EP1027434A2 - Polypeptides secretes et transmembranaires et acides nucleiques les codant - Google Patents

Polypeptides secretes et transmembranaires et acides nucleiques les codant

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Publication number
EP1027434A2
EP1027434A2 EP98946090A EP98946090A EP1027434A2 EP 1027434 A2 EP1027434 A2 EP 1027434A2 EP 98946090 A EP98946090 A EP 98946090A EP 98946090 A EP98946090 A EP 98946090A EP 1027434 A2 EP1027434 A2 EP 1027434A2
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EP
European Patent Office
Prior art keywords
seq
atcc
polypeptide
sequence
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP98946090A
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German (de)
English (en)
Inventor
William I. Wood
Austin L. Gurney
Audrey Goddard
Diane Pennica
Jian Chen
Jean Yuan
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Genentech Inc
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Genentech Inc
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Priority to EP01127791A priority Critical patent/EP1205546B1/fr
Priority to DK01127795T priority patent/DK1217006T3/da
Priority to EP04014066A priority patent/EP1464654A3/fr
Priority to DK01127792T priority patent/DK1205489T3/da
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to EP01127794A priority patent/EP1207168B1/fr
Priority to DK01127794T priority patent/DK1207168T3/da
Priority to EP01127792A priority patent/EP1205489B1/fr
Priority to ES05024504T priority patent/ES2333880T3/es
Priority to DK01127791T priority patent/DK1205546T3/da
Priority to EP01127795A priority patent/EP1217006B1/fr
Priority to EP01127793A priority patent/EP1205550B1/fr
Priority to EP05024504A priority patent/EP1659131B1/fr
Priority claimed from PCT/US1998/019330 external-priority patent/WO1999014328A2/fr
Publication of EP1027434A2 publication Critical patent/EP1027434A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides encoded by that DNA.
  • Extracellular and membrane-bound proteins play important roles in the formation, differentiation and maintenance of multicellular organisms.
  • secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment, usually at a membrane-bound receptor protein.
  • Secreted proteins have various industrial applications, including use as pharmaceuticals, diagnostics, biosensors and bioreactors.
  • most protein drugs available at present such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secretory proteins.
  • Their receptors which are membrane-bound proteins, also have potential as therapeutic or diagnostic agents.
  • Receptor immunoadhesins for instance, can be employed as therapeutic agents to block receptor-ligand interaction.
  • Membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
  • Epidermal growth factor is a conventional mitogenic factor that stimulates the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signaling and subsequent effects.
  • EGFR EGF receptor
  • the EGFR is expressed in neurons of the cerebral cortex, cerebellum, and hippocampus in addition to other regions of the central nervous system (CNS). In addition, EGF is also expressed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic neurons. In fact, many studies have indicated that EGF has neurotrophic or neuromodulatory effects on various types of neurons in the CNS.
  • EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival.
  • EGF also acts on other cell types, including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells.
  • Evidence of the effects of EGF on neurons in the CNS is accumulating, but the mechanisms of action remain essentially unknown.
  • EGF-induced signaling in mitotic cells is better understood than in postmitotic neurons .
  • Studies of cloned pheochromocytoma PC 12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF.
  • MAPK mitogen-activated protein kinase
  • EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postmitotic neurons.
  • EGF is produced by the salivary and Brunner's glands of the gastrointestinal system, kidney, pancreas, thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, Peptides 12: 653-663 (1991).
  • body fluids such as saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, Peptides 12: 653-663 (1991).
  • EGF is mediated by its membrane specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck et al., J. Cell Biochem. 3_1: 135-152 (1986). EGF is believed to function by binding to the extracellular portion of its receptor which induces a transmembrane signal that activates the intrinsic tyrosine kinase.
  • Non isolated peptides having this motif include TGF- ⁇ , amphiregulin, schwannoma-derived growth factor (SDGF), heparin-binding EGF-like growth factors and certain virally encoded peptides (e.g., Vaccinia virus, Reisner, Nature 313: 801-803 (1985), Shope fibroma virus, Chang et al., Mol Cell Biol. 7: 535-540 (1987), Molluscum contagiosum, Porter and Archard, /. Gen. Virol. 68: 673-682 (1987), and Myxoma virus, Upton et al, J. Virol. 61: 1271-1275 (1987), Prigent and Lemoine, Prog. Growth Factor Res. 4: 1-24 (1992).
  • EGF-like domains are not confined to growth factors but have been observed in a variety of cell-surface and extracellular proteins which have interesting properties in cell adhesion, protein-protein interaction and development, Laurence and Gusterson, Tumor Biol. H: 229-261 (1990). These proteins include blood coagulation factors (factors VI, IX, X, XII, protein C, protein S, protein Z, tissue plasminogen activator, urokinase), extracellular matrix components (laminin, cytotactin, entactin), cell surface receptors (LDL receptor, thrombomodulin receptor) and immunity-related proteins (complement Clr, uromodulin).
  • blood coagulation factors factors VI, IX, X, XII, protein C, protein S, protein Z, tissue plasminogen activator, urokinase
  • extracellular matrix components laminin, cytotactin, entactin
  • LDL receptor thrombomodulin receptor
  • immunity-related proteins complement Cl
  • EGF-like precursors are preserved through lower organisms as well as in mammalian cells.
  • a number of genes with developmental significance have been identified in invertebrates with EGF-like repeats.
  • the notch gene of Drosophila encodes 36 tandemly arranged 40 amino acid repeats which show homology to EGF, Wharton et al, Cell 43: 557-581 (1985).
  • Hydropathy plots indicate a putative membrane spanning domain, with the EGF-related sequences being located on the extracellular side of the membrane.
  • Other homeotic genes with EGF-like repeats include Delta, 95F and 5ZD which were identified using probes based on Notch, and the nematode gene Lin-12 which encodes a putative receptor for a developmental signal transmitted between two specified cells.
  • EGF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions, Konturek et al, Eur. J. Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulceration gastrointestinal ulcerations and congenital microvillus atrophy, Guglietta and Sullivan, Eur. J. Gastroenterol Hepatol 7(10), 945-50 (1995). Additionally, EGF has been implicated in hair follicle differentiation; du Cros, J. Invest. Dermatol 10J. (1 Suppl.), 106S-113S (1993), Hillier, Clin.
  • EGF is also implicated various skin disease characterized by abnormal keratinocyte differentiation, e.g., psoriasis, epithelial cancers such as squamous cell carcinomas of the lung, epidermoid carcinoma of the vulva and gliomas.
  • Nephritis is a condition characterized by inflammation of the kidney affecting the structure and normal function of the kidney. This condition can be chronic or acute and is generally caused by infection, degenerative process or vascular disease. In all cases, early detection is desirable so that the patient with nephritis can begin treatment of the condition.
  • TIN-ag tubulointerstitial nephritis antigen
  • the rabbit TIN-ag has a domain in the amino-terminal region containing an epidermal growth factor-like motif that shares homology with laminin A and S chains, alpha 1 chain of type I collagen, von Willebrand's factor and mucin, indicating structural and functional similarities. Studies have also been conducted in mice. However, it is desirable to identify tubulointerstitial nephritis antigens in humans to aid in the development of early detection methods and treatment of nephritis. Proteins which have homology to tubulointerstitial nephritis antigens are of particular interest to the medical and industrial communities. Often, proteins having homology to each other have similar function.
  • PRO230 hgerein as PRO230, which has homology to tubulointerstitial nephritis antigens.
  • Stem cells are undifferentiated cells capable of (a) proliferation, (b) self maintenance, (c) the production of a large number of differentiated functional progeny, (d) regeneration of tissue after injury and/or (e) a flexibility in the use of these options.
  • Stem cells often express cell surface antigens which are capable of serving as cell specific markers that can be exploited to identify stem cells, thereby providing a means for identifying and isolating specific stem cell populations.
  • stem cell populations Having possession of different stem cell populations will allow for a number of important applications. For example, possessing a specific stem cell population will allow for the identification of growth factors and other proteins which are involved in their proliferation and differentiation. In addition, there may be as yet undiscovered proteins which are associated with (1) the early steps of dedication of the stem cell to a particular lineage, (2) prevention of such dedication, and (3) negative control of stem cell proliferation, all of which may be identified if one has possession of the stem cell population. Moreover, stem cells are important and ideal targets for gene therapy where the inserted genes promote the health of the individual into whom the stem cells are transplanted. Finally, stem cells may play important roles in transplantation of organs or tissues, for example liver regeneration and skin grafting.
  • PRQ187 Growth factors are molecular signals or mediators that enhance cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. However, there are other cellular reactions than only growth upon expression to growth factors. As a result, growth factors are better characterized as multifunctional and potent cellular regulators. Their biological effects include proliferation, chemotaxis and stimulation of extracellular matrix production. Growth factors can have both stimulatory and inhibitory effects. For example, transforming growth factor (TGF- ⁇ ) is highly pleiotropic and can stimulate proliferation in some cells, especially connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.
  • TGF- ⁇ transforming growth factor
  • Peptide growth factors are elements of a complex biological language, providing the basis for intercellular communication. They permit cells to convey information between each other, mediate interaction between cells and change gene expression. The effect of these multifunctional and pluripotent factors is dependent on the presence or absence of other peptides.
  • FGF-8 is a member of the fibroblast growth factors (FGFs) which are a family of heparin-binding, potent mitogens for both normal diploid fibroblasts and established cell lines, Gospodarowicz et al. (1984L Proc. Natl.
  • the FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2 (FGF-3), K-
  • FGF/HST FGF-4
  • FGF-5 FGF-6
  • FGF-7 FGF-7
  • AIGF FGF-8 among others.
  • All FGFs have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level.
  • FGFs have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level.
  • These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells, adrenal cortical cells, chondrocytes, myoblasts, corneal and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostatic epithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblasts and osteoblasts.
  • Fibroblast growth factors can also stimulate a large number of cell types in a non-mitogenic manner. These activities include promotion of cell migration into wound area (chemotaxis
  • angiogenesis angiogenesis
  • nerve regeneration and survival neurotrophism
  • modulation of endocrine functions and stimulation or suppression of specific cellular protein expression, extracellular matrix production and cell survival.
  • Baird & Bohlen Handbook of Exp. Pharmacol. 95(1): 369418, Springer, (1990).
  • fibroblast growth factors have been suggested to rninimize myocardium damage in heart disease and surgery (U.S. P. 4,378,347).
  • FGF-8 also known as androgen-induced growth factor (AIGF)
  • AIGF androgen-induced growth factor
  • FGF-8 has been proposed to be under androgenic regulation and induction in the mouse mammary carcinoma cell line SC3. Tanaka et al., Proc. Natl. Acad. Sci. USA 89: 8928-8932 (1992); Sato et al., J. Steroid Biochem. Molec. Biol. 47: 91-98 (1993).
  • FGF-8 may have a local role in the prostate, which is known to be an androgen-responsive organ.
  • FGF-8 can also be oncogenic, as it displays transforming activity when transfected into NIH-3T3 fibroblasts. Kouhara et al, Oncogene 9455-462 (1994). While FGF-8 has been detected in heart, brain, lung, kidney, testis, prostate and ovary, expression was also detected in the absence of exogenous androgens. Schmitt et al, J. Steroid Biochem. Mol Biol. 57 (3-4): 173-78 (1996). FGF-8 shares the property with several other FGFs of being expressed at a variety of stages of murine embryogenesis, which supports the theory that the various FGFs have multiple and perhaps coordinated roles in differentiation and embryogenesis.
  • FGF-8 has also been identified as a protooncogene that cooperates with Wnt-1 in the process of mammary tumorigenesis (Shackleford et al., Proc. Natl. Acad. Sci. USA 90, 740-744 (1993); Heikinheimo et al, Mech. Dev. 48: 129-138 (1994)).
  • FGF-8 exists as three protein isoforms, as a result of alternative splicing of the primary transcript. Tanaka et al., supra.
  • FGF-8 normal adult expression of FGF-8 is weak and confined to gonadal tissue, however northern blot analysis has indicated that FGF-8 mRNA is present from day 10 through day 12 or murine gestation, which suggests that FGF-8 is important to normal development. Heikinheimo et al, Mech Dev. 48(2): 129-38 (1994). Further in situ hybridization assays between day 8 and 16 of gestation indicated initial expression in the surface ectoderm of the first bronchial arches, the frontonasal process, the forebrain and the midbrain-hindbrain junction.
  • FGF-8 was expressed in the surface ectoderm of the forelimb and hindlimb buds, the nasal its and nasopharynx, the infundibulum and in the telencephalon, diencephalon and metencephalon. Expression continues in the developing hindlimbs through day 13 of gestation, but is undetectable thereafter. The results suggest that FGF-8 has a unique temporal and spatial pattern in embryogenesis and suggests a role for this growth factor in multiple regions of ectodermal differentiation in the post-gastrulation embryo.
  • Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.
  • Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations.
  • the crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglubular shape.
  • Human matrilin-2 polypeptide is a member of the von Willebrand factor type A-like module superfamily.
