile Gly Ser Gln                 530 535 540 Asp Pro Tyr Cys Gly Trp Ala Pro Asp Gly Ser Cys Ile Phe Leu                 545 550 555 Ser Pro Gly Thr Arg Ala Ala Phe Glu Gln Asp Val Ser Gly Ala                 560 565 570 Ser Thr Ser Gly Leu Gly Asp Cys Thr Gly Leu Leu Arg Ala Ser                 575 580 585 Leu Ser Glu Asp Arg Ala Gly Leu Val Ser Val Asn Leu Leu Val                 590 595 600 Thr Ser Ser Val Ala Ala Phe Val Val Gly Ala Val Val Ser Gly                 605 610 615 Phe Ser Val Gly Trp Phe Val Gly Leu Arg Glu Arg Arg Glu Leu                 620 625 630 Ala Arg Arg Lys Asp Lys Glu Ala Ile Leu Ala His Gly Ala Gly                 635 640 645 Glu Ala Val Leu Ser Val Ser Arg Leu Gly Glu Arg Arg Ala Gln                 650 655 660 Gly Pro Gly Gly Arg Gly Gly Gly Gly Gly Gly Gly Ala Gly Val                 665 670 675 Pro Pro Glu Ala Leu Leu Ala Pro Leu Met Gln Asn Gly Trp Ala                 680 685 690 Lys Ala Thr Leu Leu Gln Gly Gly Pro His Asp Leu Asp Ser Gly                 695 700 705 Leu Leu Pro Thr Pro Glu Gln Thr Pro Leu Pro Gln Lys Arg Leu                 710 715 720 Pro Thr Pro His Pro His Pro His Ala Leu Gly Pro Arg Ala Trp                 725 730 735 Asp His Gly His Pro Leu Leu Pro Ala Ser Ala Ser Ser Ser Leu                 740 745 750 Leu Leu Leu Ala Pro Ala Arg Ala Pro Glu Gln Pro Pro Ala Pro                 755 760 765 Gly Glu Pro Thr Pro Asp Gly Arg Leu Tyr Ala Ala Arg Pro Gly                 770 775 780 Arg Ala Ser His Gly Asp Phe Pro Leu Thr Pro His Ala Ser Pro                 785 790 795 Asp Arg Arg Arg Val Val Ser Ala Pro Thr Gly Pro Leu Asp Pro                 800 805 810 Ala Ser Ala Ala Asp Gly Leu Pro Arg Pro Trp Ser Pro Pro Pro                 815 820 825 Thr Gly Ser Leu Arg Arg Pro Leu Gly Pro His Ala Pro Pro Ala                 830 835 840 Ala Thr Leu Arg Arg Thr His Thr Phe Asn Ser Gly Glu Ala Arg                 845 850 855 Pro Gly Asp Arg His Arg Gly Cys His Ala Arg Pro Gly Thr Asp                 860 865 870 Leu Ala His Leu Leu Pro Tyr Gly Gly Ala Asp Arg Thr Ala Pro                 875 880 885 Pro Val Pro <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 36 gaggacctac cggccggaca g 21 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 37 atacaccccg agtactgctg gcag 24 <210> 38 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 38 agacagggca gcggctgctg agcttggagc tggacgcagc tt 42 <210> 39 <211> 2014 <212> DNA <213> Homo Sapien <400> 39 agcaactcaa gttcatcatt gtcctgagag agaggagcag cgcggttctc 50 ggccgggaca gcagaacgcc aggggaccct cacctgggcg cgccggggca 100 cgggctttga ttgtcctggg gtcgcggaga cccgcgcgcc tgccctgcac 150 gccgggcggc aacctttgca gtcgcgttgg ctgctgcgat cggccggcgg 200 gtccctgccg aaggctcggc tgcttctgtc cacctcttac acttcttcat 250 ttatcggtgg atcatttcga gagtccgtct tgtaaatgtt tggcactttg 300 ctactttatt gcttctttct ggcgacagtt ccagcactcg ccgagaccgg 350 cggagaaagg cagctgagcc cggagaagag cgaaatatgg ggacccgggc 400 taaaagcaga cgtcgtcctt cccgcccgct atttctatat tcaggcagtg 450 gatacatcag ggaataaatt cacatcttct ccaggcgaaa aggtcttcca 500 ggtgaaagtc tcagcaccag aggagcaatt cactagagtt ggagtccagg 550 ttttagaccg aaaagatggg tccttcatag taagatacag aatgtatgca 600 agctacaaaa atctgaaggt ggaaattaaa ttccaagggc aacatgtggc 650 caaatcccca tatattttaa aagggccggt ttaccatgag aactgtgact 700 gtcctctgca agatagtgca gcctggctac gggagatgaa ctgccctgaa 750 accattgctc agattcagag agatctggca catttccctg ctgtggatcc 800 agaaaagatt gcagtagaaa tcccaaaaag atttggacag aggcagagcc 850 tatgtcacta caccttaaag gataacaagg tttatatcaa gactcatggt 900 gaacatgtag gttttagaat tttcatggat gccatactac tttctttgac 950 tagaaaggtg aagatgccag atgtggagct ctttgttaat ttgggagact 1000 ggcctttgga aaaaaagaaa tccaattcaa acatccatcc gatcttttcc 1050 tggtgtggct ccacagattc caaggatatc gtgatgccta cgtacgattt 1100 gactgattct gttctggaaa ccatgggccg ggtaagtctg gatatgatgt 1150 ccgtgcaagc taacacgggt cctccctggg aaagcaaaaa ttccactgcc 1200 gtctggagag ggcgagacag ccgcaaagag agactcgagc tggttaaact 1250 cagtagaaaa cacccagaac tcatagacgc tgctttcacc aactttttct 1300 tctttaaaca cgatgaaaac ctgtatggtc ccattgtgaa acatatttca 1350 ttttttgatt tcttcaagca taagtatcaa ataaatatcg atggcactgt 1400 agcagcttat cgcctgccat atttgctagt tggtgacagt gttgtgctga 1450 agcaggattc catctactat gaacattttt acaatgagct gcagccctgg 1500 aaacactaca ttccagttaa gagcaacctg agcgatctgc tagaaaaact 1550 taaatgggcg aaagatcacg atgaagaggc caaaaagata gcaaaagcag 1600 gacaagaatt tgcaagaaat aatctcatgg gcgatgacat attctgttat 1650 tatttcaaac ttttccagga atatgccaat ttacaagtga gtgagcccca 1700 aatccgagag ggcatgaaaa gggtagaacc acagactgag gacgacctct 1750 tcccttgtac ttgccatagg aaaaagacca aagatgaact ctgatatgca 1800 aaataacttc tattagaata atggtgctct gaagactctt cttaactaaa 1850 aagaagaatt tttttaagta ttaattccat ggacaatata aaatctgtgt 1900 gattgtttgc agtatgaaga cacatttcta cttatgcagt attctcatga 1950 ctgtacttta aagtacattt ttagaatttt ataataaaac cacctttatt 2000 ttaaaggaaa aaaa 2014 <210> 40 <211> 502 <212> PRT <213> Homo Sapien <400> 40 Met Phe Gly Thr Leu Leu Leu Tyr Cys Phe Phe Leu Ala Thr Val   1 5 10 15 Pro Ala Leu Ala Glu Thr Gly Gly Glu Arg Gln Leu Ser Pro Glu                  20 25 30 Lys Ser Glu Ile Trp Gly Pro Gly Leu Lys Ala Asp Val Val Leu                  35 40 45 Pro Ala Arg Tyr Phe Tyr Ile Gln Ala Val Asp Thr Ser Gly Asn                  50 55 60 Lys Phe Thr Ser Ser Pro Gly Glu Lys Val Phe Gln Val Lys Val                  65 70 75 Ser Ala Pro Glu Glu Gln Phe Thr Arg Val Gly Val Gln Val Leu                  80 85 90 Asp Arg Lys Asp Gly Ser Phe Ile Val Arg Tyr Arg Met Tyr Ala                  95 100 105 Ser Tyr Lys Asn Leu Lys Val Glu Ile Lys Phe Gln Gly Gln His                 110 115 120 Val Ala Lys Ser Pro Tyr Ile Leu Lys Gly Pro Val Tyr His Glu                 125 130 135 Asn Cys Asp Cys Pro Leu Gln Asp Ser Ala Ala Trp Leu Arg Glu                 140 145 150 Met Asn Cys Pro Glu Thr Ile Ala Gln Ile Gln Arg Asp Leu Ala                 155 160 165 His Phe Pro Ala Val Asp Pro Glu Lys Ile Ala Val Glu Ile Pro                 170 175 180 Lys Arg Phe Gly Gln Arg Gln Ser Leu Cys His Tyr Thr Leu Lys                 185 190 195 Asp Asn Lys Val Tyr Ile Lys Thr His Gly Glu His Val Gly Phe                 200 205 210 Arg Ile Phe Met Asp Ala Ile Leu Leu Ser Leu Thr Arg Lys Val                 215 220 225 Lys Met Pro Asp Val Glu Leu Phe Val Asn Leu Gly Asp Trp Pro                 230 235 240 Leu Glu Lys Lys Lys Ser Asn Ser Asn Ile His Pro Ile Phe Ser                 245 250 255 Trp Cys Gly Ser Thr Asp Ser Lys Asp Ile Val Met Pro Thr Tyr                 260 265 270 Asp Leu Thr Asp Ser Val Leu Glu Thr Met Gly Arg Val Ser Leu                 275 280 285 Asp Met Met Ser Val Gln Ala Asn Thr Gly Pro Pro Trp Glu Ser                 290 295 300 Lys Asn Ser Thr Ala Val Trp Arg Gly Arg Asp Ser Arg Lys Glu                 305 310 315 Arg Leu Glu Leu Val Lys Leu Ser Arg Lys His Pro Glu Leu Ile                 320 325 330 Asp Ala Ala Phe Thr Asn Phe Phe Phe Phe Lys His Asp Glu Asn                 335 340 345 Leu Tyr Gly Pro Ile Val Lys His Ile Ser Phe Phe Asp Phe Phe                 350 355 360 Lys His Lys Tyr Gln Ile Asn Ile Asp Gly Thr Val Ala Ala Tyr                 365 370 375 Arg Leu Pro Tyr Leu Leu Val Gly Asp Ser Val Val Leu Lys Gln                 380 385 390 Asp Ser Ile Tyr Tyr Glu His Phe Tyr Asn Glu Leu Gln Pro Trp                 395 400 405 Lys His Tyr Ile Pro Val Lys Ser Asn Leu Ser Asp Leu Leu Glu                 410 415 420 Lys Leu Lys Trp Ala Lys Asp His Asp Glu Glu Ala Lys Lys Ile                 425 430 435 Ala Lys Ala Gly Gln Glu Phe Ala Arg Asn Asn Leu Met Gly Asp                 440 445 450 Asp Ile Phe Cys Tyr Tyr Phe Lys Leu Phe Gln Glu Tyr Ala Asn                 455 460 465 Leu Gln Val Ser Glu Pro Gln Ile Arg Glu Gly Met Lys Arg Val                 470 475 480 Glu Pro Gln Thr Glu Asp Asp Leu Phe Pro Cys Thr Cys His Arg                 485 490 495 Lys Lys Thr Lys Asp Glu Leu                 500 <210> 41 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 41 gaaggtggaa attaaattcc aagggc 26 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 42 cgataagctg ctacagtgcc atcg 24 <210> 43 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 43 gtgactgtcc tctgcaagat agtgcagcct ggctacggga 40 <210> 44 <211> 2395 <212> DNA <213> Homo Sapien <400> 44 cctggagccg gaagcgcggc tgcagcaggg cgaggctcca ggtggggtcg 50 gttccgcatc cagcctagcg tgtccacgat gcggctgggc tccgggactt 100 tcgctacctg ttgcgtagcg atcgaggtgc tagggatcgc ggtcttcctt 150 cggggattct tcccggctcc cgttcgttcc