  • von Willebrand factor is a protein which plays an important role in the maintenence of hemostasis . More specifically, von Willebrand factor is a protein which is known to participate in platelet-vessel wall interactions at the site of vascular injury via its ability to interact and form a complex with Factor VIII. The absence of von Willebrand factor in the blood causes an abnormality with the blood platelets that prevents platelet adhesion to the vascular wall at the site of the vascular injury. The result is the propensity for brasing, nose bleeds, intestinal bleeding, and the like comprising von Willebrand's disease.
  • the cell surface protein HCAR is a membrane-bound protein that acts as a receptor for subgroup C of the adenoviruses and subgroup B of the coxsackieviruses.
  • HCAR may provide a means for mediating viral infection of cells in that the presence of the HCAR receptor on the cellular surface provides a binding site for viral particles, thereby facilitating viral infection.
  • CD97 is a seven-span transmembrane receptor which has a cellular ligand, CD55, DAF Hamann, et al , J Exp Med (U S ), 184(3) 1189 (1996) Additionally, CD97 has been reported as being a dedifferennanon marker m human thyroid carcinomas and as associated with inflammation Aust, et al , Cancer Res (U S ), 57(9) 1798 (1997), Gray, et al , J Immunol (U S ), 157(12) 5438 (1996) CD97 has also been reported as bemg related to the secretm receptor superfamily, but unlike known members of that family, CD97 and EMRl have extended extracellular regions that possess several EGF domains at the N-te ⁇ runus Hamann, et al , Geno ⁇ ucs.
  • EMRl is further described m Lm, et al , Geno ⁇ ucs. 41(3) 301 (1997) and Baud, et al , Genomics. 26(2) 334 (1995) While CD97 and EMRl appear to be related to the secretm receptors, a known member of the secretm family of G protein-coupled receptors mcludes the alpha-latroxm receptor, latrophihn, which has been described as calcium mdependent and abundant among neuronal tissues Le anova, et al , J Biol Chem .
  • TGF- ⁇ transforming growth factors
  • FGFs Fibroblast growth factors
  • Fibroblast growth factors can also stimulate a large number of cell types in a non-mitogenic manner These activities include promotion of cell migration into a wound area (chemotaxis), initiation of new blood vessel formulation (angiogenesis), modulation of nerve regeneration and survival (neurotrophism), modulation of endoc ⁇ ne functions, and stimulation or suppression of specific cellular protem expression, extracellular matrix production and cell survival Barrd, A & Bohlen, P , Handbook of Exp Phrmacol 95(1) 369-418 (1990) These properties provide a basis for usmg fibroblast growth factors m therapeutic approaches to accelerate wound healmg, nerve repair, collateral blood vessel formation, and the like For example, fibroblast growth factors, have been suggested to minimize myocardium damage m heart disease and surgery (U S P 4,378,437)
  • PRQ245 Some of the most important proteins involved in the above described regulation and modulation of cellular processes are the enzymes which regulate levels of protein phosphorylation in the cell. For example, it is known that the transduction of signals that regulate cell growth and differentiation is regulated at least in part by phosphorylation and dephosphorylation of various cellular proteins.
  • the enzymes that catalyze these processes include the protein kinases, which function to phosphorylate various cellular proteins, and the protein phosphatases, which function to remove phosphate residues from various cellular proteins. The balance of the level of protein phosphorylation in the cell is thus mediated by the relative activities of these two types of enzymes.
  • protem-protem interactions mclude receptor and antigen complexes and signaling mechanisms
  • protem-protem interacflons can be more easily manipulated to regulate the particular result of the protem-protem mteraction
  • Leucme-nch repeats are short sequence motifs present in a number of protems with diverse functions and cellular locations
  • the crystal structure of ⁇ bonuclease inhibitor protein has revealed that leucme-nch repeats correspond to beta-alpha structural units These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protem acquires an unusual, nonglubular shape
  • leucme-nch proteoglycans which serve as tissue organizers, orienting and ordermg collagen fibrils durmg ontogeny and are involved in pathological processes such as wound healing, tissue repair, and tumor stroma formation lozzo, R V , Crit Rev Biochem Mol Biol , 32(2) 141-174 (1997)
  • Others studies implicating leucme rich protems in wound healing and tissue repair are De La Salle, C , et al , Vouv Rev Fr Hematol (Germany), 37(4) 215-222 (1995), reporting mutations m the leucine rich motif m a complex associated with the bleedmg disorder Bernard-Souher syndrome and Chlemetson, K J , Thromb Haemost (Germany), 74(1) 111-116 (July 1995), reporting that platelets have leucme rich repeats
  • Another protem of particular interest which has been reported to have leucme-nch repeats is the SLIT protein which has been reported to be useful in treating neuro-degeneran
  • Efforts are therefore bemg undertaken by both industry and academia to identify new protems havmg leucme nch repeats to better understand protein-protem interactions
  • proteins having leucme rich repeats and homology to known protems havmg leucine rich repeats such as the SLIT protem and platelet glycoprotem V 12.
  • PRQ258 hnmunoglobulins are antibody molecules, the proteins that function both as receptors for antigen on the B- cell membrane and as the secreted products of the plasma cell. Like all antibody molecules, immunoglobulins perform two major functions: they bind specifically to an antigen and they participate in a limited number of biological effector functions. Therefore, new members of the Ig superfamily are always of interest.
  • Molecules which act as receptors by various viruses and those which act to regulate immune function are of particular interest. Also of particular interest are those molecules which have homology to known Ig family members which act as virus receptors or regulate immune function. Thus, molecules having homology to poliovirus receptors, CRTAM .and CD166 (a ligand for lymphocyte antigen CD6) are of particular interest.
  • Extracellular and membrane-bound proteins play important roles in the formation, differentiation and maintenance of multicellular organisms.
  • secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment, usually at a membrane-bound receptor protein.
  • PR0258 polypeptides have homology to CRTAM, designated herein as PR0258 polypeptides.
  • Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easily manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community. All proteins containing leucine-rich repeats are thought to be involved in protein-protein interactions.
  • Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations.
  • the crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape.
  • Thrombomodulin binds to and regulates the activity of thrombin. It is important in the control of blood coagulation. Thrombomodulin functions as a natural anticoagulant by accelerating the activation of protein C by thrombin. Soluble thrombomodulin may have therapeutic use as an antithrombotic agent with reduced risk for hemorrhage as compared with heparin. Thrombomodulin is a cell surface trans-membrane glycoprotein, present on endothelial cells and platelets. A smaller, functionally active form of thrombomodulin circulates in the plasma and is also found in urine. (In Haeberli, A., Human Protein Data, VCH Oub., N.Y., 1992). Peptides having homology to thrombomodulin are particularly desirable.
  • PR0269 polypeptides having homology to thrombomodulin, designated herein as PR0269 polypeptides.
  • ProcoUagen C-protemase enhancer protem binds to and enhances the activity of bone morphogenic protem
  • BMPl'Vprocollagen C-protemase (PCP). It plays a role m extracellular mat ⁇ x deposition. BMP1 protems may be used to induce bone and/or cartilage formation and m wound healmg and tissue repair Therefore, procoUagen C- protemase enhancer protein, BMP1 and protems havmg homology thereto, are of mterest to the scientific and medical communities
  • PR0287 polypeptides Having the identification and characterization of novel polypeptides havmg homology to procoUagen C-protemase enhancer protein precursor and procoUagen C-protemase enhancer protem, designated herein as PR0287 polypeptides. 16. PRQ214
  • Growth factors are molecular signals or mediators that enhances cell growth or proliferation, alone or in concert, by binding to specific cell surface receptors. However, there are other cellular reactions than only growth upon expression to growth factors. As a result, growth factors are better characterized as multifunctional and potent cellular regulators. Their biological effects include proliferation, chemotaxis and stimulation of extracellular matrix production. Growth factors can have both stimulatory and inhibitory effects.
  • TGF- ⁇ transforming growth factor ⁇
  • TGF- ⁇ is highly pleiotropic and can stimulate proliferation in some cells, especially connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and epithelial cells.
  • EGF epidermal growth factor
  • EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signaling and subsequent effects.
  • EGFR EGF receptor
  • the EGFR is expressed in neurons of the cerebral cortex, cerebellum, and hippocampus in addition to other regions of the central nervous system (CNS).
  • CNS central nervous system
  • EGF is also expressed in various regions of the CNS. Therefore, EGF acts not only on mitotic cells, but also on postmitotic neurons. In fact, many studies have indicated that EGF has neurotrophic or neuromodulatory effects on various types of neurons in the CNS. For example, EGF acts directly on cultured cerebral cortical and cerebellar neurons, enhancing neurite outgrowth and survival.
  • EGF also acts on other cell types, including septal cholinergic and mesencephalic dopaminergic neurons, indirectly through glial cells.
  • Evidence of the effects of EGF on neurons in the CNS is accumulating, but the mechanisms of action remain essentially unknown.
  • EGF-induced signaling in mitotic cells is better understood than in postmitotic neurons.
  • Studies of cloned pheochromocytoma PC 12 cells and cultured cerebral cortical neurons have suggested that the EGF-induced neurotrophic actions are mediated by sustained activation of the EGFR and mitogen-activated protein kinase (MAPK) in response to EGF.
  • MAPK mitogen-activated protein kinase
  • EGF is a multi-potent growth factor that acts upon various types of cells including mitotic cells and postmitotic neurons.
  • EGF is produced by the salivary and Brunner's glands of the gastrointestinal system, kidney, pancreas, thyroid gland, pituitary gland, and the nervous system, and is found in body fluids such as saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid, prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman, CR Peptides U: 653-663 (1991). EGF is mediated by its membrane specific receptor, which contains an intrinsic tyrosine kinase. Stoscheck
  • EGF CM et al, J. Cell Biochem. 3 135-152 (1986).
  • EGF is believed to function by binding to the extracellular portion of its receptor which induces a transmembrane signal that activates the intrinsic tyrosine kinase.
  • Purification and sequence analysis of the EGF-like domain has revealed the presence of six conserved cysteine residues which cross-bind to create three peptide loops, Savage CR et al., J. Biol. Chem. 248: 7669-7672 (1979).
  • Non isolated peptides having this motif include TGF-a, amphiregulin, schwannoma-derived growth factor (SDGF), heparin-binding EGF-like growth factors and certain virally encoded peptides (e.g., Vaccinia virus, Reisner AH, Nature 313: 801-803 (1985), Shope fibroma virus, Chang W., et al., Mol Cell Biol.
  • proteins include blood coagulation factors (factors VI, DC, X, XII, protein C, protein S, protein Z, tissue plasminogen activator, urokinase), extracellular matrix components (laminin, cytotactin, entactin), cell surface receptors (LDL receptor, thrombomodulin receptor) and immunity-related proteins (complement Clr, uromodulin).
  • EGF-like precursors are preserved through lower organisms as well as in mammalian cells.
  • a number of genes with developmental significance have been identified in invertebrates with EGF-like repeats.
  • the notch gene of Drosophila encodes 36 tandemly arranged 40 amino acid repeats which show homology to EGF, Wharton W et al, Cell 43: 557-581 (1985).
  • Hydropathy plots indicate a putative membrane spanning domain, with the EGF-related sequences being located on the extracellular side of the membrane.
  • Other homeotic genes with EGF-like repeats include Delta, 95F and 5ZD which were identified using probes based on Notch, and the nematode gene Lin-12 which encodes a putative receptor for a developmental signal transmitted between two specified cells.
  • EGF has been shown to have potential in the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions, Konturek, PC et al, Eur. J. Gastroenterol Hepatol.
  • EGF mediated blood coagulation, J. Stenflo et al, Blood 78(7): 1637-51 (1991).
  • EGF is also implicated various skin disease characterized by abnormal keratinocyte differentiation, e.g., psoriasis, epithelial cancers such as squamous cell carcinomas of the lung, epidermoid carcinoma of the vulva and gliomas. King, LE et al, Am. J. Med. Sci. 296: 154-158 (1988).
  • TGF- ⁇ supergene family or simply TGF- ⁇ superfamily, a group of secreted proteins, includes a large number of related growth and differentiation factors expressed in virtually all phyla.
  • Superfamily members bind to specific cell surface receptors that activate signal transduction mechanisms to elicit their multifunctional cytokine effects. Kolodziejczyk and Hall, Biochem. Cell. Biol.. 74: 299-314 (1996); Attisano and Wrana, Cvtokine Growth Factor Rev.. 7: 327-339 (1996); and Hill, Cellular Signaling. 8: 533-544 (1996).
  • TGF- ⁇ TGF- ⁇
  • Sporn and Roberts in Peptide Growth Factors and Their Receptors. Sporn and Roberts, eds. (Springer- Verlag: Berlin, 1990) pp. 419-472
  • the differentiation factors vgl Wedged et al , Nature. 325: 81-84 (1987)
  • DPP-C polypeptide Padgett et al , Nature. 325: 81-84 (1987)
  • the hormones activin and inhibin Mason et al , Nature. 318: 659-663 (1985); Mason et al , Growth Factors.
  • GDF mouse growth differentiation factor
  • GDF-3 and GDF-9 mouse growth differentiation factor
  • McPherron and Lee J. Biol. Chem.. 268: 3444-3449 (1993)
  • the mouse lefty/Stral mouse lefty/Stral
  • GDNF glial cell line-derived neurotrophic factor
  • neurturin Kotzbauer et al , Nature.
  • the subset BMP-2A and BMP-2B is approximately 75% homologous in sequence to DPP-C and may represent the mammalian equivalent of that protein.
  • the proteins of the TGF- ⁇ superfamily are disulfide-linked homo- or heterodimers encoded by larger precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N- terminal pro region of several hundred amino acids, a cleavage site (usually polybasic), and a shorter and more highly conserved C-terminal region.
  • This C-terminal region corresponds to the processed mature protein and contains approximately 100 amino acids with a characteristic cysteine motif, i.e. , the conservation of seven of the nine cysteine residues of TGF- ⁇ among all known family member .
  • the position of the cleavage site between the mature and pro regions varies among the family members, the C-terminus of all of the proteins is in the identical position, ending in the sequence Cys-X-Cys-X, but differing in every case from the TGF- ⁇ consensus C-terminus of Cys-Lys- Cys-Ser. Sporn and Roberts, 1990, supra.
  • TGF- ⁇ there are at least five forms of TGF- ⁇ currently identified, TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, TGF- ⁇ 4, and TGF- ⁇ 5.
  • the activated form of TGF- ⁇ l is a homodimer formed by dimerization of the carboxy-terminal 112 amino acids of a 390 amino acid precursor.
  • Recombinant TGF- ⁇ l has been cloned (Derynck et al. , Nature. 316:701-705 (1985)) and expressed in Chinese hamster ovary cells (Gentry et al, Mol. Cell. Biol.. 7: 3418-3427 (1987)). Additionally, recombinant human TGF- ⁇ 2 (deMartin et al , EMBO J..
  • TGF- ⁇ 2 has a precursor form of 414 amino acids and is also processed to a homodimer from the carboxy-terminal 112 amino acids that shares approximately 70% homology with the active form of TGF- ⁇ l nviarquardt et al. J. Biol. Chem.. 262: 12127 (1987)).
  • TGF- ⁇ 4 and TGF- ⁇ 5 were cloned from a chicken chondrocyte cDNA library (Jakowlew et al, Molec.
  • TGF- ⁇ associates non-covalently with the mature TGF- ⁇ dimer (Wakefield et al. , J. Biol.
  • TGF- ⁇ has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. TGF- ⁇ is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (Sporn and Roberts, supra).
  • EBAF TGF- ⁇ superfamily
  • the predicted protein sequence of EBAF showed a strong homology to the protein encoded by mouse lefty Istrc ⁇ of the TGF- ⁇ superfamily.
  • a motif search revealed that the predicted EBAF protein contains most of the cysteine residues which are conserved among the TGF- ⁇ -related proteins and which are necessary for the formation of the cysteine knot structure.
  • the EBAF sequence contains an additional cysteine residue, 12 amino acids upstream from the first conserved cysteine residue.
  • the only other family members known to contain an additional cysteine residue are TGF- ⁇ s, inhibins, and GDF-3.
  • EBAF similar to LEFTY, GDF-3/Vgr2, and GDF-9, lacks the cysteine residue that is known to form the intermolecular disulfide bond.
  • EBAF appears to be an additional member of the TGF- ⁇ superfamily with an unpaired cysteine residue that may not exist as a dimer.
  • hydrophobic contacts between the two monomer subunits may promote dimer formation.
  • Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Additional members of the TGF- ⁇ superfamily, such as those related to EBAF, are being searched for by industry and academics.
  • PR0317 polypeptides novel polypeptides having homology to EBAF.
  • mAbs tumor or cancer specific monoclonal antibodies
  • Such mAbs which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease.
  • Particular antigens are known to be associated with neoplastic diseases, such as colorectal cancer.
  • One particular antigen, the A33 antigen is expressed in more than 90% of primary or metastatic colon cancers as well as normal colon epithelium. Since colon cancer is a widespread disease, early diagnosis and treatment is an important medical goal.
  • Diagnosis and treatment of colon cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags. Radioactive gene, toxins and/or drug tagged mAbs can be used for treatment in situ with minimal patient description. mAbs can also be used to diagnose during the diagnosis and treatment of colon cancers. For example, when the serum levels of the A33 antigen are elevated in a patient, a drop of the levels after surgery would indicate the tumor resection was successful. On the other hand, a subsequent rise in serum A33 antigen levels after surgery would indicate that metastases of the original tumor may have formed or that new primary tumors may have appeared. Such monoclonal antibodies can be used in lieu of, or in conjunction with surgery and/or other chemotherapies.
  • U.S. P. 4,579,827 and U.S.S.N. 424,991 are directed to therapeutic administration of monoclonal antibodies, the latter of which relates to the application of anti-A33 mAb.
  • adenovirus-derived vectors have been proposed as a means of inserting antisense nucleic acids into tumors (U.S.P. 5,518,885).
  • antisense nucleic acids U.S.P. 5,518,885
  • the association of viral receptors with neoplastic tumors is not unexpected.
  • novel polypeptides having homology to certain cancer-associated antigens designated herein as PRO301 polypeptides.
  • Cholesterol uptake can have serious implications on one's health. Cholesterol uptake provides cells with most of the cholesterol they require for membrane synthesis. If this uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation of atherosclerotic plaques in blood vessel walls. Most cholesterol is transported in the blood bound to protein in the form of complexes known as low-density lipoproteins (LDLs). LDLs are endocytosed into cells via LDL receptor proteins. Therefore, LDL receptor proteins, and proteins having homology thereto, are of interest to the scientific and medical communities. Membrane-bound proteins and receptors can play an important role in the formation, differentiation and maintenance of multicellular organisms.
  • LDLs low-density lipoproteins
  • the LDL receptors are an example of membrane-bound proteins which are involved in the synthesis and formation of cell membranes, wherein the health of an individual is affected directly and indirectly by its function. Many membrane-bound proteins act as receptors such as the LDL receptor. These receptors can function to endocytose substrates or they can function as a receptor for a channel. Other membrane-bound proteins function as signals or antigens.
  • Membrane-bound proteins and receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents.
  • the membrane-bound proteins can also be employed for screening of potential peptide or small molecule regulators of the relevant receptor/ligand interaction.
  • the LDL receptor it is desirable to find molecules which enhance endocytosis so as to lower blood cholesterol levels and plaque formation.
  • Complement is a group of proteins found in the blood that are important in humoral immunity and inflammation. Complement proteins are sequentially activated by antigen-antibody complexes or by proteolytic enzymes. When activated, complement proteins kill bacteria and other microorganisms, affect vascular permeability, release histamine and attract white blood cells. Complement also enhances phagocytosis when bound to target cells.
  • the complement activation pathway is tightly regulated.
  • Deficiencies in the regulation of complement activation or in the complement proteins themselves may lead to immune-complex diseases, such as systemic lupus erythematosus, and may result in increased susceptibility to bacterial infection. In all cases, early detection of complement deficiency is desirable so that the patient can begin treatment. Thus, research efforts are currently directed toward identification of soluble and membrane proteins that regulate complement activation.
  • Factor H is a 150 kD soluble serum protein that interacts with complement protein C3b to accelerate the decay of C3 convertase and acts as a cofactor for Factor I-mediated cleavage of complement protein C4b.
  • Complement receptor type 1 is a 190-280 kD membrane bound protein found in mast cells and most blood cells.
  • CRl interacts with complement proteins C3b, C4b, and iC3b to accelerate dissociation of C3 convertases, acts as a cofactor for Factor I-mediated cleavage of C3b and C4b, and binds immune complexes and promotes their dissolution and phagocytosis.
  • Proteins which have homology to complement proteins are of particular interest to the medical and industrial communities. Often, proteins having homology to each other have similar function. It is also of interest when proteins having homology do not have similar functions, indicating that certain structural motifs identify information other than function, such as locality of function.
  • PR0222 polypeptides having homology to complement receptors, designated herein as PR0222 polypeptides.
  • oligosaccharide structures can be created through the differential activities of a smaller number of glycosyltransferases.
  • the diverse structures of oligosaccharides can be generated by transcription of relatively few gene products, which suggests that the oligosaccharides are a plausible mechanism by which is directed a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) on interacting cells have been described (J. Dodd & T.M. Jessel, . Neurosci. 5: 3278 (1985); L.J. Regan et al, Proc. Natl. Acad. Sci. USA 83: 2248 (1986); M.
  • selectins are cell adhesion molecules that are unified both structurally and functionally. Structurally, selectins are characterized by the inclusion of a domain with homology to a calcium-dependent lectin (C-lectins), an epidermal growth factor (egf)-like domain and several complement binding-like domains, Bevilacqua, M.P.
  • C-lectins calcium-dependent lectin
  • egf epidermal growth factor
  • selectins share the common property of their ability to mediate cell binding through interactions between their lectin domains and ceU surface carbohydrate Ugands (Brandley, B, et al , Cell 63., 861-863 (1990); Springer, T. and Lasky, L.A. , Nature 342, 19-197 (1991); Bevilacqua, M.P. and Nelson, R.M., J. Clin. Invest. 91 379-387 (1993) and Tedder et al, J. Exp. Med. 170: 123-133 (1989).
  • L-selectin also caUed peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1, gp90 MEL , gpl00 ME , gpl l0 MEL , MEL- 14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen
  • E-selectin LEC-CAM-2, LECAM-2, ELAM-1
  • P- selectin LEC-CAM-3, LECAM-3, GMP-140, PADGEM
  • L-selectin which comprises a lectin domain, performs its adhesive function by recognizing carbohydrate- containing ligands on endothelial cells. L-selectin is expressed on the surface of leukocytes, such as lymphocytes, neutrophUs, monocytes and eosinophils, and is involved with the trafficking of lymphocytes to peripheral lymphoid tissues (Gallatin et al, Nature 3Q3: 30-34 (1983)) and with acute neutrophil-medicated inflammatory responses (Watson, S.R., Nature 342: 164-167 (1991)).
  • leukocytes such as lymphocytes, neutrophUs, monocytes and eosinophils
  • the amino acid sequence of L-selectin and the encoding nucleic acid sequence are, for example, disclosed in U.S. patent No. 5,098,833 issued 24 March 1992.
  • L-selectin (LECAM-1) is particularly interesting because of its ability to block neutropl ⁇ l influx (Watson et al, Nature 342: 164-167 (1991). It is expressed in chronic lymphocytic leukemia cells which bind to HEV (Spertini et al, Nature 349: 691-694 (1991). It is also believed that HEV structures at sites of chronic inflammation are associated with the symptoms of diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis.
  • E-selectin is particularly interesting because of its transient expression on endothelial cells in response to E -1 or TNF. Bevilacqua et al., Science 243: 1160 (1989). The time course of this induced expression (2-8 h) suggests a role for this receptor in initial neutrophil induced extravasation in response to infection and injury. It has further been reported that anti-ELAM-1 antibody blocks the influx of neutrophUs in a primate asthma model and thus is beneficial for preventing airway obstruction resulting from the inflammatory response. Gundel et al, J. Clin. Invest. 88: 1407 (1991).
  • PR0234 polypeptides have homology to lectin proteins, herein designated as PR0234 polypeptides.
  • Some of the most important proteins involved in the above described regulation and modulation of cellular processes are the enzymes which regulate levels of protein phosphorylation in the cell.
  • the enzymes that catalyze these processes include the protem kinases, which function to phosphorylate various cellular proteins, and the protem phosphatases, which function to remove phosphate residues from various ceUular proteins. The balance of the level of protein phosphorylation in the cell is thus mediated by the relative activities of these two types of enzymes.
  • Protein phosphatases represent a growing famUy of enzymes that are found in many diverse forms, including both membrane-bound and soluble forms. While many protem phosphatases have been described, the functions of only a very few are beginning to be understood (Tonks, Semin. Cell Biol. 4:373-453 (1993) and Dixon, Recent Prog. Horm. Res. 51:405-414 (1996)). However, in general, it appears that many of the protein phosphatases function to modulate the positive or negative signals induced by various protein kinases. Therefore, it is likely that protem phosphatases play critical roles in numerous and diverse cellular processes.
  • Scavenger receptors are known to protect IgG molecules from catabolic degradation. Riechmann and Hollinger, Nature Biotechnology. 15:617 (1997). In particular, studies of the CH2 and CH3 domains have shown that specific sequences of these domains are important in dete ⁇ nrning the half-lives of antibodies. EUerson, et al. , J. Immunol.. 116: 510 (1976); Yasmeen, et al., J. Immunol. 116: 518 (1976; Pollock, et al., Eur. J. Immunol.. 20: 2021 (1990). Scavenger receptor protems and antibodies thereto are further reported in U.S. Patent No.