tctgccagag cggaacacgg 200 agcggagccc ccagcgcccg aaccctcggc tggagccagt tctaactgga 250 ccacgctgcc accacctctc ttcagtaaag ttgttattgt tctgatagat 300 gccttgagag atgattttgt gtttgggtca aagggtgtga aatttatgcc 350 ctacacaact taccttgtgg aaaaaggagc atctcacagt tttgtggctg 400 aagcaaagcc acctacagtt actatgcctc gaatcaaggc attgatgacg 450 gggagccttc ctggctttgt cgacgtcatc aggaacctca attctcctgc 500 actgctggaa gacagtgtga taagacaagc aaaagcagct ggaaaaagaa 550 tagtctttta tggagatgaa acctgggtta aattattccc aaagcatttt 600 gtggaatatg atggaacaac ctcatttttc gtgtcagatt acacagaggt 650 ggataataat gtcacgaggc atttggataa agtattaaaa agaggagatt 700 gggacatatt aatcctccac tacctggggc tggaccacat tggccacatt 750 tcagggccca acagccccct gattgggcag aagctgagcg agatggacag 800 cgtgctgatg aagatccaca cctcactgca gtcgaaggag agagagacgc 850 ctttacccaa tttgctggtt ctttgtggtg accatggcat gtctgaaaca 900 ggaagtcacg gggcctcctc caccgaggag gtgaatacac ctctgatttt 950 aatcagttct gcgtttgaaa ggaaacccgg tgatatccga catccaaagc 1000 acgtccaata gacggatgtg gctgcgacac tggcgatagc acttggctta 1050 ccgattccaa aagacagtgt agggagcctc ctattcccag ttgtggaagg 1100 aagaccaatg agagagcagt tgagattttt acatttgaat acagtgcagc 1150 ttagtaaact gttgcaagag aatgtgccgt catatgaaaa agatcctggg 1200 tttgagcagt ttaaaatgtc agaaagattg catgggaact ggatcagact 1250 gtacttggag gaaaagcatt cagaagtcct attcaacctg ggctccaagg 1300 ttctcaggca gtacctggat gctctgaaga cgctgagctt gtccctgagt 1350 gcacaagtgg cccagttctc accctgctcc tgctcagcgt cccacaggca 1400 ctgcacagaa aggctgagct ggaagtccca ctgtcatctc ctgggttttc 1450 tctgctcttt tatttggtga tcctggttct ttcggccgtt cacgtcattg 1500 tgtgcacctc agctgaaagt tcgtgctact tctgtggcct ctcgtggctg 1550 gcggcaggct gcctttcgtt taccagactc tggttgaaca cctggtgtgt 1600 gccaagtgct ggcagtgccc tggacagggg gcctcaggga aggacgtgga 1650 gcagccttat cccaggcctc tgggtgtccc gacacaggtg ttcacatctg 1700 tgctgtcagg tcagatgcct cagttcttgg aaagctaggt tcctgcgact 1750 gttaccaagg tgattgtaaa gagctggcgg tcacagagga acaagccccc 1800 cagctgaggg ggtgtgtgaa tcggacagcc tcccagcaga ggtgtgggag 1850 ctgcagctga gggaagaaga gacaatcggc ctggacactc aggagggtca 1900 aaaggagact tggtcgcacc actcatcctg ccacccccag aatgcatcct 1950 gcctcatcag gtccagattt ctttccaagg cggacgtttt ctgttggaat 2000 tcttagtcct tggcctcgga caccttcatt cgttagctgg ggagtggtgg 2050 tgaggcagtg aagaagaggc ggatggtcac actcagatcc acagagccca 2100 ggatcaaggg acccactgca gtggcagcag gactgttggg cccccacccc 2150 aaccctgcac agccctcatc ccctcttggc ttgagccgtc agaggccctg 2200 tgctgagtgt ctgaccgaga cactcacagc tttgtcatca gggcacaggc 2250 ttcctcggag ccaggatgat ctgtgccacg cttgcacctc gggcccatct 2300 gggctcatgc tctctctcct gctattgaat tagtacctag ctgcacacag 2350 tatgtagtta ccaaaagaat aaacggcaat aattgagaaa aaaaa 2395 <210> 45 <211> 310 <212> PRT <213> Homo Sapien <400> 45 Met Arg Leu Gly Ser Gly Thr Phe Ala Thr Cys Cys Val Ala Ile   1 5 10 15 Glu Val Leu Gly Ile Ala Val Phe Leu Arg Gly Phe Phe Pro Ala                  20 25 30 Pro Val Arg Ser Ser Ala Arg Ala Glu His Gly Ala Glu Pro Pro                  35 40 45 Ala Pro Glu Pro Ser Ala Gly Ala Ser Ser Asn Trp Thr Thr Leu                  50 55 60 Pro Pro Pro Leu Phe Ser Lys Val Val Ile Val Leu Ile Asp Ala                  65 70 75 Leu Arg Asp Asp Phe Val Phe Gly Ser Lys Gly Val Lys Phe Met                  80 85 90 Pro Tyr Thr Thr Tyr Leu Val Glu Lys Gly Ala Ser His Ser Phe                  95 100 105 Val Ala Glu Ala Lys Pro Pro Thr Val Thr Met Pro Arg Ile Lys                 110 115 120 Ala Leu Met Thr Gly Ser Leu Pro Gly Phe Val Asp Val Ile Arg                 125 130 135 Asn Leu Asn Ser Pro Ala Leu Leu Glu Asp Ser Val Ile Arg Gln                 140 145 150 Ala Lys Ala Ala Gly Lys Arg Ile Val Phe Tyr Gly Asp Glu Thr                 155 160 165 Trp Val Lys Leu Phe Pro Lys His Phe Val Glu Tyr Asp Gly Thr                 170 175 180 Thr Ser Phe Phe Val Ser Asp Tyr Thr Glu Val Asp Asn Asn Val                 185 190 195 Thr Arg His Leu Asp Lys Val Leu Lys Arg Gly Asp Trp Asp Ile                 200 205 210 Leu Ile Leu His Tyr Leu Gly Leu Asp His Ile Gly His Ile Ser                 215 220 225 Gly Pro Asn Ser Pro Leu Ile Gly Gln Lys Leu Ser Glu Met Asp                 230 235 240 Ser Val Leu Met Lys Ile His Thr Ser Leu Gln Ser Lys Glu Arg                 245 250 255 Glu Thr Pro Leu Pro Asn Leu Leu Val Leu Cys Gly Asp His Gly                 260 265 270 Met Ser Glu Thr Gly Ser His Gly Ala Ser Ser Thr Glu Glu Val                 275 280 285 Asn Thr Pro Leu Ile Leu Ile Ser Ser Ala Phe Glu Arg Lys Pro                 290 295 300 Gly Asp Ile Arg His Pro Lys His Val Gln                 305 310 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 46 cgggactttc gctacctgtt gc 22 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 47 catcatattc cacaaaatgc tttggg 26 <210> 48 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 48 ccttcgggga ttcttcccgg ctcccgttcg ttcctctg 38 <210> 49 <211> 918 <212> DNA <213> Homo Sapien <400> 49 agccaggcag cacatcacag cgggaggagc tgtcccaggt ggcccagctc 50 agcaatggca atgggggtcc ccagagtcat tctgctctgc ctctttgggg 100 ctgcgctctg cctgacaggg tcccaagccc tgcagtgcta cagctttgag 150 cacacctact ttggcccctt tgacctcagg gccatgaagc tgcccagcat 200 ctcctgtcct catgagtgct ttgaggctat cctgtctctg gacaccgggt 250 atcgcgcgcc ggtgaccctg gtgcggaagg gctgctggac cgggcctcct 300 gcgggccaga cgcaatcgaa cccggacgcg ctgccgccag actactcggt 350 ggtgcgcggc tgcacaactg acaaatgcaa cgcccacctc atgactcatg 400 acgccctccc caacctgagc caagcacccg acccgccgac gctcagcggc 450 gccgagtgct acgcctgtat cggggtccac caggatgact gcgctatcgg 500 caggtcccga cgagtccagt gtcaccagga ccagaccgcc tgcttccagg 550 gcagtggcag aatgacagtt ggcaatttct cagtccctgt gtacatcaga 600 acctgccacc ggccctcctg caccaccgag ggcaccacca gcccctggac 650 agccatcgac ctccagggct cctgctgtga ggggtacctc tgcaacagga 700 aatccatgac ccagcccttc accagtgctt cagccaccac ccctccccga 750 gcactacagg tcctggccct gctcctccca gtcctcctgc tggtggggct 800 ctcagcatag accgcccctc caggatgctg gggacagggc tcacacacct 850 cattcttgct gcttcagccc ctatcacata gctcactgga aaatgatgtt 900 aaagtaagaa ttgcaaaa 918 <210> 50 <211> 251 <212> PRT <213> Homo Sapien <400> 50 Met Ala Met Gly Val Pro Arg Val Ile Leu Leu Cys Leu Phe Gly   1 5 10 15 Ala Ala Leu Cys Leu Thr Gly Ser Gln Ala Leu Gln Cys Tyr Ser                  20 25 30 Phe Glu His Thr Tyr Phe Gly Pro Phe Asp Leu Arg Ala Met Lys                  35 40 45 Leu Pro Ser Ile Ser Cys Pro His Glu Cys Phe Glu Ala Ile Leu                  50 55 60 Ser Leu Asp Thr Gly Tyr Arg Ala Pro Val Thr Leu Val Arg Lys                  65 70 75 Gly Cys Trp Thr Gly Pro Pro Ala Gly Gln Thr Gln Ser Asn Pro                  80 85 90 Asp Ala Leu Pro Pro Asp Tyr Ser Val Val Arg Gly Cys Thr Thr                  95 100 105 Asp Lys Cys Asn Ala His Leu Met Thr His Asp Ala Leu Pro Asn                 110 115 120 Leu Ser Gln Ala Pro Asp Pro Pro Thr Leu Ser Gly Ala Glu Cys                 125 130 135 Tyr Ala Cys Ile Gly Val His Gln Asp Asp Cys Ala Ile Gly Arg                 140 145 150 Ser Arg Arg Val Gln Cys His Gln Asp Gln Thr Ala Cys Phe Gln                 155 160 165 Gly Ser Gly Arg Met Thr Val Gly Asn Phe Ser Val Pro Val Tyr                 170 175 180 Ile Arg Thr Cys His Arg Pro Ser Cys Thr Thr Glu Gly Thr Thr                 185 190 195 Ser Pro Trp Thr Ala Ile Asp Leu Gln Gly Ser Cys Cys Glu Gly                 200 205 210 Tyr Leu Cys Asn Arg Lys Ser Met Thr Gln Pro Phe Thr Ser Ala                 215 220 225 Ser Ala Thr Thr Pro Pro Arg Ala Leu Gln Val Leu Ala Leu Leu                 230 235 240 Leu Pro Val Leu Leu Leu Val Gly Leu Ser Ala                 245 250 <210> 51 <211> 3288 <212> DNA <213> Homo Sapien <400> 51 cccacgcgtc cgggacagat gaacttaaaa gagaagcttt agctgccaaa 50 gattgggaaa gggaaaggac aaaaaagacc cctgggctac acggcgtagg 100 tgcagggttt cctactgctg ttcttttatg ctgggagctg tggctgtaac 150 caactaggaa ataacgtatg cagcagctat ggctgtcaga gagttgtgct 200 tcccaagaca aaggcaagtc ctgtttcttt ttcttttttg gggagtgtcc 250 ttggcaggtt ctgggtttgg acgttattcg gtgactgagg aaacagagaa 300 aggatccttt gtggtcaatc tggcaaagga tctgggacta gcagaggggg 350 agctggctgc aaggggaacc agggtggttt ccgatgataa caaacaatac 400 ctgctcctgg attcacatac cgggaatttg ctcacaaatg agaaactgga 450 ccgagagaag ctgtgtggcc ctaaagagcc ctgtatgctg tatttccaaa 500 ttttaatgga tgatcccttt cagatttacc gggctgagct gagagtcagg 550 gatataaatg atcacgcgcc agtatttcag gacaaagaaa cagtcttaaa 600 aatatcagaa aatacagctg aagggacagc atttagacta gaaagagcac 650 aggatccaga tggaggactt aacggtatcc aaaactacac gatcagcccc 700 aactcttttt tccatattaa cattagtggc ggtgatgaag gcatgatata 750 tccagagcta gtgttggaca aagcactgga tcgggaggag cagggagagc 800 tcagcttaac cctcacagcg ctggatggtg ggtctccatc caggtctggg 850 acctctactg tacgcatcgt tgtcttggac gtcaatgaca atgccccaca 900 gtttgcccag gctctgtatg agacccaggc tccagaaaac agccccattg 950 ggttccttat tgttaaggta tgggcagaag atgtagactc tggagtcaac 1000 gcggaagtat cctattcatt ttttgatgcc tcagaaaata ttcgaacgac 1050 ctttcaaatc aatccttttt ctggggaaat ctttctcaga gaattgcttg 1100 attatgagtt agtaaattct tacaaaataa atatacaggc aatggacggt 1150 ggaggccttt ctgcaagatg tagggtttta gtggaagtat tggacaccaa 1200 tgacaatccc cctgaactga tcgtatcatc attttccaac tctgttgctg 1250 agaattctcc tgagacgccg ctggctgttt ttaagattaa tgacagagac 1300 tctggagaaa atggaaagat ggtttgctac attcaagaga atctgccatt 1350 cctactaaaa ccttctgtgg agaattttta catcctaatt acagaaggcg 1400 cgctggacag agagatcaga gccgagtaca acatcactat caccgtcact 1450 gacttgggga cacccaggct gaaaaccgag cacaacataa cggtcctggt 1500 ctccgacgtc aatgacaacg cccccgcctt cacccaaacc tcctacaccc 1550 tgttcgtccg cgagaacaac agccccgccc tgcacatcgg cagcgtcagc 1600 gccacagaca gagactcggg caccaacgcc caggtcacct actcgctgct 1650 gccgccccaa gacccgcacc tgcccctcgc ctccctggtc tccatcaacg 1700 cggacaacgg ccacctgttc gccctcaggt cgctggacta cgaggccctg 1750 caggctttcg agttccgcgt gggcgccaca gaccgcggct cccccgcgct 1800 gagcagagag gcgctggtgc gcgtgctggt gctggacgcc aacgacaact 1850 cgcccttcgt gctgtacccg ctgcagaacg gctccgcgcc ctgcaccgag 1900 ctggtgcccc gggcggccga gccgggctac ctggtgacca aggtggtggc 1950 ggtggacggc gactcgggcc agaacgcctg gctgtcgtac cagctgctca 2000 aggccacgga gcccgggctg ttcggtgtgt gggcgcacaa tggggaggtg 2050 cgcaccgcca ggctgctgag cgagcgcgac gcagccaagc acaggctcgt 2100 ggtgcttgtc aaggacaatg gcgagcctcc tcgctcggcc accgccacgc 2150 tgcacttgct cctggtggac ggcttctccc agccctacct gcctctcccg 2200 gaggcggccc cggcccaggc ccaggccgag gccgacttgc tcaccgtcta 2250 cctggtggtg gcgttggcct cggtgtcttc gctcttcctc ctctcggtgc 2300 tcctgttcgt ggcggtgcgg ctgtgcagga ggagcagggc ggcctcggtg 2350 ggtcgctgct cggtgcccga gggtcctttt ccagggcatc tggtggacgt 2400 gaggggcgct gagaccctgt cccagagcta ccagtatgag gtgtgtctga 2450 cgggaggccc cgggaccagt gagttcaagt tcttgaaacc agttatttcg 2500 gatattcagg cacagggccc tgggaggaag ggtgaagaaa attccacctt 2550 ccgaaatagc tttggattta atattcagta aagtctgttt ttagtttcat 2600 atacttttgg tgtgttacat agccatgttt ctattagttt acttttaaat 2650 ctcaaattta agttattatg caacttcaag cattattttc aagtagtata 2700 cccctgtggt tttacaatgt ttcatcattt ttttgcatta ataacaactg 2750 ggtttaattt aatgagtatt tttttctaaa tgatagtgtt aaggttttaa 2800 ttctttccaa ctgcccaagg aattaattac tattatatct cattacagaa 2850 atctgaggtt ttgattcatt tcagagcttg catctcatga ttctaatcac 2900 ttctgtctat agtgtacttg ctctatttaa gaaggcatat ctacatttcc 2950 aaactcattc taacattcta tatattcgtg tttgaaaacc atgtcattta 3000 tttctacatc atgtatttaa aaagaaatat ttctctacta ctatgctcat 3050 gacaaaatga aacaaagcat attgtgagca atactgaaca tcaataatac 3100 ccttagttta tatacttatt attttatctt taagcatgct acttttactt 3150 ggccaatatt ttcttatgtt aacttttgct gatgtataaa acagactatg 3200 ccttataatt gaaataaaat tataatctgc ctgaaaatga ataaaaataa 3250 aacattttga aatgtgaaaa aaaaaaaaaa aaaaaaaa 3288 <210> 52 <211> 800 <212> PRT <213> Homo Sapien <400> 52 Met Ala Val Arg Glu Leu Cys Phe Pro Arg Gln Arg Gln Val Leu   1 5 10 15 Phe Leu Phe Leu Phe Trp Gly Val Ser Leu Ala Gly Ser Gly Phe                  20 25 30 Gly Arg Tyr Ser Val Thr Glu Glu Thr Glu Lys Gly Ser Phe Val                  35 40 45 Val Asn Leu Ala Lys Asp Leu Gly Leu Ala Glu Gly Glu Leu Ala                  50 55 60 Ala Arg Gly Thr Arg Val Val Ser Asp Asp Asn Lys Gln Tyr Leu                  65 70 75 Leu Leu Asp Ser His Thr Gly Asn Leu Leu Thr Asn Glu Lys Leu                  80 85 90 Asp Arg Glu Lys Leu Cys Gly Pro Lys Glu Pro Cys Met Leu Tyr                  95 100 105 Phe Gln Ile Leu Met Asp Asp Pro Phe Gln Ile Tyr Arg Ala Glu                 110 115 120 Leu Arg Val Arg Asp Ile Asn Asp His Ala Pro Val Phe Gln Asp                 125 130 135 Lys Glu Thr Val Leu Lys Ile Ser Glu Asn Thr Ala Glu Gly Thr                 140 145 150 Ala Phe Arg Leu Glu Arg Ala Gln Asp Pro Asp Gly Gly Leu Asn                 155 160 165 Gly Ile Gln Asn Tyr Thr Ile Ser Pro Asn Ser Phe Phe His Ile                 170 175 180 Asn Ile Ser Gly Gly Asp Glu Gly Met Ile Tyr Pro Glu Leu Val                 185 190 195 Leu Asp Lys Ala Leu Asp Arg Glu Glu Gln Gly Glu Leu Ser Leu                 200 205 210 Thr Leu Thr Ala Leu Asp Gly Gly Ser Pro Ser Arg Ser Gly Thr                 215 220 225 Ser Thr Val Arg Ile Val Val Leu Asp Val Asn Asp Asn Ala Pro                 230 235 240 Gln Phe Ala Gln Ala Leu Tyr Glu Thr Gln Ala Pro Glu Asn Ser                 245 250 255 Pro Ile Gly Phe Leu Ile Val Lys Val Trp Ala Glu Asp Val Asp                 260 265 270 Ser Gly Val Asn Ala Glu Val Ser Tyr Ser Phe Phe Asp Ala Ser                 275 280 285 Glu Asn Ile Arg Thr Thr Phe Gln Ile Asn Pro Phe Ser Gly Glu                 290 295 300 Ile Phe Leu Arg Glu Leu Leu Asp Tyr Glu Leu Val Asn Ser Tyr                 305 310 315 Lys Ile Asn Ile Gln Ala Met Asp Gly Gly Gly Leu Ser Ala Arg                 320 325 330 Cys Arg Val Leu Val Glu Val Leu Asp Thr Asn Asp Asn Pro Pro                 335 340 345 Glu Leu Ile Val Ser Ser Phe Ser Asn Ser Val Ala Glu Asn Ser                 350 355 360 Pro Glu Thr Pro Leu Ala Val Phe Lys Ile Asn Asp Arg Asp Ser                 365 370 375 Gly Glu Asn Gly Lys Met Val Cys Tyr Ile Gln Glu Asn Leu Pro                 380 385 390 Phe Leu Leu Lys Pro Ser Val Glu Asn Phe Tyr Ile Leu Ile Thr                 395 400 405 Glu Gly Ala Leu Asp Arg Glu Ile Arg Ala Glu Tyr Asn Ile Thr                 410 415 420 Ile Thr Val Thr Asp Leu Gly Thr Pro Arg Leu Lys Thr Glu His                 425 430 435 Asn Ile Thr Val Leu Val Ser Asp Val Asn Asp Asn Ala Pro Ala                 440 445 450 Phe Thr Gln Thr Ser Tyr Thr Leu Phe Val Arg Glu Asn Asn Ser                 455 460 465 Pro Ala Leu His Ile Gly Ser Val Ser Ala Thr Asp Arg Asp Ser                 470 475 480 Gly Thr Asn Ala Gln Val Thr Tyr Ser Leu Leu Pro Pro Gln Asp                 485 490 495 Pro His Leu Pro Leu Ala Ser Leu Val Ser Ile Asn Ala Asp Asn                 500 505 510 Gly His Leu Phe Ala Leu Arg Ser Leu Asp Tyr Glu Ala Leu Gln                 515 520 525 Ala Phe Glu Phe Arg Val Gly Ala Thr Asp Arg Gly Ser Pro Ala                 530 535 540 Leu Ser Arg Glu Ala Leu Val Arg Val Leu Val Leu Asp Ala Asn                 545 550 555 Asp Asn Ser Pro Phe Val Leu Tyr Pro Leu Gln Asn Gly Ser Ala                 560 565 570 Pro Cys Thr Glu Leu Val Pro Arg Ala Ala Glu Pro Gly Tyr Leu                 575 580 585 Val Thr Lys Val Val Ala Val Asp Gly Asp Ser Gly Gln Asn Ala                 590 595 600 Trp Leu Ser Tyr Gln Leu Leu Lys Ala Thr Glu Pro Gly Leu Phe                 605 610 615 Gly Val Trp Ala His Asn Gly Glu Val Arg Thr Ala Arg Leu Leu                 620 625 630 Ser Glu Arg Asp Ala Ala Lys His Arg Leu Val Val Leu Val Lys                 635 640 645 Asp Asn Gly Glu Pro Pro Arg Ser Ala Thr Ala Thr Leu His Leu                 650 655 660 Leu Leu Val Asp Gly Phe Ser Gln Pro Tyr Leu Pro Leu Pro Glu                 665 670 675 Ala Ala Pro Ala Gln Ala Gln Ala Glu Ala Asp Leu Leu Thr Val                 680 685 690 Tyr Leu Val Val Ala Leu Ala Ser Val Ser Ser Leu Phe Leu Leu                 695 700 705 Ser Val Leu Leu Phe Val Ala Val Arg Leu Cys Arg Arg Ser Arg                 710 715 720 Ala Ala Ser Val Gly Arg Cys Ser Val Pro Glu Gly Pro Phe Pro                 725 730 735 Gly His Leu Val Asp Val Arg Gly Ala Glu Thr Leu Ser Gln Ser                 740 745 750 Tyr Gln Tyr Glu Val Cys Leu Thr Gly Gly Pro Gly Thr Ser Glu                 755 760 765 Phe Lys Phe Leu Lys Pro Val Ile Ser Asp Ile Gln Ala Gln Gly                 770 775 780 Pro Gly Arg Lys Gly Glu Glu Asn Ser Thr Phe Arg Asn Ser Phe                 785 790 795 Gly Phe Asn Ile Gln                 800 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 53 ctggggagtg tccttggcag gttc 24 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 54 cagcatacag ggctctttag ggcacac 27 <210> 55 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 55 cggtgactga ggaaacagag aaaggatcct ttgtggtcaa tctggc 46 <210> 56 <211> 2242 <212> DNA <213> Homo Sapien <220> <221> unsure <222> 2181 <223> unknown base <400> 56 gaatgaatac ctccgaagcc gctttgttct ccagatgtga atagctccac 50 tataccagcc tcgtcttcct tccgggggac aacgtgggtc agggcacaga 100 gagatattta atgtcaccct cttggggctt tcatgggact ccctctgcca 150 cattttttgg