  • Oxygen free radicals and antioxidants appear to play an important role m the central nervous system after cerebral ischemia and reperfusion Moreover, cardiac injury, related to lschaemia and reperfusion has been reported to be caused by the action of free radicals Additionally, studies have reported that the redox state of the cell is a pivotal determinant of the fate of the ceUs Furthermore, reactive oxygen species have been reported to be cytotoxic, causing inflammatory disease, including tissue necrosis, organ failure, atherosclerosis, infertility, birth defects, premature aging, mutauons and mahgnancy Thus, the control of oxidation and reduction is important for a number of reasons mcludmg for control and prevention of strokes, heart attacks, oxidative stress and hypertension In this regard, reductases, and particularly, oxidoreductases, are of mterest Publications further descnbmg this subject matter mclude Kelsey, et al , Br J Cancer. 76(7) 8524 (1997), F ⁇
  • carboxypeptidase family of exopeptidases constitutes a diverse group of enzymes that hydrolyze carboxyl-terminal amide bonds m polypeptides, wherein a large number of mammalian tissues produce these enzymes
  • carboxypeptidase enzymes that have been identified to date exhibit rather strong cleavage specificities for certain ammo acids m polypeptides
  • carboxypeptidase enzymes have been identified which prefer lysine, arginine, se ⁇ ne or ammo acids with either aromatic or branched aliphatic side chains as substrates at the carboxyl terminus of the polypeptide
  • serme carboxypeptidases such am o acid specific enzymes have been identified from a vanety of different mammalian and non-mammalian organisms
  • the mammalian serme carboxypeptidase enzymes play important roles in many different biological processes including, for example, protem digestion, activation, inactivation, or modulation of peptide hormone activity, and alteration of the physical properties of proteins and enzymes
  • Plexin was first identified m Xenopus tadpole nervous system as a membrane glycoprotem which was shown to mediate cell adhesion via a homophihc bmdmg mechamsm m the presence of calcium ions
  • Strong evolutionary conservation between Xenopus, mouse and human homologs of plexin has been observed [Kaneyama et al , Biochem And Biophys Res Comm 226 524-529 (1996)]
  • efforts are currently bemg under taken to identify new, native proteins which are mvolved m ceU adhesion
  • PR0235 novel polypeptide which has homology to plexin
  • PRQ236 and PRQ262 ⁇ -galactosidase is a well known enzymatic protein which functions to hydrolyze ⁇ -galactoside molecules ⁇ -galactosidase has been employed for a variety of different applications, both in vitro and in vivo and has proven to be an extremely useful research tool As such, there is an interest in obtaimng novel polypeptides which exhibit homology to the ⁇ -galactosidase polypeptide Given the strong mterest in obtaining novel polypeptides having homology to ⁇ -galactosidase, efforts are currently bemg undertaken by both industry and proficient to identify new, native ⁇ -galactosidase homolog proteins.
  • Densin is a glycoprotein which has been isolated from the brain which has all the hallmarks of an adhesion molecule. It is highly concentrated at synaptic sites in the brain and is expressed prominently in dendritic processes in developing neurons. Densin has been characterized as a member of the O-linked sialoglycoproteins. Densin has relevance to medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell adhesion mechanisms in vivo, efforts are currently being under taken to identify new, native protems which are involved in synaptic machinery and cell adhesion. We describe herein the identification of novel polypeptides which have homology to densin, designated herem as PR0239 polypeptides.
  • Ebnerin is a ceU surface protein associated with von Ebner glands in mammals. Efforts are being undertaken by both industry and proficient to identify new, native cell surface receptor protems and specifically those which possess sequence homology to cell surface proteins such as ebnerin. Many of these efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel receptor proteins. We herein describe the identification of novel polypeptides having significant homology to the von Ebner's gland- associated protein ebnerin, designated herem as PR0257 polypeptides.
  • Fucosidases are enzymes that remove fucose residues from fucose containing proteoglycans. In some pathological conditions, such as cancer, rheumatoid arthritis, and diabetes, there is an abnormal fucosylation of serum proteins. Therefore, fucosidases, and proteins havmg homology to fucosidase, are of importance to the study and abrogation of these conditions. In particular, proteins havmg homology to the alpha-l-fiicosidase precursor are of interest. Fucosidases and fucosidase inhibitors are further described in U.S. Patent Nos.
  • PRO260 polypeptides Having a homology to fucosidases, designated herein as PRO260 polypeptides
  • CD44 is a ceU surface adhesion molecule mvolved m ceU-cell and cell-matrix interactions Hyaluromc acid, a component of the extracellular matrix is a major ligand Other ligands include collagen, fibronectin, laminin, chrondroitm sulfate, mucosal addressm, serglycm and osteopomn CD44 is also important m regulating cell traffic, lymph node homing, transmission of growth signals, and presentation of chemokmes and growth factors to travelmg cells
  • CD44 surface protems are associated with metastatic tumors and CD44 has been used as a marker for HIV infection Certain splice variants are associated with metastasis and poor prognosis of cancer patients Therefore, molecules having homology with CD44 are of particular interest, as their homology mdicates that they may have functions related to those functions of CD44 CD44 is further described in U S Patent Nos 5,506,119, 5,504,194 and 5, 108,904, Gerbe ⁇ ck,
  • Thioredoxins effect reduction-oxidation (redox) state
  • redox reactive oxygen species
  • the transc ⁇ ption factors, NF -kappa B and AP- 1 are regulated by redox state and are known to affect the expression of a large va ⁇ ety of genes thought to be mvolved m the pathogenesis of AIDS, cancer, atherosclerosis and diabetic complications
  • protems may also play a role in cellular antioxidant defense, and m pathological conditions mvolvmg oxidative stress such as stroke and inflammation m addition to having a role m apoptosis Therefore, thioredoxins, and proteins havmg homology thereto, are of interest to the scientific and medical communities
  • PRO270 polypeptides 34 PRQ271
  • the proteoglycan link protein is a protein which is Ultimately associated with various extracellular matrix proteins and more specifically with proteins such as collagen.
  • one primary component of collagen is a large proteoglycan called aggrecan.
  • aggrecan a large proteoglycan called aggrecan.
  • This molecule is retained by binding to the glycosaminoglycan hyaluronan through the amino terminal Gl globular domain of the core protein. This binding is stabilized by the proteoglycan link protein which is a protein that is also associated with other tissues containing hyaluronan binding proteoglycans such as versican.
  • Link protein has been identified as a potential target for autoimmune antibodies in individuals who suffer fromjuvenUe rheumatoid arthritis (see Guerassimov et al., . Rheumatology 24(5):959-964 (1997)). As such, there is strong interest in identifying novel protems havmg homology to link protem. We herein describe the identification and characterization of novel polypeptides having such homology, designated herein as PR0271 polypeptides.
  • Reticulocalbin is an endoplasmic reticular protein which may be involved in protein transport and luminal protem processing. Reticulocalbin resides in the lumen of the endopladsmic rerticulum, is known to bind calcium, and may be mvolved in a luminal retention mechamsm of the endoplasmic reticulum. It contains six domains of the EF-hand motif associated with high affinity calcium binding. We describe herein the identification and characterization of a novel polypeptide which has homology to the reticulocalbin protein, designated herem as PR0272.
  • CoUagen a naturaUy occurring protein, finds wide application in industry. Chemically hydrolyzed natural collagen can be denatured and renatured by heating and cooling to produce gelatin, which is used in photographic and medical, among other applications. Collagen has important properties such as the ability to form interchain aggregates having a conformation designated as a triple helix. We herein describe the identification and characterization of a novel polypeptide which has homology to portions of the collagen molecule, designated herem as PR0294.
  • the integrins comprise a supergene family of cell-surface glycoprotein receptors that promote cellular adhesion. Each ceU has numerous receptors that define its cell adhesive capabilities. Integrins are involved in a wide variety of interaction between ceUs and other cells or matrix components. The integrins are of particular importance in regulating movement and function of immune system cells The platelet Ilb/IIIA integrin complex is of particular importance in regulating platelet aggregation. A member of the integrin family, integrin ⁇ -6, is expressed on epitheUal ceUs and modulates epithelial inflammation. Another integrin, leucocyte-associated antigen-1 (LFA-1) is important in the adhesion of lymphocytes during an immune response.
  • LFA-1 leucocyte-associated antigen-1
  • the integrins are expressed as heterodimers of non- covalently associated alpha and beta subunits. Given the physiological importance of cell adhesion mechanisms in vivo, efforts are currentiy bemg under taken to identify new, native protems which are involved in cell adhesion. We describe herein the identification and characterization of a novel polypeptide which has homology to integrin, designated herem as PR0295.
  • Protein-protein interactions mclude receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protem-protem interactions can be more easUy manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of mterest to the scientific and medical community.
  • Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations.
  • the crystal structore of ribonuclease inhibitor protem has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protem acquires an unusual, nonglubular shape.
  • Protein-protein interactions mclude receptor and antigen complexes and signaling mechamsms As more is known about the structural and functional mechamsms underlying protein-protem interactions, protein-protem interactions can be more easUy manipulated to regulate the particular result of the protem-protem interaction Thus, the underlying mechamsms of protein-protem interactions are of mterest to the scientific and medical community
  • Leucme-nch repeats are short sequence motifs present m a number of proteins with diverse functions and cellular locations
  • the crystal structure of ribonuclease inhibitor protem has revealed that leucme-nch repeats correspond to beta-alpha structural units These umts are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protem acquires an unusual, nonglubular shape
  • Densin is a glycoprotem which has been isolated from the bram which has all the hallmarks of an adhesion molecule It is highly concentrated at synaptic sites in the bram and is expressed prominently m dendntic processes m developing neurons Densin has been characterized as a member of the O-linked sialoglycoproteins Densm has relevance to medically important processes such as regeneration Given the physiological importance of synaptic processes and cell adhesion mechamsms in vivo, efforts are currently bemg under taken to identify new, native proteins which are mvolved in synaptic machinery and cell adhesion Densm is further described m Kennedy, M B, Trends Neurosci (England), 20(6) 264 (1997) and Apperson, et al , J Neurosci .
  • PR0247 leucine rich repeat protems
  • protease enzymes are enzymatic proteins which are mvolved in a large number of very important biological processes in mammalian and non-mammalian organisms. Numerous different protease enzymes from a variety of different mammalian and non-mammalian organisms have been both identified and characterized. The mammalian protease enzymes play important roles in many different biological processes including, for example, protein digestion, activation, inactivation, or modulation of peptide hormone activity, and alteration of the physical properties of protems and enzymes.
  • the GLIP protein family has been characterized as comprising zinc-finger proteins which play important roles fn embryogenesis. These protems may function as transcriptional regulatory protems and are known to be amplified in a subset of human tumors.
  • Glioma pathogenesis protem is structurally related to a group of plant pathogenesis-related protems. It is highly expressed in glioblastoma. See US Pat. Nos. 5,582,981 (issued Dec. 10, 1996) and 5,322,801 (issued June 21, 1996), EUmgton, A.D. et al., Nature. 346:818 (1990), Grindley, J.C. et al., Dev. Biol..
  • Protein-protein interactions include receptor and antigen complexes and signaling mechanisms. As more is known about the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be more easUy manipulated to regulate the particular result of the protein-protein interaction. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community. All protems containing leucine-rich repeats are thought to be involved in protein-protein interactions.
  • Leucine-rich repeats are short sequence motifs present in a number of protems with diverse functions and cellular locations.
  • the crystal structore of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglubular shape.
  • IGF insulin like growth factor
  • ALS acid labile subunit of IGF
  • IGF insulin receptor gammase
  • SLIT prote n Another protem which has been reported to have leucine-rich repeats is the SLIT prote n which has been reported to be useful fn treating neuro-degenerative diseases such as Alzheimer's disease, nerve damage such as in
  • LIG-1 a membrane glycoprotem that is expressed specifically in glial cells m the mouse bram, and has leucme rich repeats and immunoglobulin-like domains. Suzuki, et al., J. Biol. Chem. (U.S.), 271(37):22522 (1996). Other stodies reporting on the biological functions of protems havmg leucme rich repeats mclude: Tayar, N., et al., Mol. Cell Endocrmol..
  • PR0335 novel polypeptides which have homology to protems of the leucine rich repeat superfamily, designated herein as PR0335, PR0331 and PR0326 polypeptides.
  • proteoglycans comprising a repeat characterized by an arrangement of conserved leucine residues (leucine-rich repeat motif) Uave diverse biological roles.
  • Certain proteoglycans such as biglycan, fibromodulin and decorin, are, for example, characterized by the presence of a leucine-rich repeat of about 24 ammo acids [Ruoslahti, Ann. Rev. CeU. Biol. 4229-255 (1988); Oldberg et al. , EMBO J. 8, 2601-2604 (1989)].
  • proteoglycans are believed to play a role in regulating extracellular matrix, cartilage or bone function.
  • the proteoglycan decorin binds to collagen type I and II and affects the rate of fibril formation. Fibromodulin also binds coUagen and delays fibril formation. Both fibromodulin and decorin inhibit the activity of transforming growth factor beta (TGF- ⁇ ) (U.S. Patent No. 5,583,103 issued December 10, 1996). TGF- ⁇ is known to play a key role in the induction of extracellular matrix and has been implicated in the development of fibrotic diseases, such as cancer and glomerulonephritis. Accordingly, proteoglycans have been proposed for the treatment of fibrotic cancer, based upon their abUity to inhibit TGF- ⁇ 's growth stimulating activity on the cancer cell.
  • TGF- ⁇ transforming growth factor beta
  • Proteoglycans have also been described as potentiaUy useful in the treatment of other proUferative pathologies, including rheumatoid arthritis, arteriosclerosis, adult respiratory distress syndrome, cirrhosis of the liver, fibrosis of the lungs, post-myocardial infarction, cardiac fibrosis, post-angioplasty restenosis, renal interstitial fibrosis and certain dermal fibrotic conditions, such as keloids and scarring, which might result from burn injuries, other invasive skin injuries, or cosmetic or reconstructive surgery (U.S. Patent No. 5,654,270, issued August 5, 1997).