aggttgggaa agttgctaga ggcttcagaa ctccagccta 200 atggatccca aactcgggag aatggctgcg tccctgctgg ctgtgctgct 250 gctgctgctg gagcgcggca tgttctcctc accctccccg cccccggcgc 300 tgttagagaa agtcttccag tacattgacc tccatcagga tgaatttgtg 350 cagacgctga aggagtgggt ggccatcgag agcgactctg tccagcctgt 400 gcctcgcttc agacaagagc tcttcagaat gatggccgtg gctgcggaca 450 cgctgcagcg cctgggggcc cgtgtggcct cggtggacat gggtcctcag 500 cagctgcccg atggtcagag tcttccaata cctcccgtca tcctggccga 550 actggggagc gatcccacga aaggcaccgt gtgcttctac ggccacttgg 600 acgtgcagcc tgctgaccgg ggcgatgggt ggctcacgga cccctatgtg 650 ctgacggagg tagacgggaa actttatgga cgaggagcga ccgacaacaa 700 aggccctgtc ttggcttgga tcaatgctgt gagcgccttc agagccctgg 750 agcaagatct tcctgtgaat atcaaattca tcattgaggg gatggaagag 800 gctggctctg ttgccctgga ggaacttgtg gaaaaagaaa aggaccgatt 850 cttctctggt gtggactaca ttgtaatttc agataacctg tggatcagcc 900 aaaggaagcc agcaatcact tatggaaccc gggggaacag ctacttcatg 950 gtggaggtga aatgcagaga ccaggatttt cactcaggaa cctttggtgg 1000 catccttcat gaaccaatgg ctgatctggt tgctcttctc ggtagcctgg 1050 tagactcgtc tggtcatatc ctggtccctg gaatctatga tgaagtggtt 1100 cctcttacag aagaggaaat aaatacatac aaagccatcc atctagacct 1150 agaagaatac cggaatagca gccgggttga gaaatttctg ttcgatacta 1200 aggaggagat tctaatgcac ctctggaggt acccatctct ttctattcat 1250 gggatcgagg gcgcgtttga tgagcctgga actaaaacag tcatacctgg 1300 ccgagttata ggaaaatttt caatccgtct agtccctcac atgaatgtgt 1350 ctgcggtgga aaaacaggtg acacgacatc ttgaagatgt gttctccaaa 1400 agaaatagtt ccaacaagat ggttgtttcc atgactctag gactacaccc 1450 gtggattgca aatattgatg acacccagta tctcgcagca aaaagagcga 1500 tcagaacagt gtttggaaca gaaccagata tgatccggga tggatccacc 1550 attccaattg ccaaaatgtt ccaggagatc gtccacaaga gcgtggtgct 1600 aattccgctg ggagctgttg atgatggaga acattcgcag aatgagaaaa 1650 tcaacaggtg gaactacata gagggaacca aattatttgc tgcctttttc 1700 ttagagatgg cccagctcca ttaatcacaa gaaccttcta gtctgatctg 1750 atccactgac agattcacct cccccacatc cctagacagg gatggaatgt 1800 aaatatccag agaatttggg tctagtatag tacattttcc cttccattta 1850 aaatgtcttg ggatatctgg atcagtaata aaatatttca aaggcacaga 1900 tgttggaaat ggtttaaggt cccccactgc acaccttcct caagtcatag 1950 ctgcttgcag caacttgatt tccccaagtc ctgtgcaata gccccaggat 2000 tggattcctt ccaacctttt agcatatctc caaccttgca atttgattgg 2050 cataatcact ccggtttgct ttctaggtcc tcaagtgctc gtgacacata 2100 atcattccat ccaatgatcg cctttgcttt accactcttt ccttttatct 2150 tattaataaa aatgttggtc tccaccactg nctcccaaaa aaaaaaaaaa 2200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2242 <210> 57 <211> 507 <212> PRT <213> Homo Sapien <400> 57 Met Asp Pro Lys Leu Gly Arg Met Ala Ala Ser Leu Leu Ala Val   1 5 10 15 Leu Leu Leu Leu Leu Glu Arg Gly Met Phe Ser Ser Pro Ser Pro                  20 25 30 Pro Pro Ala Leu Leu Glu Lys Val Phe Gln Tyr Ile Asp Leu His                  35 40 45 Gln Asp Glu Phe Val Gln Thr Leu Lys Glu Trp Val Ala Ile Glu                  50 55 60 Ser Asp Ser Val Gln Pro Val Pro Arg Phe Arg Gln Glu Leu Phe                  65 70 75 Arg Met Met Ala Val Ala Ala Asp Thr Leu Gln Arg Leu Gly Ala                  80 85 90 Arg Val Ala Ser Val Asp Met Gly Pro Gln Gln Leu Pro Asp Gly                  95 100 105 Gln Ser Leu Pro Ile Pro Pro Val Ile Leu Ala Glu Leu Gly Ser                 110 115 120 Asp Pro Thr Lys Gly Thr Val Cys Phe Tyr Gly His Leu Asp Val                 125 130 135 Gln Pro Ala Asp Arg Gly Asp Gly Trp Leu Thr Asp Pro Tyr Val                 140 145 150 Leu Thr Glu Val Asp Gly Lys Leu Tyr Gly Arg Gly Ala Thr Asp                 155 160 165 Asn Lys Gly Pro Val Leu Ala Trp Ile Asn Ala Val Ser Ala Phe                 170 175 180 Arg Ala Leu Glu Gln Asp Leu Pro Val Asn Ile Lys Phe Ile Ile                 185 190 195 Glu Gly Met Glu Glu Ala Gly Ser Val Ala Leu Glu Glu Leu Val                 200 205 210 Glu Lys Glu Lys Asp Arg Phe Phe Ser Gly Val Asp Tyr Ile Val                 215 220 225 Ile Ser Asp Asn Leu Trp Ile Ser Gln Arg Lys Pro Ala Ile Thr                 230 235 240 Tyr Gly Thr Arg Gly Asn Ser Tyr Phe Met Val Glu Val Lys Cys                 245 250 255 Arg Asp Gln Asp Phe His Ser Gly Thr Phe Gly Gly Ile Leu His                 260 265 270 Glu Pro Met Ala Asp Leu Val Ala Leu Leu Gly Ser Leu Val Asp                 275 280 285 Ser Ser Gly His Ile Leu Val Pro Gly Ile Tyr Asp Glu Val Val                 290 295 300 Pro Leu Thr Glu Glu Glu Ile Asn Thr Tyr Lys Ala Ile His Leu                 305 310 315 Asp Leu Glu Glu Tyr Arg Asn Ser Ser Arg Val Glu Lys Phe Leu                 320 325 330 Phe Asp Thr Lys Glu Glu Ile Leu Met His Leu Trp Arg Tyr Pro                 335 340 345 Ser Leu Ser Ile His Gly Ile Glu Gly Ala Phe Asp Glu Pro Gly                 350 355 360 Thr Lys Thr Val Ile Pro Gly Arg Val Ile Gly Lys Phe Ser Ile                 365 370 375 Arg Leu Val Pro His Met Asn Val Ser Ala Val Glu Lys Gln Val                 380 385 390 Thr Arg His Leu Glu Asp Val Phe Ser Lys Arg Asn Ser Ser Asn                 395 400 405 Lys Met Val Val Ser Met Thr Leu Gly Leu His Pro Trp Ile Ala                 410 415 420 Asn Ile Asp Asp Thr Gln Tyr Leu Ala Ala Lys Arg Ala Ile Arg                 425 430 435 Thr Val Phe Gly Thr Glu Pro Asp Met Ile Arg Asp Gly Ser Thr                 440 445 450 Ile Pro Ile Ala Lys Met Phe Gln Glu Ile Val His Lys Ser Val                 455 460 465 Val Leu Ile Pro Leu Gly Ala Val Asp Asp Gly Glu His Ser Gln                 470 475 480 Asn Glu Lys Ile Asn Arg Trp Asn Tyr Ile Glu Gly Thr Lys Leu                 485 490 495 Phe Ala Ala Phe Phe Leu Glu Met Ala Gln Leu His                 500 505 <210> 58 <211> 1470 <212> DNA <213> Homo Sapien <400> 58 ctcggctgga tttaaggttg ccgctagccg cctgggaatt taagggaccc 50 acactacctt cccgaagttg aaggcaagcg gtgattgttt gtagacggcg 100 ctttgtcatg ggacctgtgc ggttgggaat attgcttttc ctttttttgg 150 ccgtgcacga ggcttgggct gggatgttga aggaggagga cgatgacaca 200 gaacgcttgc ccagcaaatg cgaagtgtgt aagctgctga gcacagagct 250 acaggcggaa ctgagtcgca ccggtcgatc tcgagaggtg ctggagctgg 300 ggcaggtgct ggatacaggc aagaggaaga gacacgtgcc ttacagcgtt 350 tcagagacaa ggctggaaga ggccttagag aatttatgtg agcggatcct 400 ggactatagt gttcacgctg agcgcaaggg ctcactgaga tatgccaagg 450 gtcagagtca gaccatggca acactgaaag gcctagtgca gaagggggtg 500 aaggtggatc tggggatccc tctggagctt tgggatgagc ccagcgtgga 550 ggtcacatac ctcaagaagc agtgtgagac catgttggag gagtttgaag 600 acattgtggg agactggtac ttccaccatc aggagcagcc cctacaaaat 650 tttctctgtg aaggtcatgt gctcccagct gctgaaactg catgtctaca 700 ggaaacttgg actggaaagg agatcacaga tggggaagag aaaacagaag 750 gggaggaaga gcaggaggag gaggaggaag aggaggaaga ggaaggggga 800 gacaagatga ccaagacagg aagccacccc aaacttgacc gagaagatct 850 ttgacccttg cctttgagcc cccaggaggg gaagggatca tggagagccc 900 tctaaagcct gcactctccc tgctccacag ctttcagggt gtgtttatga 950 gtgactccac ccaagcttgt agctgttctc tcccatctaa cctcaggcaa 1000 gatcctggtg aaacagcatg acatggcttc tggggtggag ggtgggggtg 1050 gaggtcctgc tcctagagat gaactctatc cagcccctta attggcaggt 1100 gtatgtgctg acagtactga aagctttcct ctttaactga tcccaccccc 1150 acccaaaagt cagcagtggc actggagctg tgggctttgg ggaagtcact 1200 tagctcctta aggtctgttt ttagaccctt ccaaggaaga ggccagaacg 1250 gacattctct gcgatctata tacattgcct gtatccagga ggctacacac 1300 cagcaaaccg tgaaggagaa tgggacactg ggtcatggcc tggagttgct 1350 gataatttag gtgggataga tacttggtct acttaagctc aatgtaaccc 1400 agagcccacc atatagtttt ataggtgctc aactttctat atcgctatta 1450 aacttttttc tttttttcta 1470 <210> 59 <211> 248 <212> PRT <213> Homo Sapien <400> 59 Met Gly Pro Val Arg Leu Gly Ile Leu Leu Phe Leu Phe Leu Ala   1 5 10 15 Val His Glu Ala Trp Ala Gly Met Leu Lys Glu Glu Asp Asp Asp                  20 25 30 Thr Glu Arg Leu Pro Ser Lys Cys Glu Val Cys Lys Leu Leu Ser                  35 40 45 Thr Glu Leu Gln Ala Glu Leu Ser Arg Thr Gly Arg Ser Arg Glu                  50 55 60 Val Leu Glu Leu Gly Gln Val Leu Asp Thr Gly Lys Arg Lys Arg                  65 70 75 His Val Pro Tyr Ser Val Ser Glu Thr Arg Leu Glu Glu Ala Leu                  80 85 90 Glu Asn Leu Cys Glu Arg Ile Leu Asp Tyr Ser Val His Ala Glu                  95 100 105 Arg Lys Gly Ser Leu Arg Tyr Ala Lys Gly Gln Ser Gln Thr Met                 110 115 120 Ala Thr Leu Lys Gly Leu Val Gln Lys Gly Val Lys Val Asp Leu                 125 130 135 Gly Ile Pro Leu Glu Leu Trp Asp Glu Pro Ser Val Glu Val Thr                 140 145 150 Tyr Leu Lys Lys Gln Cys Glu Thr Met Leu Glu Glu Phe Glu Asp                 155 160 165 Ile Val Gly Asp Trp Tyr Phe His His Gln Glu Gln Pro Leu Gln                 170 