  • PR0332 polypeptides which have homology to proteins of the leucine rich repeat superfamily, designated herein as PR0332 polypeptides.
  • Fibrillin microfibrils define the continuous elastic network of skin, and are present in dermis as microfibril bundles devoid of measurable elastin extending from the dermal-epithelial junction and as components of the thick elastic fibres present in the deep reticular dermis.
  • Marfan syndrome has been Unked to mutations which interfere with multimerization of fibrillin monomers or other connective tissue elements.
  • Fibulin-1 is a modular glycoprotein with amUio-terminal anaphlatoxin-like modules foUowed by nine epidermal growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl terrnini.
  • Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin.
  • fibrillin, fibulin, and molecules related thereto are of interest, particularly for the use of preventing skin from being damaged from aging, injuries or the sun, or for restoring skin damaged from same.
  • these molecules are generaUy of interest in the study of connective tissue and attachment molecules and related mechanisms.
  • Fibrillin, fibulin and related molecules are further described in Adams, et al. , J. Mol. Biol. , 272(2):226-36 (1997); Kielty and Shuttleworth, Microsc. Res. Tech.. 38(4)413-27 (1997); and Child. J. Card. Surg.. 12(2Supp.): 131-5 (1997).
  • mAbs tumor or cancer specific monoclonal antibodies
  • Such mAbs which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease.
  • Particular antigens are known to be associated with neoplastic diseases, such as colorectal and breast cancer. Since colon cancer is a widespread disease, early diagnosis and treatment is an important medical goal.
  • Diagnosis and treatment of cancer can be implemented using monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs can be used for treatment in situ with minimal patient description.
  • CEA Carcinoembryonic antigen
  • CEA is a glycoprotein found in human colon cancer and the digestive organs of a 2-6 month human embryos.
  • CEA is a known human tumor marker and is widely used in the diagnosis of neoplastic diseases, such as colon cancer. For example, when the serum levels of CEA are elevated in a patient, a drop of CEA levels after surgery would indicate the tumor resection was successful. On the other hand, a subsequent rise in serum CEA levels after surgery would indicate that metastases of the original tumor may have formed or that new primary tumors may have appeared.
  • CEA may also be a target for mAb, antisense nucleotides
  • Protein disulfide isomerase is an enzymatic protein which is mvolved in the promotion of correct refolding of proteins through the establishment of correct disulfide bond formation. Protein disulfide isomerase was initially identified based upon its abUity to catalyze the renatoration of reduced denatured RNAse (Goldberger et al. , J. Biol Chem. 239:1406-1410 (1964) and Epstein et al., Cold Spring Harbor Symp. Quant. Biol. 28:439449 (1963)).
  • Protein disulfide isomerase has been shown to be a resident enzyme of the endoplasmic reticulum which is retained in the endoplasmic reticulum via a -KDEL or -HDEL amino acid sequence at its C-terminus.
  • disulfide bond-forming enzymes and their potential uses in a number of different appMcations, for example in increasing the yield of correct refolding of recombinantly produced proteins, efforts are currently being undertaken by both industry and proficient to identify new, native proteins having homology to protein disulfide isomerase. Many of these efforts are focused on the screemng of mammalian recombinant DNA libraries to identify the coding sequences for novel protein disulfide isomerase homologs.
  • PR0268 a novel polypeptide having homology to protein disulfide isomerase
  • Prolyl 4-hydroxylase is an enzyme which functions to post-translationally hydroxylate proline residues at the Y position of the ammo acid sequence Gly-X-Y, which is a repeating three ammo acid sequence found in both coUagen and procoUagen. Hydroxylation of proline residues at the Y position of the Gly-X-Y amino acid triplet to form 4-hydroxyproline residues at those positions is required before newly synthesized collagen polypeptide chains may fold into their proper three-dimensional triple-heUcal conformation. If hydroxylation does not occur, synthesized collagen polypeptides remain non-helical, are poorly secreted by cells and cannot assemble into stable functional coUagen fibrils. Vuorio et al., Proc. Natl. Acad. Sci. USA 89:7467-7470 (1992). Prolyl 4-hydroxylase is comprised of at least two different polypeptide subunits, alpha and beta.
  • PRQ339 and PRO310 Fringe is a protein which specificaUy blocks serrate-mediated activation of notch in the dorsal compartment of die DrosophUa wing imaginal disc. Fleming, et al., Development. 124(15):2973-81 (1997). Therefore, fringe is of interest for both its role in development as well as its ability to regulate serrate, particularly senate's signaling abilities . Also of interest are novel polypeptides which may have a role in development and/or the regulation of serrate-like molecules. Of particular interest are novel polypeptides having homology to fringe as identified and described herein, designated herein as PR0339 and PRO310 polypeptides.
  • Lectins are a class of proteins comprising a region that binds carbohydrates specifically and non-covalently. Numerous lectins have been identified in higher animals, both membrane-bound and soluble, and have been implicated in a variety of cell-recognition phenomena and tumor metastasis.
  • lectins can be classified as either C-type (calcium-dependent) or S-type (thiol-dependent).
  • Lectins are thought to play a role in regulating cellular events that are initiated at the level of the plasma membrane.
  • plasma membrane associated molecules are involved in the activation of various subsets of lymphoid cells, e.g. T-lymphocytes, and it is known that cell surface molecules are responsible for activation of these cells and consequently their response during an immune reaction.
  • a particular group of cell adhesion molecules, selectins belong n the superfamily of C-type lectins.
  • This group includes L-selectin (peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1, gp90 ME , gplOO MEL , gpllO MEL , MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECAM-2, ELAM-1), and P-selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM).
  • pnHR peripheral lymph node homing receptor
  • LEC-CAM-1 peripheral lymph node homing receptor
  • LAM-1 LAM-1
  • gp90 ME gplOO MEL
  • gpllO MEL MEL-14 antigen
  • Leu-8 antigen Leu-8 antigen
  • TQ-1 antigen TQ-1 antigen
  • the structore of selectins consists of a C-type lectin (carbohydrate binding) domain, an epidermal growth factor-like (EGF-like) motif, and variable numbers of complement regulatory (CR) motifs.
  • Selectins are associated with leukocyte adhesion, e.g. the attachment of neutrophUs to venular endothelial cells adjacent to Uiflammation (E-selectin), or with the trafficking of lymphocytes from blood to secondary lymphoid organs, e.g. lymph nodes and Peyer's patches (L-selectin).
  • Another exemplary lectin is the cell-associated macrophage antigen, Mac-2 that is believed to be involved in cell adhesion and immune responses. Macrophages also express a lectin that recognizes Tn Ag, a human carcinoma-associated epitope.
  • Another C-type lectin is CD95 (Fas antigen/APO-1) that is an important mediator of immunologically relevant regulated or programmed cell death (apoptosis). "Apoptosis" is a non-necrotic cell death that takes place in metazoan animal cells following activation of an intrinsic cell suicide program. The cloning of Fas antigen is described in PCT pubUcation WO 91/10448, and European patent application EP510691.
  • the mature Fas molecule consists of 319 amino acids of which 157 are extracellular, 17 constitute the transmembrane domain, and 145 are intracellular. Increased levels of Fas expression at T cell surface have been associated with tumor cells and HIV- infected ceUs. Ligation of CD95 triggers apoptosis in the presence of interleukin-1 (IL-2).
  • IL-1 interleukin-1
  • C-type lectins also mclude receptors for oxidized low-density lipoprotein (LDL). This suggests a possible role in the pathogenesis of atherosclerosis.
  • LDL low-density lipoprotein
  • PR0244 polypeptides having homology to C-type lectins, designated herein as PR0244 polypeptides.
  • AppUcants have identified cDNA clones that encode novel polypeptides having homology to EGF, designated in the present application as "PR0211 " and "PR0217” polypeptides.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0211 or PR0217 polypeptide.
  • the isolated nucleic acid comprises DNA encoding EGF-like homologue PR0211 and PR0217 polypeptides of Fig. 2 (SEQ ID NO:2) and/or 4 (SEQ ID NO:4) indicated in Fig. 1 (SEQ ID NO: 1) and/or Fig. 3 (SEQ ID NO:3), respectively, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0211 and PR0217 EGF-like homologue PR0211 and PR0217 polypeptides.
  • the invention provides isolated native sequence PR0211 and PR0217 EGF- like homologue polypeptides, which in one embodiment, includes an amino acid sequence comprising residues: 1 to 353 of Fig. 2 (SEQ ID NO:2) or (2) 1 to 379 of Fig. 4 (SEQ ID NO: 4).
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PRO230" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PRO230 polypeptide having amino acid residues 1 through 467 of Figure 6 (SEQ ID NO: 12), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PRO230 polypeptide.
  • the invention provides isolated native sequence PRO230 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 through 467 of Figure 6 (SEQ ID NO: 12).
  • the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO: 13 ( Figure 7) which is herein designated as DNA20088.
  • EST expressed sequence tag
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0232 polypeptide havmg amino acid residues 1 to 114 of Figure 9 (SEQ ID NO:18), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0232 polypeptide.
  • the invention provides isolated native sequence PR0232 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 114 of Figure 9 (SEQ ID NO:18).
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0187 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0187 polypeptide of Figure 11 (SEQ ID NO:23), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides a nucleic acid comprising the coding sequence of Figure 10 (SEQ ID NO:22) or its complement.
  • the invention provides a nucleic acid of the full length protein of clone DNA27864-1155, deposited with the ATCC under accession number ATCC 209375, alternatively the coding sequence of clone DNA27864-1155, deposited under accession number ATCC 209375.
  • the invention provides isolated PR0187 polypeptide.
  • the invention provides isolated native sequence PRO 187 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 205 of Figure 11 (SEQ ID NO:23).
  • the invention provides a polypeptide encoded by the nucleic acid deposited under accession number ATCC 209375.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0265 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0265 polypeptide havmg amino acid residues 1 to 660 of Figure 13 (SEQ ID NO:28), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0265 polypeptide.
  • the invention provides isolated native sequence PR0265 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 660 of Figure 13 (SEQ ID NO:28).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0265 polypeptide.
  • PRQ219 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0219" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0219 polypeptide having amino acid residues 1 to 915 of Figure 15 (SEQ ID NO:34), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0219 polypeptide.
  • the invention provides isolated native sequence PR0219 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 915 of Figure 15 (SEQ ID NO:34).
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0246" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0246 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0246 polypeptide havmg ammo acid residues 1 to 390 of Figure 17 (SEQ ID NO:39), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0246 polypeptide.
  • the invention provides isolated native sequence PR0246 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 390 of Figure 17 (SEQ ID NO:39).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0246 polypeptide.
  • AppUcants have identified a cDNA clone that encodes a novel polypeptide havmg homology to CD97, EMRl and latrophilin, wherein the polypeptide is designated in the present application as "PR0228" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0228 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0228 polypeptide having amino acid residues 1 to 690 of Figure 19 (SEQ ID N049), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0228 polypeptide.
  • the invention provides isolated native sequence PR0228 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 690 of Figure 19 (SEQ ID NO:49).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0228 polypeptide.
  • the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO: 50, designated herein as DNA21951.
  • EST expressed sequence tag
  • AppUcants have identified a cDNA clone (DNA49435-1219) that encodes a novel polypeptide, designated in the present application as PR0533.
  • the invention provides an isolated nucleic acid molecule havmg at least about 80% sequence identity to (a) a DNA molecule encoding a PR0533 polypeptide comprising the sequence of amino acids 23 to 216 of Figure 22 (SEQ ID NO:59), or (b) the complement of the DNA molecule of (a).
  • the sequence identity preferably is about 85 % , more preferably about 90 % , most preferably about 95 % .
  • the isolated nucleic acid has 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 a polypeptide havmg amino acid residues 23 to 216 of Figure 22 (SEQ ID NO:59).
  • the highest degree of sequence identity occurs within the secreted portion (ammo acids 23 to 216 of Figure 22, SEQ ID NO:59).
  • the isolated nucleic acid molecule comprises DNA encoding a PR0533 polypeptide having amino acid residues 1 to 216 of Figure 22 (SEQ ID NO:59), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionaUy, under high stringency conditions.
  • the invention provides a nucleic acid of the full length protein of clone DNA49435-1219, deposited with the ATCC under accession number ATCC 209480.
  • the invention provides isolated PR0533 polypeptide.
  • the invention provides isolated native sequence PR0533 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 23 to 216 of Figure 22 (SEQ ID NO:59).
  • Native PR0533 polypeptides with or without the native signal sequence (amino acids 1 to 22 in Figure 22 (SEQ ID NO:59)), and with or without the initiating methionine are specifically included.
  • the invention provides a PR0533 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209480.
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0245" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0245 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0245 polypeptide havmg amino acid residues 1 to 312 of Fig. 24 (SEQ ID NO: 64), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0245 polypeptide.
  • the invention provides isolated native sequence PR0245 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 312 of Figure 24 (SEQ ID NO:64).
  • AppUcants have identified cDNA clones that each encode novel polypeptides, all havmg leucme rich repeats. These polypeptides are designated in the present application as PRO220, PR0221 and PR0227.
  • the invention provides isolated nucleic acid molecules comprising DNA respectively encoding PRO220, PR0221 and PR0227, respectively.
  • an isolated nucleic acid comprises DNA encoding the PRO220 polypeptide havmg amino acid residues 1 through 708 of Figure 26 (SEQ ID NO:69), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • an isolated nucleic acid comprises DNA encoding die PR0221 polypeptide having amino acid residues 1 through 259 of Figure 28 (SEQ ID NO:71), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • an isolated nucleic acid comprises DNA encodmg the PR0227 polypeptide having amino acid residues 1 through 620 of Figure 30 (SEQ ID NO:73), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PRO220, PR0221 and PR0227 polypeptides.
  • the isolated native sequence for the PRO220 polypeptide which in one embodiment, includes an amino acid sequence comprising residues 1 to 708 of Figure 26 (SEQ ID NO: 69).
  • the isolated native sequence for die PR0221 polypeptide which in one embodiment, includes an amino acid sequence comprising residues 1 to 259 of Figure 28 (SEQ ID NO:71).
  • the isolated native sequence for the PR0227 polypeptide which in one embodiment, includes an amino acid sequence comprising residues 1 to 620 of Figure 30 (SEQ ID NO:73). 12.
  • AppUcants have identified a cDNA clone that encodes a novel polypeptide having homology to CRTAM and poliovirus receptor precursors, wherein the polypeptide is designated in the present application as "PR0258" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encodmg a
  • the isolated nucleic acid comprises DNA encoding the PR0258 polypeptide having amino acid residues 1 to 398 of Figure 32 (SEQ ID NO:84), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0258 polypeptide.
  • the invention provides isolated native sequence PR0258 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 398 of Figure 32 (SEQ ID N0:84).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0258 polypeptide.
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0266" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0266 polypeptide havmg amino acid residues 1 to 696 of Figure 34 (SEQ ID NO:91), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionaUy, under high stringency conditions.
  • the invention provides isolated PR0266 polypeptide.
  • the invention provides isolated native sequence PR0266 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 696 of Figure 34 (SEQ ID NO:91).
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as PR0269.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encodmg the PR0269 polypeptide having amino acid residues 1 to 490 of Fig. 36 (SEQ ID NO:96), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0269 polypeptide.
  • the invention provides isolated native sequence PR0269 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 490 of Figure 36 (SEQ ID NO:96).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0269 polypeptide. 15. PRQ287
  • the invention provides an isolated nucleic acid molecule compnsmg DNA encodmg a PR0287 polypeptide
  • the isolated nucleic acid compnses DNA encodmg the PR0287 polypeptide havmg ammo acid residues 1 to 415 of Fig 38 (SEQ ID NO 104), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high st ⁇ ngency conditions
  • the invention provides isolated PR0287 polypeptide in particular, the mvention provides isolated native sequence PR0287 polypeptide, which m one embodiment, mcludes an ammo acid sequence compnsmg residues 1 to 415 of Figure 38 (SEQ ID NO 104)
  • the mvention provides an isolated nucleic acid molecule comprising DNA encodmg a PR0214 polypeptide.
  • the isolated nucleic acid comprises DNA encodmg the PR0214 polypeptide of Fig. 40 (SEQ ID NO: 109), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides a nucleic acid comprising the coding sequence of Fig. 39 (SEQ ID NO: 108) or its complement.
  • the mvention provides a nucleic acid of the full length protein of clone DNA32286-1191, deposited with ATCC under accession number ATCC 209385.
  • the invention provides isolated PR0214 polypeptide.
  • the invention provides isolated native sequence PR0214 polypeptide, which in one embodiment, includes an amino acid sequence comprising the residues of Figure 40 (SEQ ID NO: 109).
  • the invention provides a polypeptide encoded by the nucleic acid deposited under accession number ATCC 209385.
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, designated in the present application as "PR0317" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding PR0317 polypeptide.
  • die isolated nucleic acid comprises DNA (SEQ ID NO: 113) encoding PR0317 polypeptide having amino acid residues 1 to 366 of Fig. 42, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0317 polypeptide.
  • the mvention provides isolated native-sequence PR0317 polypeptide, which in one embodiment, mcludes an ammo acid sequence comprismg residues 1 to 366 of Figure 42 (SEQ ID NO: 114).
  • the invention supplies a method of detecting the presence of PR0317 in a sample, the method comprising: a) contacting a detectable anti-PR0317 antibody witii a sample suspected of containing PR0317; and b) detecting binding of the antibody to the sample; wherein the sample is selected from the group consisting of a body fluid, a tissue sample, a cell extract, and a cell culture medium.
  • a method for determining the presence of PR0317 mRNA in a sample comprising: a) contacting a sample suspected of containing PR0317 mRNA with a detectable nucleic acid probe that hybridizes under moderate to stringent conditions to PR0317 mRNA; and b) detecting hybridization of the probe to the sample.
  • the sample is a tissue sample and the detecting step is by in situ hybridization, or the sample is a cell extract and detection is by Northern analysis.
  • the invention provides a method for treating a PR0317-associated disorder comprising administering to a mammal an effective amount of the PR0317 polypeptide or a composition thereof containing a carrier, or with an effective amount of a PR0317 agonist or PR0317 antagonist, such as an antibody which binds specifically to PR0317.
  • AppUcants have identified a cDNA clone (DNA40628-1216) that encodes a novel polypeptide, designated in the present application as "PRO301 " .
  • the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encoding a PRO301 polypeptide comprising die sequence of amino acids 28 to 258 of Fig. 44 (SEQ ID NO: 119), or (b) the complement of the DNA molecule of (a).
  • the sequence identity preferably is about 85 % , more preferably about 90 % , most preferably about 95 % .
  • the isolated nucleic acid has 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 a polypeptide having amino acid residues 28 to 258 of Fig. 44 (SEQ ID NO:119).
  • the highest degree of sequence identity occurs within the extracellular domains (ammo acids 28 to 258 of Fig. -44, SEQ ID NO: 119).
  • the isolated nucleic acid molecule comprises DNA encoding a PRO301 polypeptide havmg amino acid residues 28 to 299 of Fig. 44 (SEQ ID NO.119), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionaUy, under high stringency conditions.
  • the invention provides a nucleic acid of the full length protein of clone DNA40628-1216, deposited with the ATCC under accession number ATCC 209432, alternatively the coding sequence of clone DNA40628-1216, deposited under accession number ATCC 209432.
  • the invention provides isolated PRO301 polypeptide.
  • the invention provides isolated native sequence PRO301 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising the extraceUular domain residues 28 to 258 of Figure 44 (SEQ ID NO: 119).
  • Native PRO301 polypeptides with or without the native signal sequence amino acids 1 to 27 in Figure 44 (SEQ ID NO: 119), and with or without the initiating methionine are specifically included.
  • sequences of the mvention may also comprise the transmembrane domain (residues 236 to about 258 in Figure 44; SEQ ID NO: 119) and/or the UitraceUular domain (about residue 259 to 299 in Figure 44; SEQ ID NO: 119).
  • the invention provides a PRO301 polypeptide encoded by die nucleic acid deposited under accession number ATCC 209432.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0224 polypeptide havmg amino acid residues 1 to 282 of Figure 46 (SEQ ID NO: 127), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0224 polypeptide.
  • the invention provides isolated native sequence PR0224 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 282 of Figure 46 (SEQ ID NO: 127).
  • PRQ222 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0222" .
  • the mvention provides an isolated nucleic acid molecule comprising DNA encodmg a PR0222 polypeptide.
  • die isolated nucleic acid comprises DNA encoding the PR0222 polypeptide having amino acid residues 1 to 490 of Fig. 48 (SEQ ID NO:132), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • die invention provides isolated PR0222 polypeptide.
  • the mvention provides isolated native sequence PR0222 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 490 of Figure 48 (SEQ ID NO: 132).
  • Applicants have identified a cDNA clone that encodes a novel lectin polypeptide molecule, designated in die present application as "PR0234" .
  • the invention provides an isolated nucleic acid encoding a novel lectin comprising DNA encoding a PR0234 polypeptide.
  • the isolated nucleic acid comprises the DNA encodmg PR0234 polypeptides having amino acid residues 1 to 382 of Fig. 50 (SEQ ID NO:137), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of Fig. 49 (SEQ ID NO: 136).
  • the invention provides isolated novel PR0234 polypeptides.
  • the invention provides isolated native sequence PR0234 polypeptide, which in one embodiment, includes an ammo acid sequence comprising residues 1 to 382 of Figure 50 (SEQ ID NO:137).
  • the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences.
  • Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to a putative acid phosphatase, wherein the polypeptide is designated in the present application as "PR0231 " .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0231 polypeptide.
  • me isolated nucleic acid comprises DNA encoding the PR0231 polypeptide havmg amino acid residues 1 to 428 of Fig. 52 (SEQ ID NO: 142), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0231 polypeptide.
  • the mvention provides isolated native sequence PR0231 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 428 of Figure 52 (SEQ ID NO: 142).
  • PRQ229 Applicants have identified a cDNA clone that encodes a novel polypeptide havmg homology to scavenger receptors wherein the polypeptide is designated in the present application as "PR0229" .
  • the mvention provides an isolated nucleic acid molecule comprising DNA encodmg a PR0229 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0229 polypeptide having ammo acid residues 1 to 347 of Figure 54 (SEQ ID NO: 148), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0229 polypeptide.
  • the invention provides isolated native sequence PR0229 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 347 of Figure 54 (SEQ ID NO: 148).
  • AppUcants have identified a cDNA clone that encodes a novel polypeptide having homology to reductase, wherein the polypeptide is designated in the present application as "PR0238" .
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a PR0238 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0238 polypeptide havmg amino acid residues 1 to 310 of Figure 56 (SEQ ID NO:153), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0238 polypeptide.
  • the invention provides isolated native sequence PR0238 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 310 of Figure 56 (SEQ ID NO: 153). 25. PRQ233
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0233 polypeptide havmg amino acid residues 1 to 300 of Figure 58 (SEQ ID NO: 159), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0233 polypeptide.
  • the invention provides isolated native sequence PR0233 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 300 of Figure 58 (SEQ ID NO: 159).
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0223 polypeptide having amino acid residues 1 to 476 of Figure 60 (SEQ ID NO: 164), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0223 polypeptide.
  • the mvention provides isolated native sequence PR0223 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 476 of Figure 60 (SEQ ID NO: 164).
  • PRQ235 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated hi the present application as "PR0235".
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0235 polypeptide having amino acid residues 1 to 552 of Figure 62 (SEQ ID NO: 170), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0235 polypeptide.
  • the invention provides isolated native sequence PR0235 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 552 of Figure 62 (SEQ ID NO: 170). 28. PRQ236 and PRQ262
  • AppUcants have identified cDNA clones that encode novel polypeptides havmg homology to ⁇ -galactosidase, wherein those polypeptides are designated in the present application as "PR0236" and "PR0262” .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0236 polypeptide having amino acid residues 1 to 636 of Figure 64 (SEQ ID NO: 175), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • me invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0262 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0262 polypeptide havmg amino acid residues 1 to 654 of Figure 66 (SEQ ID NO: 177), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0236 polypeptide.
  • the mvention provides isolated native sequence PR0236 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 636 of Figure 64 (SEQ ID NO: 175).
  • the mvention provides isolated PR0262 polypeptide.
  • the invention provides isolated native sequence PR0262 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 654 of Figure 66 (SEQ ID NO: 177).
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0239 polypeptide having amino acid residues 1 to 501 of Figure 68 (SEQ ID NO: 185), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0239 polypeptide.
  • the invention provides isolated native sequence PR0239 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 501 of Figure 68 (SEQ ID NO: 185).
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0257 polypeptide having amino acid residues 1 to 607 of Figure 70 (SEQ ID NO:190), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0257 polypeptide.
  • the invention provides isolated native sequence PR0257 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 607 of Figure 70 (SEQ ID NO: 190).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0257 polypeptide.
  • the invention provides an isolated nucleic acid molecule comprising DNA encodmg a
  • the isolated nucleic acid comprises DNA encoding the PRO260 polypeptide having amino acid residues 1 to 467 of Figure 72 (SEQ ID NO: 195), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PRO260 polypeptide.
  • the invention provides isolated native sequence PRO260 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 467 of Figure 72 (SEQ ID NO: 195).
  • PRQ263 Applicants have identified a cDNA clone that encodes a novel polypeptide havmg homology to CD44 antigen, wherein the polypeptide is designated in die present application as "PR0263" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0263 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0263 polypeptide havmg amino acid residues 1 to 322 of Figure 74 (SEQ ID NO:201), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0263 polypeptide.
  • the invention provides isolated native sequence PR0263 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 322 of Figure 74 (SEQ ID NO:201).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0263 polypeptide.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA whivch mcludes the sequence encodmg the PRO270 polypeptide havmg amino acid residues 1 to 296 of Fig. 76 (SEQ ID NO:207), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PRO270 polypeptide.
  • the mvention provides isolated native sequence PRO270 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 296 of Figure 76 (SEQ ID NO:207).
  • Applicants have identified a cDNA clone that encodes a novel polypeptide having homology to the proteoglycan link protein, wherein the polypeptide is designated m the present application as "PR0271 " .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0271 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0271 polypeptide havmg amino acid residues 1 to 360 of Figure 78 (SEQ ID NO:213), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0271 polypeptide.