175 180 Asn Phe Leu Cys Glu Gly His Val Leu Pro Ala Ala Glu Thr Ala                 185 190 195 Cys Leu Gln Glu Thr Trp Thr Gly Lys Glu Ile Thr Asp Gly Glu                 200 205 210 Glu Lys Thr Glu Gly Glu Glu Glu Gln Glu Glu Glu Glu Glu Glu                 215 220 225 Glu Glu Glu Glu Gly Gly Asp Lys Met Thr Lys Thr Gly Ser His                 230 235 240 Pro Lys Leu Asp Arg Glu Asp Leu                 245 <210> 60 <211> 890 <212> DNA <213> Homo Sapien <400> 60 aagtacttgt gtccgggtgg tggactggat tagctgcgga gccctggaag 50 ctgcctgtcc ttctccctgt gcttaaccag aggtgcccat gggttggaca 100 atgaggctgg tcacagcagc actgttactg ggtctcatga tggtggtcac 150 tggagacgag gatgagaaca gcccgtgtgc ccatgaggcc ctcttggacg 200 aggacaccct cttttgccag ggccttgaag ttttctaccc agagttgggg 250 aacattggct gcaaggttgt tcctgattgt aacaactaca gacagaagat 300 cacctcctgg atggagccga tagtcaagtt cccgggggcc gtggacggcg 350 caacctatat cctggtgatg gtggatccag atgcccctag cagagcagaa 400 cccagacaga gattctggag acattggctg gtaacagata tcaagggcgc 450 cgacctgaag aaagggaaga ttcagggcca ggagttatca gcctaccagg 500 ctccctcccc accggcacac agtggcttcc atcgctacca gttctttgtc 550 tatcttcagg aaggaaaagt catctctctc cttcccaagg aaaacaaaac 600 tcgaggctct tggaaaatgg acagatttct gaaccgcttc cacctgggcg 650 aacctgaagc aagcacccag ttcatgaccc agaactacca ggactcacca 700 accctccagg ctcccagagg aagggccagc gagcccaagc acaaaaccag 750 gcagagatag ctgcctgcta gatagccggc tttgccatcc gggcatgtgg 800 ccacactgct caccaccgac gatgtgggta tggaaccccc tctggataca 850 gaaccccttc ttttccaaat taaaaaaaaa aatcatcaaa 890 <210> 61 <211> 223 <212> PRT <213> Homo Sapien <400> 61 Met Gly Trp Thr Met Arg Leu Val Thr Ala Ala Leu Leu Leu Gly   1 5 10 15 Leu Met Met Val Val Thr Gly Asp Glu Asp Glu Asn Ser Pro Cys                  20 25 30 Ala His Glu Ala Leu Leu Asp Glu Asp Thr Leu Phe Cys Gln Gly                  35 40 45 Leu Glu Val Phe Tyr Pro Glu Leu Gly Asn Ile Gly Cys Lys Val                  50 55 60 Val Pro Asp Cys Asn Asn Tyr Arg Gln Lys Ile Thr Ser Trp Met                  65 70 75 Glu Pro Ile Val Lys Phe Pro Gly Ala Val Asp Gly Ala Thr Tyr                  80 85 90 Ile Leu Val Met Val Asp Pro Asp Ala Pro Ser Arg Ala Glu Pro                  95 100 105 Arg Gln Arg Phe Trp Arg His Trp Leu Val Thr Asp Ile Lys Gly                 110 115 120 Ala Asp Leu Lys Lys Gly Lys Ile Gln Gly Gln Glu Leu Ser Ala                 125 130 135 Tyr Gln Ala Pro Ser Pro Pro Ala His Ser Gly Phe His Arg Tyr                 140 145 150 Gln Phe Phe Val Tyr Leu Gln Glu Gly Lys Val Ile Ser Leu Leu                 155 160 165 Pro Lys Glu Asn Lys Thr Arg Gly Ser Trp Lys Met Asp Arg Phe                 170 175 180 Leu Asn Arg Phe His Leu Gly Glu Pro Glu Ala Ser Thr Gln Phe                 185 190 195 Met Thr Gln Asn Tyr Gln Asp Ser Pro Thr Leu Gln Ala Pro Arg                 200 205 210 Gly Arg Ala Ser Glu Pro Lys His Lys Thr Arg Gln Arg                 215 220 <210> 62 <211> 1321 <212> DNA <213> Homo Sapien <400> 62 gtcgacccac gcgtccgaag ctgctggagc cacgattcag tcccctggac 50 tgtagataaa gaccctttct tgccaggtgc tgagacaacc acactatgag 100 aggcactcca ggagacgctg atggtggagg aagggccgtc tatcaatcaa 150 tcactgttgc tgttatcaca tgcaagtatc cagaggctct tgagcaaggc 200 agaggggatc ccatttattt gggaatccag aatccagaaa tgtgtttgta 250 ttgtgagaag gttggagaac agcccacatt gcagctaaaa gagcagaaga 300 tcatggatct gtatggccaa cccgagcccg tgaaaccctt ccttttctac 350 cgtgccaaga ctggtaggac ctccaccctt gagtctgtgg ccttcccgga 400 ctggttcatt gcctcctcca agagagacca gcccatcatt ctgacttcag 450 aacttgggaa gtcatacaac actgcctttg aattaaatat aaatgactga 500 actcagccta gaggtggcag cttggtcttt gtcttaaagt ttctggttcc 550 caatgtgttt tcgtctacat tttcttagtg tcattttcac gctggtgctg 600 agacaggagc aaggctgctg ttatcatctc attttataat gaagaagaag 650 caattacttc atagcaactg aagaacagga tgtggcctca gaagcaggag 700 agctgggtgg tataaggctg tcctctcaag ctggtgctgt gtaggccaca 750 aggcatctgc atgagtgact ttaagactca aagaccaaac actgagcttt 800 cttctagggg tgggtatgaa gatgcttcag agctcatgcg cgttacccac 850 gatggcatga ctagcacaga gctgatctct gtttctgttt tgctttattc 900 cctcttggga tgatatcatc cagtctttat atgttgccaa tatacctcat 950 tgtgtgtaat agaaccttct tagcattaag accttgtaaa caaaaataat 1000 tcttggggtg ggtatgaaga tgcttcagag ctcatgcgcg ttacccacga 1050 tggcatgact agcacagagc tgatctctgt ttctgttttg ctttattccc 1100 tcttgggatg atatcatcca gtctttatat gttgccaata tacctcattg 1150 tgtgtaatag aaccttctta gcattaagac cttgtaaaca aaaataattc 1200 ttgtgttaag ttaaatcatt tttgtcctaa ttgtaatgtg taatcttaaa 1250 gttaaataaa ctttgtgtat ttatataata ataaagctaa aactgatata 1300 aaataaagaa agagtaaact g 1321 <210> 63 <211> 134 <212> PRT <213> Homo Sapien <400> 63 Met Arg Gly Thr Pro Gly Asp Ala Asp Gly Gly Gly Arg Ala Val   1 5 10 15 Tyr Gln Ser Ile Thr Val Ala Val Ile Thr Cys Lys Tyr Pro Glu                  20 25 30 Ala Leu Glu Gln Gly Arg Gly Asp Pro Ile Tyr Leu Gly Ile Gln                  35 40 45 Asn Pro Glu Met Cys Leu Tyr Cys Glu Lys Val Gly Glu Gln Pro                  50 55 60 Thr Leu Gln Leu Lys Glu Gln Lys Ile Met Asp Leu Tyr Gly Gln                  65 70 75 Pro Glu Pro Val Lys Pro Phe Leu Phe Tyr Arg Ala Lys Thr Gly                  80 85 90 Arg Thr Ser Thr Leu Glu Ser Val Ala Phe Pro Asp Trp Phe Ile                  95 100 105 Ala Ser Ser Lys Arg Asp Gln Pro Ile Ile Leu Thr Ser Glu Leu                 110 115 120 Gly Lys Ser Tyr Asn Thr Ala Phe Glu Leu Asn Ile Asn Asp                 125 130 <210> 64 <211> 999 <212> DNA <213> Homo Sapien <400> 64 gcgaggctgc accagcgcct ggcaccatga ggacgcctgg gcctctgccc 50 gtgctgctgc tgctcctggc gggagccccc gccgcgcggc ccactccccc 100 gacctgctac tcccgcatgc gggccctgag ccaggagatc acccgcgact 150 tcaacctcct gcaggtctcg gagccctcgg agccatgtgt gagatacctg 200 cccaggctgt acctggacat acacaattac tgtgtgctgg acaagctgcg 250 ggactttgtg gcctcgcccc cgtgttggaa agtggcccag gtagattcct 300 tgaaggacaa agcacggaag ctgtacacca tcatgaactc gttctgcagg 350 agagatttgg tattcctgtt ggatgactgc aatgccttgg aatacccaat 400 cccagtgact acggtcctgc cagatcgtca gcgctaaggg aactgagacc 450 agagaaagaa cccaagagaa ctaaagttat gtcagctacc cagacttaat 500 gggccagagc catgaccctc acaggtcttg tgttagttgt atctgaaact 550 gttatgtatc tctctacctt ctggaaaaca gggctggtat tcctacccag 600 gaacctcctt tgagcataga gttagcaacc atgcttctca ttcccttgac 650 tcatgtcttg ccaggatggt tagatacaca gcatgttgat ttggtcacta 700 aaaagaagaa aaggactaac aagcttcact tttatgaaca actattttga 750 gaacatgcac aatagtatgt ttttattact ggtttaatgg agtaatggta 800 cttttattct ttcttgatag aaacctgctt acatttaacc aagcttctat 850 tatgcctttt tctaacacag actttcttca ctgtctttca tttaaaaaga 900 aattaatgct cttaagatat atattttacg tagtgctgac aggacccact 950 ctttcattga aaggtgatga aaatcaaata aagaatctct tcacatgga 999 <210> 65 <211> 136 <212> PRT <213> Homo Sapien <400> 65 Met Arg Thr Pro Gly Pro Leu Pro Val Leu Leu Leu Leu Leu Ala   1 5 10 15 Gly Ala Pro Ala Ala Arg Pro Thr Pro Pro Thr Cys Tyr Ser Arg                  20 25 30 Met Arg Ala Leu Ser Gln Glu Ile Thr Arg Asp Phe Asn Leu Leu                  35 40 45 Gln Val Ser Glu Pro Ser Glu Pro Cys Val Arg Tyr Leu Pro Arg                  50 55 60 Leu Tyr Leu Asp Ile His Asn Tyr Cys Val Leu Asp Lys Leu Arg                  65 70 75 Asp Phe Val Ala Ser Pro Pro Cys Trp Lys Val Ala Gln Val Asp                  80 85 90 Ser Leu Lys Asp Lys Ala Arg Lys Leu Tyr Thr Ile Met Asn Ser                  95 100 105 Phe Cys Arg Arg Asp Leu Val Phe Leu Leu Asp Asp Cys Asn Ala                 110 115 120 Leu Glu Tyr Pro Ile Pro Val Thr Thr Val Leu Pro Asp Arg Gln                 125 130 135 Arg <210> 66 <211> 1893 <212> DNA <213> Homo Sapien <400> 66 gtctccgcgt cacaggaact tcagcaccca cagggcggac agcgctcccc 50 tctacctgga gacttgactc ccgcgcgccc caaccctgct tatcccttga 100 ccgtcgagtg tcagagatcc tgcagccgcc cagtcccggc ccctctcccg 150 ccccacaccc accctcctgg ctcttcctgt ttttactcct ccttttcatt 200 cataacaaaa gctacagctc caggagccca gcgccgggct gtgacccaag 250 ccgagcgtgg aagaatgggg ttcctcggga ccggcacttg gattctggtg 300 ttagtgctcc cgattcaagc tttccccaaa cctggaggaa gccaagacaa 350 atctctacat aatagagaat taagtgcaga aagacctttg aatgaacaga 400 ttgctgaagc agaagaagac aagattaaaa aaacatatcc tccagaaaac 450 aagccaggtc agagcaacta ttcttttgtt gataacttga acctgctaaa 500 ggcaataaca gaaaaggaaa aaattgagaa agaaagacaa tctataagaa 550 gctccccact tgataataag ttgaatgtgg aagatgttga ttcaaccaag 600 aatcgaaaac tgatcgatga ttatgactct actaagagtg gattggatca 650 taaatttcaa gatgatccag atggtcttca tcaactagac gggactcctt 700 taaccgctga agacattgtc cataaaatcg ctgccaggat ttatgaagaa 750 aatgacagag ccgtgtttga caagattgtt tctaaactac ttaatctcgg 800 ccttatcaca