  • the invention provides isolated native sequence PR0271 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 360 of Figure 78 (SEQ ID NO:213).
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0272 polypeptide having amino acid residues 1 to 328 of Figure 80 (SEQ ID NO:221), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0272 polypeptide.
  • the invention provides isolated native sequence PR0272 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 328 of Figure 80 (SEQ ID NO:211).
  • Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0294" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0294 polypeptide havmg ammo acid residues 1 to 550 of Figure 82 (SEQ ID NO:227), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0294 polypeptide.
  • the invention provides isolated native sequence PR0294 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 550 of Figure 82 (SEQ ID NO:227).
  • PRQ295 Applicants have identified a cDNA clone that encodes a novel polypeptide, wherein the polypeptide is designated in the present application as "PR0295" .
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • die isolated nucleic acid comprises DNA encoding the PR0295 polypeptide having amino acid residues 1 to 350 of Figure 84 (SEQ ID NO:236), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0295 polypeptide.
  • the invention provides isolated native sequence PR0295 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 350 of Figure 84 (SEQ ID NO:236).
  • Applicants have identified a cDNA clone that encodes a novel human neuronal leucine rich repeat polypeptide, wherein the polypeptide is designated in the present application as "PR0293" .
  • die invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0293 polypeptide.
  • the isolated nucleic acid comprises DNA encoding d e PR0293 polypeptide having amino acid residues 1 to 713 of Figure 86 (SEQ ID NO:245), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0293 polypeptide.
  • the invention provides isolated native sequence PR0293 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 713 of Figure 86 (SEQ ID NO:245).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0293 polypeptide.
  • PRQ247 Applicants have identified a cDNA clone that encodes a novel polypeptide havmg leucme rich repeats wherein the polypeptide is designated in the present application as "PR0247" .
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encodmg the PR0247 polypeptide having amino acid residues 1 to 546 of Figure 88 (SEQ ID NO:250), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0247 polypeptide.
  • the invention provides isolated native sequence PR0247 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 546 of Figure 88 (SEQ ID NO:250).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0247 polypeptide.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO302 polypeptide.
  • me isolated nucleic acid comprises DNA encoding the PRO302 polypeptide having amino acid residues 1 to 452 of Figure 90 (SEQ ID NO:255), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO303 polypeptide.
  • die isolated nucleic acid comprises DNA encodmg the PRO303 polypeptide having amino acid residues 1 to 314 of Figure 92 (SEQ ID NO:257), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO304 polypeptide.
  • the isolated nucleic acid comprises DNA encodmg the PRO304 polypeptide having amino acid residues 1 to 556 of Figure 94 (SEQ ID NO:259), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • me invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO307 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PRO307 polypeptide having amino acid residues 1 to 383 of Figure 96 (SEQ ID NO:261), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides an isolated nucleic acid molecule comprising DNA encodmg a PR0343 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0343 polypeptide having amino acid residues 1 to 317 of Figure 98 (SEQ ID NO:263), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PRO302 polypeptide.
  • the invention provides isolated native sequence PRO302 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 452 of Figure 90 (SEQ ID NO:255).
  • the invention provides isolated PRO303 polypeptide.
  • the invention provides isolated native sequence PRO303 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 314 of Figure 92 (SEQ ID NO:257).
  • the invention provides isolated PRO304 polypeptide.
  • the invention provides isolated native sequence PRO304 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 556 of Figure 94 (SEQ ID NO:259).
  • the invention provides isolated PRO307 polypeptide.
  • the invention provides isolated native sequence PRO307 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 383 of Figure 96 (SEQ ID NO:261).
  • the invention provides isolated PR0343 polypeptide.
  • the mvention provides isolated native sequence PR0343 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 317 of Figure 98 (SEQ ID NO:263).
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a
  • the isolated nucleic acid comprises DNA encoding the PR0328 polypeptide havmg ammo acid residues 1 to 463 of Figure 100 (SEQ ID NO:285), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0328 polypeptide.
  • the invention provides isolated native sequence PR0328 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 463 of Figure 100 (SEQ ID NO:285).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PRO306 polypeptide.
  • AppUcants have identified three cDNA clones that respectively encode three novel polypeptides, each having leucme rich repeats and homology to LIG-1 and ALS. These polypeptides are designated in the present application as PR0335, PR0331 and PR0326, respectively.
  • die mvention provides three isolated nucleic acid molecules comprising DNA respectively encoding PR0335, PR0331 and PR0326, respectively.
  • an isolated nucleic acid comprising DNA encoding the PR0335 polypeptide having ammo acid residues 1 through 1059 of Figure 102 (SEQ ID NO:290), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. Also provided herein is an isolated nucleic acid comprises DNA encodmg die PR0331 polypeptide havmg amino acid residues 1 through 640 of Figure 104 (SEQ ID NO:292), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • an isolated nucleic acid comprises DNA encoding the PR0326 polypeptide having amino acid residues 1 through 1119 of Figure 106 (SEQ ID NO:294), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0335, PR0331 and PR0326 polypeptides or extracellular domains thereof.
  • the invention provides isolated native sequence for the PR0335 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 through 1059 of Figure 102 (SEQ ID NO:290).
  • the isolated native sequence for the PR0331 polypeptide which in one embodiment, includes an amino acid sequence comprising residues 1 through 640 of Figure 104 (SEQ ID NO:292).
  • the isolated native sequence for the PR0326 polypeptide which in one embodiment, includes an amino acid sequence comprising residues 1 through 1119 of Figure 106 (SEQ ID NO:294).
  • the mvention provides an isolated nucleic acid molecule comprising DNA having at least about 80% sequence identity to (a) a DNA molecule encodmg a PR0358 polypeptide comprising the sequence of amino acids 49 to 642 of Fig. 108 (SEQ ID NO:310), or (b) the complement of the DNA molecule of (a).
  • the sequence identity preferably is about 85%, more preferably about 90%, most preferably about 95%.
  • me isolated nucleic acid has 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 a polypeptide having amino acid residues 1 to 642 of Fig. 108 (SEQ ID NO:310).
  • the highest degree of sequence identity occurs within the leucme-rich repeat domains (amino acids 116 to 624 of Fig. 108, SEQ ID NO:310).
  • the isolated nucleic acid molecule comprises DNA encodmg a PR0332 polypeptide havmg amino acid residues 49 to 642 of Fig. 108 (SEQ ID NO:310), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • me invention provides isolated PR0332 polypeptides.
  • the invention provides isolated native sequence PR0332 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 49 to 624 of Figure 108 (SEQ ID NO:310).
  • Native PR0332 polypeptides with or without the native signal sequence amino acids 1 to 48 in Figure 108, SEQ ID NO:310, and with or without the initiating methionine are specifically included.
  • AppUcants have identified a cDNA clone tiiat encodes a novel polypeptide havmg homology to fibulin and fibrillin, wherein the polypeptide is designated in the present application as "PR0334".
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0334 polypeptide.
  • the isolated nucleic acid comprises DNA encoding die PR0334 polypeptide having amino acid residues 1 to 509 of Figure 110 (SEQ ID NO:315), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the mvention provides isolated PR0334 polypeptide.
  • the invention provides isolated native sequence PR0334 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 509 of Figure 110 (SEQ ID NO:315).
  • AppUcants have identified a cDNA clone (DNA44167-1243) that encodes a novel polypeptide, designated in die present application as "PR0346.”
  • the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity to (a) a DNA molecule encodmg a PR0346 polypeptide comprising the sequence of amino acids 19 to 339 of Fig. 112 (SEQ ID NO: 320), or (b) the complement of the DNA molecule of (a).
  • the sequence identity preferably is about 85 % , more preferably about 90% , most preferably about 95 % .
  • the isolated nucleic acid has 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 a polypeptide havmg amino acid residues 19 to 339 of Fig. 112 (SEQ ID NO:320).
  • the highest degree of sequence identity occurs within die extracellular domains (amino acids 19 to 339 of Fig. 112, SEQ ID NO.320).
  • die polypeptide by which me homology is measured comprises the residues 1-339, 19-360 or 19450 of Fig. 112, SEQ ID NO:320).
  • the isolated nucleic acid molecule comprises DNA encoding a PR0346 polypeptide havmg ammo acid residues 19 to 339 of Fig. 112 (SEQ ID NO:320), alternatively residues 1-339, 19-360 or 19450 of Fig.
  • the mvention provides a nucleic acid of the full length protein of clone DNA44167-1243, deposited with e ATCC under accession number ATCC 209434, alternatively the coding sequence of clone DNA44167-1243, deposited under accession number ATCC 209434.
  • me invention provides isolated PR0346 polypeptide.
  • the invention provides isolated native sequence PR0346 polypeptide, which hi one embodiment, includes an amino acid sequence comprising residues 19 to 339 of Figure 112 (SEQ ID NO: 320).
  • Native PR0346 polypeptides with or without the native signal sequence (residues 1 to 18 in Figure 112 (SEQ ID NO:320), with or without the initiating methionine, with or without the transmembrane domain (residues 340 to 360) and with or without the mtraceUular domain (residues 361 to 450) are specificaUy included.
  • the invention provides a PR0346 polypeptide encoded by the nucleic acid deposited under accession number ATCC 209434.
  • Applicants have identified a cDNA clone that encodes a novel polypeptide havmg homology to protein disulfide isomerase, wherein the polypeptide is designated in the present application as "PR0268".
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PR0268 polypeptide.
  • the isolated nucleic acid comprises DNA encoding the PR0268 polypeptide havmg amino acid residues 1 to 280 of Figure 114 (SEQ ID NO:325), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0268 polypeptide.
  • the invention provides isolated native sequence PR0268 polypeptide, which in one embodiment, includes an amino acid sequence compnsmg residues 1 to 280 of Figure 114 (SEQ ID NO:325).
  • An additional embodiment of the present invention is directed to an isolated extracellular domain of a PR0268 polypeptide.
  • cDNA clone tiiat encodes a novel polypeptide havmg homology to the alpha subunit of prolyl 4-hydroxylase, wherein the polypeptide is designated in the present application as "PRO330".
  • the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO330 polypeptide.
  • the isolated nucleic acid comprises DNA encoding die PRO330 polypeptide having amino acid residues 1 to 533 of Figure 116 (SEQ ID NO:332), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PRO330 polypeptide.
  • the invention provides isolated native sequence PRO330 polypeptide, which in one embodiment, mcludes an amino acid sequence comprising residues 1 to 533 of Figure 116 (SEQ ID NO:332).
  • each clone encodes a novel polypeptide having homology to fringe, wherein the polypeptides are designated in d e present application as "PR0339" and "PRO310".
  • the mvention provides isolated nucleic acid molecules comprising DNA encodmg a
  • me isolated nucleic acid comprises DNA encoding me
  • PR0339 polypeptide having amino acid residues 1 to 772 of Figure 118 (SEQ ID NO:339), or is complementary to such encodmg nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the isolated nucleic acid comprises DNA encoding me PRO310 polypeptide having amino acid residues 1 to 318 of Figure 120 (SEQ ID NO:341), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionaUy, under high stringency conditions.
  • the invention provides isolated PR0339 as well as isolated PRO310 polypeptides.
  • the invention provides isolated native sequence PR0339 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 772 of Figure 118 (SEQ ID NO:339).
  • the invention further provides isolated native sequence PRO310 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 318 of Figure 120 (SEQ ID NO:341).
  • the mvention provides an isolated nucleic acid molecule comprising DNA encoding PR0244 polypeptide.
  • the isolated nucleic acid comprises DNA encoding PR0244 polypeptide havmg amino acid residues 1 to 219 of Fig. 122 (SEQ ID NO:377), or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
  • the invention provides isolated PR0244 polypeptide.
  • the invention provides isolated native sequence PR0244 polypeptide, which in one embodiment, includes an amino acid sequence comprising residues 1 to 219 of Figure 122 (SEQ ID NO:377).
  • the invention provides vectors comprising DNA encoding any of the above or below described polypeptides.
  • a host cell comprising any such vector is also provided.
  • the host cells may be CHO cells, E. coli, or yeast.
  • a process for producing any of the above or below described polypeptides is further provided and comprises cultoring host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
  • the invention provides chimeric molecules comprising any of the above or below described polypeptides fused to a heterologous polypeptide or amino acid sequence.
  • An example of such a chimeric molecule comprises any of the above or below described polypeptides fused to an epitope tag sequence or a Fc region of an immunoglobulin.
  • the mvention provides an antibody which specifically binds to any of the above or below described polypeptides.
  • die antibody is a monoclonal antibody.
  • the mvention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences, wherein those probes may be derived from any of the above or below described nucleotide sequences.
  • Figure 1 shows a nucleotide sequence (SEQ ID NO:l) of a native sequence PR0211 cDNA, wherein SEQ ID NO:l
  • ID NO:l is a clone designated herein as "UNQ185” and/or "DNA32292-1131".
  • Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding sequence of SEQ ID NO: 1 shown in Figure 1.
  • Figure 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequence PR0217 cDNA, wherein SEQ ID NO:3 is a clone designated herein as "UNQ191 " and/or "DNA33094-1131 " .
  • Figure 4 shows die amino acid sequence (SEQ ID NO:4) derived from the coding sequence of SEQ ID NO:3 shown Ui Figure 3.