gaaagccaag cacatacact ggaagatgaa gtagcagagg 850 ttttacaaaa attaatctca aaggaagcca acaattatga ggaggatccc 900 aataagccca caagctggac tgagaatcag gctggaaaaa taccagagaa 950 agtgactcca atggcagcaa ttcaagatgg tcttgctaag ggagaaaacg 1000 atgaaacagt atctaacaca ttaaccttga caaatggctt ggaaaggaga 1050 actaaaacct acagtgaaga caactttgag gaactccaat atttcccaaa 1100 tttctatgcg ctactgaaaa gtattgattc agaaaaagaa gcaaaagaga 1150 aagaaacact gattactatc atgaaaacac tgattgactt tgtgaagatg 1200 atggtgaaat atggaacaat atctccagaa gaaggtgttt cctaccttga 1250 aaacttggat gaaatgattg ctcttcagac caaaaacaag ctagaaaaaa 1300 atgctactga caatataagc aagcttttcc cagcaccatc agagaagagt 1350 catgaagaaa cagacagtac caaggaagaa gcagctaaga tggaaaagga 1400 atatggaagc ttgaaggatt ccacaaaaga tgataactcc aacccaggag 1450 gaaagacaga tgaacccaaa ggaaaaacag aagcctattt ggaagccatc 1500 agaaaaaata ttgaatggtt gaagaaacat gacaaaaagg gaaataaaga 1550 agattatgac ctttcaaaga tgagagactt catcaataaa caagctgatg 1600 cttatgtgga gaaaggcatc cttgacaagg aagaagccga ggccatcaag 1650 cgcatttata gcagcctgta aaaatggcaa aagatccagg agtctttcaa 1700 ctgtttcaga aaacataata tagcttaaaa cacttctaat tctgtgatta 1750 aaattttttg acccaagggt tattagaaag tgctgaattt acagtagtta 1800 accttttaca agtggttaaa acatagcttt cttcccgtaa aaactatctg 1850 aaagtaaagt tgtatgtaag ctgaaaaaaa aaaaaaaaaa aaa 1893 <210> 67 <211> 468 <212> PRT <213> Homo Sapien <400> 67 Met Gly Phe Leu Gly Thr Gly Thr Trp Ile Leu Val Leu Val Leu   1 5 10 15 Pro Ile Gln Ala Phe Pro Lys Pro Gly Gly Ser Gln Asp Lys Ser                  20 25 30 Leu His Asn Arg Glu Leu Ser Ala Glu Arg Pro Leu Asn Glu Gln                  35 40 45 Ile Ala Glu Ala Glu Glu Asp Lys Ile Lys Lys Thr Tyr Pro Pro                  50 55 60 Glu Asn Lys Pro Gly Gln Ser Asn Tyr Ser Phe Val Asp Asn Leu                  65 70 75 Asn Leu Leu Lys Ala Ile Thr Glu Lys Glu Lys Ile Glu Lys Glu                  80 85 90 Arg Gln Ser Ile Arg Ser Ser Pro Leu Asp Asn Lys Leu Asn Val                  95 100 105 Glu Asp Val Asp Ser Thr Lys Asn Arg Lys Leu Ile Asp Asp Tyr                 110 115 120 Asp Ser Thr Lys Ser Gly Leu Asp His Lys Phe Gln Asp Asp Pro                 125 130 135 Asp Gly Leu His Gln Leu Asp Gly Thr Pro Leu Thr Ala Glu Asp                 140 145 150 Ile Val His Lys Ile Ala Ala Arg Ile Tyr Glu Glu Asn Asp Arg                 155 160 165 Ala Val Phe Asp Lys Ile Val Ser Lys Leu Leu Asn Leu Gly Leu                 170 175 180 Ile Thr Glu Ser Gln Ala His Thr Leu Glu Asp Glu Val Ala Glu                 185 190 195 Val Leu Gln Lys Leu Ile Ser Lys Glu Ala Asn Asn Tyr Glu Glu                 200 205 210 Asp Pro Asn Lys Pro Thr Ser Trp Thr Glu Asn Gln Ala Gly Lys                 215 220 225 Ile Pro Glu Lys Val Thr Pro Met Ala Ala Ile Gln Asp Gly Leu                 230 235 240 Ala Lys Gly Glu Asn Asp Glu Thr Val Ser Asn Thr Leu Thr Leu                 245 250 255 Thr Asn Gly Leu Glu Arg Arg Thr Lys Thr Tyr Ser Glu Asp Asn                 260 265 270 Phe Glu Glu Leu Gln Tyr Phe Pro Asn Phe Tyr Ala Leu Leu Lys                 275 280 285 Ser Ile Asp Ser Glu Lys Glu Ala Lys Glu Lys Glu Thr Leu Ile                 290 295 300 Thr Ile Met Lys Thr Leu Ile Asp Phe Val Lys Met Met Val Lys                 305 310 315 Tyr Gly Thr Ile Ser Pro Glu Glu Gly Val Ser Tyr Leu Glu Asn                 320 325 330 Leu Asp Glu Met Ile Ala Leu Gln Thr Lys Asn Lys Leu Glu Lys                 335 340 345 Asn Ala Thr Asp Asn Ile Ser Lys Leu Phe Pro Ala Pro Ser Glu                 350 355 360 Lys Ser His Glu Glu Thr Asp Ser Thr Lys Glu Glu Ala Ala Lys                 365 370 375 Met Glu Lys Glu Tyr Gly Ser Leu Lys Asp Ser Thr Lys Asp Asp                 380 385 390 Asn Ser Asn Pro Gly Gly Lys Thr Asp Glu Pro Lys Gly Lys Thr                 395 400 405 Glu Ala Tyr Leu Glu Ala Ile Arg Lys Asn Ile Glu Trp Leu Lys                 410 415 420 Lys His Asp Lys Lys Gly Asn Lys Glu Asp Tyr Asp Leu Ser Lys                 425 430 435 Met Arg Asp Phe Ile Asn Lys Gln Ala Asp Ala Tyr Val Glu Lys                 440 445 450 Gly Ile Leu Asp Lys Glu Glu Ala Glu Ala Ile Lys Arg Ile Tyr                 455 460 465 Ser Ser Leu <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 68 cgtcacagga acttcagcac cc 22 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 69 gtcttggctt cctccaggtt tgg 23 <210> 70 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide probe <400> 70 ggacagcgct cccctctacc tggagacttg actcccgc 38 <210> 71 <211> 2379 <212> DNA <213> Homo Sapien <400> 71 gttgctccgg cggcgctcgg ggagggagcc agcagcctag ggcctaggcc 50 cgggccacca tggcgctgcc tccaggccca gccgccctcc ggcacacact 100 gctgctcctg ccagcccttc tgagctcagg ttggggggag ttggagccac 150 aaatagatgg tcagacctgg gctgagcggg cacttcggga gaatgaacgc 200 cacgccttca cctgccgggt ggcagggggg cctggcaccc ccagattggc 250 ctggtatctg gatggacagc tgcaggaggc cagcacctca agactgctga 300 gcgtgggagg ggaggccttc tctggaggca ccagcacctt cactgtcact 350 gcccatcggg cccagcatga gctcaactgc tctctgcagg accccagaag 400 tggccgatca gccaacgcct ctgtcatcct taatgtgcaa ttcaagccag 450 agattgccca agtcggcgcc aagtaccagg aagctcaggg cccaggcctc 500 ctggttgtcc tgtttgccct ggtgcgtgcc aacccgccgg ccaatgtcac 550 ctggatcgac caggatgggc cagtgactgt caacacctct gacttcctgg 600 tgctggatgc gcagaactac ccctggctca ccaaccacac ggtgcagctg 650 cagctccgca gcctggcaca caacctctcg gtggtggcca ccaatgacgt 700 gggtgtcacc agtgcgtcgc ttccagcccc aggcccctcc cggcacccat 750 ctctgatatc aagtgactcc aacaacctaa aactcaacaa cgtgcgcctg 800 ccacgggaga acatgtccct cccgtccaac cttcagctca atgacctcac 850 tccagattcc agagcagtga aaccagcaga ccggcagatg gctcagaaca 900 acagccggcc agagcttctg gacccggagc ccggcggcct cctcaccagc 950 caaggtttca tccgcctccc agtgctgggc tatatctatc gagtgtccag 1000 cgtgagcagt gatgagatct ggctctgagc cgagggcgag acaggagtat 1050 tctcttggcc tctggacacc ctcccattcc tccaaggcat cctctaccta 1100 gctaggtcac caacgtgaag aagttatgcc actgccactt ttgcttgccc 1150 tcctggctgg ggtgccctcc atgtcatgca cgtgatgcat ttcactgggc 1200 tgtaacccgc aggggcacag gtatctttgg caaggctacc agttggacgt 1250 aagcccctca tgctgactca gggtgggccc tgcatgtgat gactgggccc 1300 ttccagaggg agctctttgg ccaggggtgt tcagatgtca tccagcatcc 1350 aagtgtggca tggcctgctg tataccccac cccagtactc cacagcacct 1400 tgtacagtag gcatgggggc gtgcctgtgt gggggacagg gagggccctg 1450 catggatttt cctccttcct atgctatgta gccttgttcc ctcaggtaaa 1500 atttaggacc ctgctagctg tgcagaaccc aattgccctt tgcacagaaa 1550 ccaacccctg acccagcggt accggccaag cacaaacgtc ctttttgctg 1600 cacacgtctc tgcccttcac ttcttctctt ctgtccccac ctcctcttgg 1650 gaattctagg ttacacgttg gaccttctct actacttcac tgggcactag 1700 acttttctat tggcctgtgc catcgcccag tattagcaca agttagggag 1750 gaagaggcag gcgatgagtc tagtagcacc caggacggct tgtagctatg 1800 catcattttc ctacggcgtt agcactttaa gcacatcccc taggggaggg 1850 ggtgagtgag gggcccagag ccctctttgt ggcttcccca cgtttggcct 1900 tctgggattc actgtgagtg tcctgagctc tcggggttga tggtttttct 1950 ctcagcatgt ctcctccacc acgggacccc agccctgacc aacccatggt 2000 tgcctcatca gcaggaaggt gcccttcctg gaggatggtc gccacaggca 2050 cataattcaa cagtgtggaa gctttagggg aacatggaga aagaaggaga 2100 ccacataccc caaagtgacc taagaacact ttaaaaagca acatgtaaat 2150 gattggaaat taatatagta cagaatatat ttttcccttg ttgagatctt 2200 cttttgtaat gtttttcatg ttactgccta gggcggtgct gagcacacag 2250 caagtttaat aaacttgact gaattcattt aaaaaaaaaa aaaaaaaaaa 2300 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2350 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2379 <210> 72 <211> 322 <212> PRT <213> Homo Sapien <400> 72 Met Ala Leu Pro Pro Gly Pro Ala Ala Leu Arg His Thr Leu Leu   1 5 10 15 Leu Leu Pro Ala Leu Leu Ser Ser Gly Trp Gly Glu Leu Glu Pro                  20 25 30 Gln Ile Asp Gly Gln Thr Trp Ala Glu Arg Ala Leu Arg Glu Asn                  35 40 45 Glu Arg His Ala Phe Thr Cys Arg Val Ala Gly Gly Pro Gly Thr                  50 55 60 Pro Arg Leu Ala Trp Tyr Leu Asp Gly Gln Leu Gln Glu Ala Ser                  65 70 75 Thr Ser Arg Leu Leu Ser Val Gly Gly Glu Ala Phe Ser Gly Gly                  80 85 90 Thr Ser Thr Phe Thr Val Thr Ala His Arg Ala Gln His Glu Leu                  95 100 105 Asn Cys Ser Leu Gln Asp Pro Arg Ser Gly Arg Ser Ala Asn Ala                 110 115 120 Ser Val Ile Leu Asn Val Gln Phe Lys Pro Glu Ile Ala Gln Val                 125 130 135 Gly Ala Lys Tyr Gln Glu Ala Gln Gly Pro Gly Leu Leu Val Val                 140 145 150 Leu Phe Ala Leu Val Arg Ala Asn Pro Pro Ala Asn Val Thr Trp                 155 160 165 Ile Asp Gln Asp Gly Pro Val Thr Val Asn Thr Ser Asp Phe Leu                 170 175 180 Val Leu Asp Ala Gln Asn Tyr Pro Trp Leu Thr Asn His Thr Val                 185 190 195 Gln Leu Gln Leu Arg Ser Leu Ala His Asn Leu Ser Val Val Ala                 200 205 210 Thr Asn Asp Val Gly Val Thr Ser Ala Ser Leu Pro Ala Pro Gly                 215 220 225 Pro Ser Arg His Pro Ser Leu Ile Ser Ser Asp Ser Asn Asn Leu                 230 235 240 Lys Leu Asn Asn Val Arg Leu Pro Arg Glu Asn Met Ser Leu Pro                 245 250 255 Ser Asn Leu Gln Leu Asn Asp Leu Thr Pro Asp Ser Arg Ala Val                 260 265 270 Lys Pro Ala Asp Arg Gln Met Ala Gln Asn Asn Ser Arg Pro Glu                 275 280 285 Leu Leu Asp Pro Glu Pro Gly Gly Leu Leu Thr Ser Gln Gly Phe                 290 295 300 Ile Arg Leu Pro Val Leu Gly Tyr Ile Tyr Arg Val Ser Ser Val                 305 310 315 Ser Ser Asp Glu Ile Trp Leu                 320 <210> 73 <211> 843 <212> DNA <213> Homo Sapien <400> 73 cggggacgga agcggcccct gggcccgagg ggctggagcc gggccggggc 50 gatgtggagc gcgggccgcg gcggggctgc ctggccggtg ctgttggggc 100 tgctgctggc gctgttagtg ccgggcggtg gtgccgccaa gaccggtgcg 150 gagctcgtga cctgcgggtc ggtgctgaag ctgctcaata cgcaccaccg 200 cgtgcggctg cactcgcacg acatcaaata cggatccggc agcggccagc 250 aatcggtgac cggcgtagag gcgtcggacg acgccaatag ctactggcgg 300 atccgcggcg gctcggaggg cgggtgcccg cgcgggtccc cggtgcgctg 350 cgggcaggcg gtgaggctca cgcatgtgct tacgggcaag aacctgcaca 400 cgcaccactt cccgtcgccg ctgtccaaca accaggaggt gagtgccttt 450 ggggaagacg gcgagggcga cgacctggac ctatggacag tgcgctgctc 500 tggacagcac tgggagcgtg aggctgctgt gcgcttccag catgtgggca 550 cctctgtgtt cctgtcagtc acgggtgagc agtatggaag ccccatccgt 600 gggcagcatg aggtccacgg catgcccagt gccaacacgc acaatacgtg 650 gaaggccatg gaaggcatct tcatcaagcc tagtgtggag ccctctgcag 700 gtcacgatga actctgagtg tgtggatgga tgggtggatg gagggtggca 750 ggtggggcgt ctgcagggcc actcttggca gagactttgg gtttgtaggg 800 gtcctcaagt gcctttgtga ttaaagaatg ttggtctatg aaa 843 <210> 74 <211> 221 <212> PRT <213> Homo Sapien <400> 74 Met Trp Ser Ala Gly Arg Gly Gly Ala Ala Trp Pro Val Leu Leu   1 5 10 15 Gly Leu Leu Leu Ala Leu Leu Val Pro Gly Gly Gly Ala Ala Lys                  20 25 30 Thr Gly Ala Glu Leu Val Thr Cys Gly Ser Val Leu Lys Leu Leu                  35 40 45 Asn Thr His His Arg Val Arg Leu His Ser His Asp Ile Lys Tyr                  50 55 60 Gly Ser Gly Ser Gly Gln Gln Ser Val Thr Gly Val Glu Ala Ser                  65 70 75 Asp Asp Ala Asn Ser Tyr Trp Arg Ile Arg Gly Gly Ser Glu Gly                  80 85 90 Gly Cys Pro Arg Gly Ser Pro Val Arg Cys Gly Gln Ala Val Arg                  95 100 105 Leu Thr His Val Leu Thr Gly Lys Asn Leu His Thr His His Phe                 110 115 120 Pro Ser Pro Leu Ser Asn Asn Gln Glu Val Ser Ala Phe Gly Glu                 125 130 135 Asp Gly Glu Gly Asp Asp Leu Asp Leu Trp Thr Val Arg Cys Ser                 140 145 150 Gly Gln His Trp Glu Arg Glu Ala Ala Val Arg Phe Gln His Val                 155 160 165 Gly Thr Ser Val Phe Leu Ser Val Thr Gly Glu Gln Tyr Gly Ser                 170 175 180 Pro Ile Arg Gly Gln His Glu Val His Gly Met Pro Ser Ala Asn                 185 190 195 Thr His Asn Thr Trp Lys Ala Met Glu Gly Ile Phe Ile Lys Pro                 200 205 210 Ser Val Glu Pro Ser Ala Gly His Asp Glu Leu                 215 220 <210> 75 <211> 1049 <212> DNA <213> Homo Sapien <400> 75 gttgctatgt tgcccaggct ggtcttgaag tgccttgacc tcctaaagtg 50 ttggaaccac agacgtgagc cactccaccc agcctaaaac ttcatcttct 100 ttggatgaga tgaacacttt taacaagaga acaggactct atataaatcg 150 ctgtgggctc accacctcta aggaggagca ctgactgaag acagaaaaat 200 tgatgaactg aagaagacat ggtccattat gccttacaaa cttacacagt 250 gctttgggaa ttccaaagta ctcagtggag agaggtgttt caggagccgt 300 agagccagat cgtcatcatg tctgcattgt ggctgctgct gggcctcctt 350 gccctgatgg acttgtctga aagcagcaac tggggatgct atggaaacat 400 ccaaagcctg gacacccctg gagcatcttg tgggattgga agacgtcacg 450 gcctgaacta ctgtggagtt cgtgcttctg aaaggctggc tgaaatagac 500 atgccatacc tcctgaaata tcaacccatg atgcaaacca ttggccaaaa 550 gtactgcatg gatcctgccg tgatcgctgg tgtcttgtcc aggaagtctc 600 ccggtgacaa aattctggtc aacatgggcg ataggactag catggtgcag 650 gaccctggct ctcaagctcc cacatcctgg attagtgagt ctcaggtttc 700 ccagacaact gaagttctga ctactagaat caaagaaatc cagaggaggt 750 ttccaacctg gacccctgac cagtacctga gaggtggact ctgtgcctac 800 agtgggggtg ctggctatgt ccgaagcagc caggacctga gctgtgactt 850 ctgcaatgat gtccttgcac gagccaagta cctcaagaga catggcttct 900 aacatctcag atgaaaccca agaccatgat cacatatgca gcctcaaatg 950 ttacacagat aaaactagcc aagggcacct gtaactggga atctgagttt 1000 gacctaaaag tcattaaaat aacatgaatc ccattaaaaa aaaaaaaaa 1049 <210> 76 <211> 194 <212> PRT <213> Homo Sapien <400> 76 Met Ser Ala Leu Trp Leu Leu Leu Gly Leu Leu Ala Leu Met Asp   1 5 10 15 Leu Ser Glu Ser Ser Asn Trp Gly Cys Tyr Gly Asn Ile Gln Ser                  20 25 30 Leu Asp Thr Pro Gly Ala Ser Cys Gly Ile Gly Arg Arg His Gly                  35 40 45 Leu Asn Tyr Cys Gly Val Arg Ala Ser Glu Arg Leu Ala Glu Ile                  50 55 60 Asp Met Pro Tyr Leu Leu Lys Tyr Gln Pro Met Met Gln Thr Ile                  65 70 75 Gly Gln Lys Tyr Cys Met Asp Pro Ala Val Ile Ala Gly Val Leu                  80 85 90 Ser Arg Lys Ser Pro Gly Asp Lys Ile Leu Val Asn Met Gly Asp                  95 100 105 Arg Thr Ser Met Val Gln Asp Pro Gly Ser Gln Ala Pro Thr Ser                 110 115 120 Trp Ile Ser Glu Ser Gln Val Ser Gln Thr Thr Glu Val Leu Thr                 125 130 135 Thr Arg Ile Lys Glu Ile Gln Arg Arg Phe Pro Thr Trp Thr Pro                 140 145 150 Asp Gln Tyr Leu Arg Gly Gly Leu Cys Ala Tyr Ser Gly Gly Ala                 155 160 165 Gly Tyr Val Arg Ser Ser Gln Asp Leu Ser Cys Asp Phe Cys Asn                 170 175 180 Asp Val Leu Ala Arg Ala Lys Tyr Leu Lys Arg His Gly Phe                 185 190 <210> 77 <211> 899 <212> DNA <213> Homo Sapien <400> 77 ttgaaaatct actctatcag ctgctgtggt tgccaccatt ctcaggaccc 50 tcgccatgaa agcccttatg ctgctcaccc tgtctgttct gctctgctgg 100 gtctcagctg acattcgctg tcactcctgc tacaaggtcc ctgtgctggg 150 ctgtgtggac cggcagtcct gccgcctgga gccaggacag caatgcctga 200 caacacatgc ataccttggt aagatgtggg ttttctccaa tctgcgctgt 250 ggcacaccag aagagccctg tcaggaggcc ttcaaccaaa ccaaccgcaa 300 gctgggtctg acatataaca ccacctgctg caacaaggac aactgcaaca 350 gcgcaggacc ccggcccact ccagccctgg gccttgtctt ccttacctcc 400 ttggctggcc ttggcctctg gctgctgcac tgagactcat tccattggct 450 gcccctcctc ccacctgcct tggcctgagc ctctctccct gtgtctctgt 500 atcccctggc tttacagaat cgtctctccc tagctcccat ttctttaatt 550 aaacactgtt ccgagtggtc tcctcatcca tccttcccac ctcacaccct 600 tcactctcct ttttctgggt cccttcccac ttccttccag gacctccatt 650 ggctcctaga agggctcccc actttgcttc ctatactctg ctgtccccta 700 cttgaggagg gattgggatc tgggcctgaa atggggcttc tgtgttgtcc 750 ccagtgaagg ctcccacaag gacctgatga cctcactgta cagagctgac 800 tccccaaacc caggctccca tatgtacccc atcccccata ctcacctctt 850 tccattttga gtaataaatg tctgagtctg gaaaaaaaaa aaaaaaaaa 899 <210> 78 <211> 125 <212> PRT <213> Homo Sapien <400> 78 Met Lys Ala Leu Met Leu Leu Thr Leu Ser Val Leu Leu Cys Trp   1 5 10 15 Val Ser Ala Asp Ile Arg Cys His Ser Cys Tyr Lys Val Pro Val                  20 25 30 Leu Gly Cys Val Asp Arg Gln Ser Cys Arg Leu Glu Pro Gly Gln                  35 40 45 Gln Cys Leu Thr Thr His Ala Tyr Leu Gly Lys Met Trp Val Phe                  50 55 60 Ser Asn Leu Arg Cys Gly Thr Pro Glu Glu Pro Cys Gln Glu Ala                  65 70 75 Phe Asn Gln Thr Asn Arg Lys Leu Gly Leu Thr Tyr Asn Thr Thr                  80 85 90 Cys Cys Asn Lys Asp Asn Cys Asn Ser Ala Gly Pro Arg Pro Thr                  95 100 105 Pro Ala Leu Gly Leu Val Phe Leu Thr Ser Leu Ala Gly Leu Gly                 110 115 120 Leu Trp Leu Leu His                 125 <210> 79 <211> 1977 <212> DNA <213> Homo Sapien <400> 79 acgggccgca gcggcagtga cgtagggttg gcgcacggat ccgttgcggc 50 tgcagctctg cagtcgggcc gttccttcgc cgccgccagg ggtagcggtg 100 tagctgcgca gcgtcgcgcg cgctaccgca cccaggttcg gcccgtaggc 150 gtctggcagc ccggcgccat cttcatcgag cgccatggcc gcagcctgcg 200 ggccgggagc ggccgggtac tgcttgctcc tcggcttgca tttgtttctg 250 ctgaccgcgg gccctgccct gggctggaac gaccctgaca gaatgttgct 300 gcgggatgta aaagctctta ccctccacta tgaccgctat accacctccc 350 gcaggctgga tcccatccca cagttgaaat gtgttggagg cacagctggt 400 tgtgattctt ataccccaaa agtcatacag tgtcagaaca aaggctggga 450 tgggtatgat gtacagtggg aatgtaagac ggacttagat attgcataca 500 aatttggaaa aactgtggtg agctgtgaag gctatgagtc ctctgaagac 550 cagtatgtac taagaggttc ttgtggcttg gagtataatt tagattatac 600 agaacttggc ctgcagaaac tgaaggagtc tggaaagcag cacggctttg 650 cctctttctc tgattattat tataagtggt cctcggcgga ttcctgtaac 700 atgagtggat tgattaccat cgtggtactc cttgggatcg cctttgtagt 750 ctataagctg ttcctgagtg acgggcagta ttctcctcca ccgtactctg 800 agtatcctcc attttcccac cgttaccaga gattcaccaa ctcagcagga 850 cctcctcccc caggctttaa gtctgagttc acaggaccac agaatactgg 900 ccatggtgca acttctggtt ttggcagtgc ttttacagga caacaaggat 950 atgaaaattc aggaccaggg ttctggacag gcttgggaac tggtggaata 1000 ctaggatatt tgtttggcag caatagagcg gcaacaccct tctcagactc 1050 gtggtactac ccgtcctatc ctccctccta ccctggcacg tggaataggg 1100 cttactcacc ccttcatgga ggctcgggca gctattcggt atgttcaaac 1150 tcagacacga aaaccagaac tgcatcagga tatggtggta ccaggagacg 1200 ataaagtaga aagttggagt caaacactgg atgcagaaat tttggatttt 1250 tcatcacttt ctctttagaa aaaaagtact acctgttaac aattgggaaa 1300 aggggatatt caaaagttct gtggtgttat gtccagtgta gctttttgta 1350 ttctattatt tgaggctaaa agttgatgtg tgacaaaata cttatgtgtt 1400 gtatgtcagt gtaacatgca gatgtatatt gcagtttttg aaagtgatca 1450 ttactgtgga atgctaaaaa tacattaatt tctaaaacct gtgatgccct 1500 aagaagcatt aagaatgaag gtgttgtact aatagaaact aagtacagaa 1550 aatttcagtt ttaggtggtt gtagctgatg agttattacc tcatagagac 1600 tataatattc tatttggtat tatattattt gatgtttgct gttcttcaaa 1650 catttaaatc aagctttgga ctaattatgc taatttgtga gttctgatca 1700 cttttgagct ctgaagcttt gaatcattca gtggtggaga tggccttctg 1750 gtaactgaat attaccttct gtaggaaaag gtggaaaata agcatctaga 1800 aggttgttgt gaatgactct gtgctggcaa aaatgcttga aacctctata 1850 tttctttcgt tcataagagg taaaggtcaa atttttcaac aaaagtcttt 1900 taataacaaa agcatgcagt tctctgtgaa atctcaaata ttgttgtaat 1950 agtctgtttc aatcttaaaa agaatca 1977 <210> 80 <211> 339 <212> PRT <213> Homo Sapien <400> 80 Met Ala Ala Ala Cys Gly Pro Gly Ala Ala Gly Tyr Cys Leu Leu   1 5 10 15 Leu Gly Leu His Leu Phe Leu Leu Thr Ala Gly Pro Ala Leu Gly                  20 25 30 Trp Asn Asp Pro Asp Arg Met Leu Leu Arg Asp Val Lys Ala Leu                  35 40 45 Thr Leu His Tyr Asp Arg Tyr Thr Thr Ser Arg Arg Leu Asp Pro                  50 55 60 Ile Pro Gln Leu Lys Cys Val Gly Gly Thr Ala Gly Cys Asp Ser                  65 70 75 Tyr Thr Pro Lys Val Ile Gln Cys Gln Asn Lys Gly Trp Asp Gly                  80 85 90 Tyr Asp Val Gln Trp Glu Cys Lys Thr Asp Leu Asp Ile Ala Tyr                  95 100 105 Lys Phe Gly Lys Thr Val Val Ser Cys Glu Gly Tyr Glu Ser Ser                 110 115 120 Glu Asp Gln Tyr Val Leu Arg Gly Ser Cys Gly Leu Glu Tyr Asn                 125 130 135 Leu Asp Tyr Thr Glu Leu Gly Leu Gln Lys Leu Lys Glu Ser Gly                 140 145 150 Lys Gln His Gly Phe Ala Ser Phe Ser Asp Tyr Tyr Tyr Lys Trp                 155 160 165 Ser Ser Ala Asp Ser Cys Asn Met Ser Gly Leu Ile Thr Ile Val                 170 175 180 Val Leu Leu Gly Ile Ala Phe Val Val Tyr Lys Leu Phe Leu Ser                 185 190 195 Asp Gly Gln Tyr Ser Pro Pro Pro Tyr Ser Glu Tyr Pro Pro Phe                 200 205 210 Ser His Arg Tyr Gln Arg Phe Thr Asn Ser Ala Gly Pro Pro Pro                 215 220 225 Pro Gly Phe Lys Ser Glu Phe Thr Gly Pro Gln Asn Thr Gly His                 230 235 240 Gly Ala Thr Ser Gly Phe Gly Ser Ala Phe Thr Gly Gln Gln Gly                 245 250 255 Tyr Glu Asn Ser Gly Pro Gly Phe Trp Thr Gly Leu Gly Thr Gly                 260 265 270 Gly Ile Leu Gly Tyr Leu Phe Gly Ser Asn Arg Ala Ala Thr Pro                 275 280 285 Phe Ser Asp Ser Trp Tyr Tyr Pro Ser Tyr Pro Pro Ser Tyr Pro                 290 295 300 Gly Thr Trp Asn Arg Ala Tyr Ser Pro Leu His Gly Gly Ser Gly                 305 310 315 Ser Tyr Ser Val Cys Ser Asn Ser Asp Thr Lys Thr Arg Thr Ala                 320 325 330 Ser Gly Tyr Gly Gly Thr Arg Arg Arg                 335

权利要求:
Claims (22)
[1" claim-type="Currently amended] Isolated nucleic acid having the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 1).
[2" claim-type="Currently amended] An isolated nucleic acid having the full length coding sequence of the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 1).
[3" claim-type="Currently amended] Isolated nucleic acid with full length coding sequence of DNA deposited with ATCC Accession No. 203581
[4" claim-type="Currently amended] An isolated nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in FIG. 2 (SEQ ID NO: 2).
[5" claim-type="Currently amended] An isolated nucleic acid having at least 80% amino acid sequence identity with the amino acid sequence set forth in FIG. 2 (SEQ ID NO: 2) and having a nucleotide sequence encoding an amino acid sequence having the ability to induce cell regeneration.
[6" claim-type="Currently amended] A vector comprising the nucleic acid according to any one of claims 1 to 5.
[7" claim-type="Currently amended] The vector of claim 6 operably linked with regulatory sequences recognized by the host cell transformed with the vector.
[8" claim-type="Currently amended] An isolated host cell comprising the vector of claim 6.
[9" claim-type="Currently amended] The host cell of claim 8, which is a CHO cell.
[10" claim-type="Currently amended] The method of claim 8, wherein: Choline host cell.
[11" claim-type="Currently amended] The host cell of claim 8, which is a yeast cell.
[12" claim-type="Currently amended] An isolated polypeptide having the amino acid sequence set forth in FIG. 2 (SEQ ID NO: 2).
[13" claim-type="Currently amended] An isolated polypeptide having at least 80% amino acid sequence identity with the amino acid sequence set forth in FIG. 2 (SEQ ID NO: 2) and having the ability to induce cell regeneration.
[14" claim-type="Currently amended] An isolated polypeptide having an amino acid sequence encoded by the full length coding sequence of DNA deposited with ATCC Accession No. 203581.
[15" claim-type="Currently amended] An isolated polypeptide having at least 80% amino acid sequence identity with an amino acid sequence encoded by the full length coding sequence of a DNA deposited with ATCC Accession No. 203581 and having the ability to induce cell regeneration.
[16" claim-type="Currently amended] A chimeric molecule comprising a polypeptide according to any one of claims 12 to 15 fused with a heterologous amino acid sequence.
[17" claim-type="Currently amended] The chimeric molecule of claim 16, wherein the heterologous amino acid sequence is an epitope tag sequence.
[18" claim-type="Currently amended] The chimeric molecule of claim 16, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin.
[19" claim-type="Currently amended] An antibody that specifically binds to a polypeptide of any one of claims 12-15.
[20" claim-type="Currently amended] The antibody of claim 19, which is a monoclonal antibody, humanized antibody or single chain antibody.
[21" claim-type="Currently amended] Isolated polypeptide having at least 80% amino acid sequence identity with one of the following sequences and the ability to induce cell regeneration:
(a) the polypeptide described in FIG. 2 (SEQ ID NO: 2), lacking a bound signal peptide;
(b) the extracellular domain of the polypeptide described in FIG. 2 (SEQ ID NO: 2), with the bound signal peptide; or
(c) the extracellular domain of the polypeptide described in FIG. 2 (SEQ ID NO: 2), with no signal peptide bound.
[22" claim-type="Currently amended] An isolated nucleic acid having a nucleotide sequence encoding the amino acid sequence of an isolated polypeptide according to claim 21.

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同族专利:

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公开号 | 公开日

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KR100532172B1|2005-12-01|

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KR100473125B1|2005-03-14|

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KR20050009279A|2005-01-24|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-03-01|Priority to PCT/US2000/005601

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2004-04-16|Application filed by 제넨테크, 인크.

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2004-06-23|Publication of KR20040053176A

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2005-03-14|Application granted

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2005-03-14|Publication of KR100473125B1

优先权:

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申请号 | 申请日 | 专利标题

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PCT/US2000/005601|WO2000056889A2|1999-03-23|2000-03-01|Secreted and transmembrane polypeptides and nucleic acids encoding the same|