  • Figure 5 shows a nucleotide sequence (SEQ ID NO: 11) of a native sequence PRO230 cDNA, wherein SEQ ID NO: 11 is a clone designated herein as "UNQ204" and/or "DNA33223-1136".
  • Figure 6 shows the amino acid sequence (SEQ ID NO: 12) derived from the coding sequence of SEQ ID NO:
  • Figure 7 shows a nucleotide sequence designated herein as DNA20088 (SEQ ID NO: 13).
  • Figure 8 shows a nucleotide sequence (SEQ ID NO: 17) of a native sequence PR0232 cDNA, wherein SEQ ID NO: 17 is a clone designated herein as "UNQ206" and/or "DNA34435-1140" .
  • Figure 9 shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 8.
  • Figure 10 shows a nucleotide sequence (SEQ ED NO:22) of a native sequence PR0187 cDNA, wherein SEQ ID NO:22 is a clone designated herein as "UNQ161 " and/or "DNA27864-1155 " .
  • Figure 11 shows the amino acid sequence (SEQ ID NO:23) derived from the coding sequence of SEQ ID NO:22 shown in Figure 10.
  • Figure 12 shows a nucleotide sequence (SEQ ID NO:27) of a native sequence PR0265 cDNA, wherein SEQ ID NO:27 is a clone designated herein as "UNQ232" and/or "DNA36350-1158" .
  • Figure 13 shows the amino acid sequence (SEQ ID NO:28) derived from the coding sequence of SEQ ID NO:
  • Figures 14A-B show a nucleotide sequence (SEQ ID NO:33) of a native sequence PR0219 cDNA, wherein SEQ ID NO:33 is a clone designated herein as "UNQ193" and/or "DNA32290-1164" .
  • Figure 15 shows the amino acid sequence (SEQ ID NO: 34) derived from the coding sequence of SEQ ID NO:33 shown in Figures 14A-B.
  • Figure 16 shows a nucleotide sequence (SEQ ID NO:38) of a native sequence PR0246 cDNA, wherein SEQ ID NO:38 is a clone designated herein as "UNQ220" and/or "DNA35639-1172" .
  • Figure 17 shows the amino acid sequence (SEQ ID NO:39) derived from the coding sequence of SEQ ID NO:38 shown n Figure 16.
  • Figure 18 shows a nucleotide sequence (SEQ ED NO:48) of a native sequence PR0228 cDNA, wherein SEQ
  • ID NO:48 is a clone designated herein as "UNQ202" and/or "DNA33092-1202" .
  • Figure 19 shows the amino acid sequence (SEQ ID NO:49) derived from the coding sequence of SEQ ID NO:48 shown in Figure 18.
  • Figure 20 shows a nucleotide sequence designated herein as DNA21951 (SEQ ID NO:50).
  • Figure 21 shows a nucleotide sequence (SEQ ID NO:58) of a native sequence PR0533 cDNA, wherein SEQ
  • ID NO:58 is a clone designated herein as "UNQ344" and/or "DNA49435-1219".
  • Figure 22 shows the ammo acid sequence (SEQ ID NO: 59) derived from the coding sequence of SEQ ID NO:58 shown in Figure 21.
  • Figure 23 shows a nucleotide sequence (SEQ ID NO:63) of a native sequence PR0245 cDNA, wherein SEQ ID NO:63 is a clone designated herein as "UNQ219" and/or "DNA35638-1141" .
  • Figure 24 shows the amino acid sequence (SEQ ID NO:64) derived from the coding sequence of SEQ ID NO: 63 shown in Figure 23.
  • Figure 25 shows a nucleotide sequence (SEQ ID NO:68) of a native sequence PRO220 cDNA, wherein SEQ ID NO:68 is a clone designated herein as "UNQ194" and/or "DNA32298-1132” .
  • Figure 26 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ID NO:
  • FIG. 70 shows a nucleotide sequence (SEQ ID NO:70) of a native sequence PR0221 cDNA. wherein SEQ ID NO:70 is a clone designated herein as "UNQ195" and/or "DNA33089-1132" .
  • Figure 28 shows the ammo acid sequence (SEQ ID NO:71) derived from the coding sequence of SEQ ID NO:70 shown in Figure 27.
  • Figure 29 shows a nucleotide sequence (SEQ ID NO:72) of a native sequence PR0227 cDNA, wherein SEQ ID NO:72 is a clone designated herein as "UNQ201 " and/or "DNA33786-1132" .
  • Figure 30 shows the ammo acid sequence (SEQ ID NO:73) derived from the coding sequence of SEQ ID NO: 72 shown in Figure 29.
  • Figure 31 shows a nucleotide sequence (SEQ ID NO:83) of a native sequence PR0258 cDNA, wherein SEQ ID NO:83 is a clone designated herein as "UNQ225" and/or "DNA35918-1174" .
  • Figure 32 shows the amino acid sequence (SEQ ID NO: 84) derived from the coding sequence of SEQ ID NO:
  • Figure 33 shows a nucleotide sequence (SEQ ID NO: 90) of a native sequence PR0266 cDNA, wherein SEQ ID NO:90 is a clone designated herein as "UNQ233" and/or "DNA37150-1178" .
  • Figure 34 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:90 shown in Figure 33.
  • Figure 35 shows a nucleotide sequence (SEQ ID NO:95) of a native sequence PR0269 cDNA, wherein SEQ ID NO:95 is a clone designated herein as "UNQ236" and/or "DNA38260-1180".
  • Figure 36 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID NO: 95 shown in Figure 35.
  • Figure 37 shows a nucleotide sequence (SEQ ID NO: 103) of a native sequence PR0287 cDNA, wherein
  • SEQ ID NO:103 is a clone designated herein as "UNQ250" and/or “DNA39969-1185” .
  • Figure 38 shows the amino acid sequence (SEQ ID NO: 104) derived from the coding sequence of SEQ ID NO:103 shown in Figure 37.
  • Figure 39 shows a nucleotide sequence (SEQ ID NO: 108) of a native sequence PR0214 cDNA, wherein SEQ ID NO:108 is a clone designated herein as "UNQ188" and/or “DNA32286-1191 " .
  • Figure -40 shows die amino acid sequence (SEQ ID NO: 109) derived from the coding sequence of SEQ ID NO: 108 shown in Figure 39.
  • Figure 41 shows a nucleotide sequence (SEQ ID NO: 113) of a native sequence PR0317 cDNA, wherein SEQ ID NO: 113 is a clone designated herein as "UNQ278" and/or "DNA33461-1199".
  • Figure 42 shows the amino acid sequence (SEQ ID NO: 114) derived from the coding sequence of SEQ ID NO:
  • Figure 43 shows a nucleotide sequence (SEQ ID NO: 118) of a native sequence PRO301 cDNA, wherein SEQ ID NO:118 is a clone designated herein as "UNQ264" and/or "DNA40628-1216" .
  • Figure 44 shows the amino acid sequence (SEQ ID NO: 119) derived from the coding sequence of SEQ ID NO:118 shown hi Figure 43.
  • Figure 45 shows a nucleotide sequence (SEQ ID NO: 126) of a native sequence PR0224 cDNA, wherein SEQ ID NO:126 is a clone designated herein as "UNQ198" and/or "DNA33221-1133” .
  • Figure 46 shows the amino acid sequence (SEQ ID NO: 127) derived from the coding sequence of SEQ ID NO: 126 shown in Figure 45.
  • Figure 47 shows a nucleotide sequence (SEQ ID NO: 131) of a native sequence PR0222 cDNA, wherein SEQ ID NO:131 is a clone designated herein as "UNQ196" and/or "DNA33107-1135” .
  • Figure 48 shows die amino acid sequence (SEQ ID NO: 132) derived from the coding sequence of SEQ ID NO : 131 shown in Figure 47.
  • Figure 49 shows a nucleotide sequence (SEQ ID NO: 136) of a native sequence PR0234 cDNA, wherein SEQ ID NO: 136 is a clone designated herein as "UNQ208" and/or "DNA35557-1137".
  • Figure 50 shows the amino acid sequence (SEQ ID NO: 137) derived from the coding sequence of SEQ ID NO: 136 shown in Figure 49.
  • Figure 51 shows a nucleotide sequence (SEQ ID NO: 141) of a native sequence PR0231 cDNA, wherein
  • SEQ ID NO: 141 is a clone designated herein as "UNQ205" and/or “DNA34434-1139” .
  • Figure 52 shows die amino acid sequence (SEQ ID NO: 142) derived from the coding sequence of SEQ ID NO: 141 shown in Figure 51.
  • Figure 53 shows a nucleotide sequence (SEQ ID NO: 147) of a native sequence PR0229 cDNA, wherein SEQ ID NO: 147 is a clone designated herein as "UNQ203 " and/or "DNA33100-1159” .
  • Figure 54 shows the amino acid sequence (SEQ ID NO: 148) derived from the coding sequence of SEQ ID NO: 147 shown in Figure 53.
  • Figure 55 shows a nucleotide sequence (SEQ ID NO: 152) of a native sequence PR0238 cDNA, wherein SEQ ID NO: 152 is a clone designated herein as "UNQ212" and/or "DNA35600-1162" .
  • Figure 56 shows the amino acid sequence (SEQ ID NO: 153) derived from the coding sequence of SEQ ID NO:
  • Figure 57 shows a nucleotide sequence (SEQ ID NO: 158) of a native sequence PR0233 cDNA, wherein SEQ ID NO: 158 is a clone designated herein as "UNQ207" and/or "DNA34436-1238".
  • Figure 58 shows the amino acid sequence (SEQ ID NO: 159) derived from the coding sequence of SEQ ID NO: 158 shown m Figure 57.
  • Figure 59 shows a nucleotide sequence (SEQ ID NO: 163) of a native sequence PR0223 cDNA, wherein SEQ ID NO:163 is a clone designated herein as "UNQ197" and/or "DNA33206-1165” .
  • Figure 60 shows the amino acid sequence (SEQ ID NO: 164) derived from the coding sequence of SEQ ID NO: 163 shown in Figure 59.
  • Figure 61 shows a nucleotide sequence (SEQ ID NO: 169) of a native sequence PR0235 cDNA, wherein
  • SEQ ID NO:169 is a clone designated herein as "UNQ209" and/or “DNA35558-1167” .
  • Figure 62 shows the amino acid sequence (SEQ ID NO: 170) derived from the coding sequence of SEQ ID NO: 169 shown in Figure 61.
  • Figure 63 shows a nucleotide sequence (SEQ ID NO: 174) of a native sequence PR0236 cDNA, wherein SEQ ID NO: 174 is a clone designated herein as "UNQ210" and/or "DNA35599-1168" .
  • Figure 64 shows the amino acid sequence (SEQ ID NO: 175) derived from the coding sequence of SEQ ID NO: 174 shown in Figure 63.
  • Figure 65 shows a nucleotide sequence (SEQ ID NO: 176) of a native sequence PR0262 cDNA, wherein SEQ ID NO: 176 is a clone designated herein as "UNQ229" and/or "DNA36992-1168" .
  • Figure 66 shows the ammo acid sequence (SEQ ID NO: 177) derived from the coding sequence of SEQ ID NO: 176 shown in Figure 65.
  • Figure 67 shows a nucleotide sequence (SEQ ID NO: 184) of a native sequence PR0239 cDNA, wherein SEQ ID NO:184 is a clone designated herein as "UNQ213" and/or "DNA34407-1169".
  • Figure 68 shows die amino acid sequence (SEQ ID NO: 185) derived from the coding sequence of SEQ ID NO: 184 shown in Figure 67.
  • Figure 69 shows a nucleotide sequence (SEQ ID NO: 189) of a native sequence PR0257 cDNA, wherein SEQ ID NO: 189 is a clone designated herein as "UNQ224" and/or "DNA35841-1173” .
  • Figure 70 shows die amino acid sequence (SEQ ID NO: 190) derived from the coding sequence of SEQ ID NO:
  • Figure 71 shows a nucleotide sequence (SEQ ID NO: 194) of a native sequence PRO260 cDNA, wherein SEQ ID NO: 194 is a clone designated herein as "UNQ227" and/or "DNA33470-1175” .
  • Figure 72 shows me amino acid sequence (SEQ ID NO: 195) derived from the coding sequence of SEQ ID NO : 194 shown in Figure 71.
  • Figure 73 shows a nucleotide sequence (SEQ ID NO:200) of a native sequence PR0263 cDNA, wherein SEQ ID NO:200 is a clone designated herein as "UNQ230" and/or "DNA34431-1177".
  • Figure 74 shows the amino acid sequence (SEQ ID NO:201) derived from the coding sequence of SEQ ID NO:200 shown in Figure 73.
  • Figure 75 shows a nucleotide sequence (SEQ ID NO:206) of a native sequence PRO270 cDNA, wherein
  • SEQ ID NO:206 is a clone designated herein as "UNQ237” and/or "DNA39510-1181".
  • Figure 76 shows the amino acid sequence (SEQ ID NO:207) derived from the coding sequence of SEQ ID NO:206 shown in Figure 75.
  • Figure 77 shows a nucleotide sequence (SEQ ID NO:212) of a native sequence PR0271 cDNA, wherein SEQ ID NO:212 is a clone designated herein as "UNQ238" and/or "DNA39423-1182" .
  • Figure 78 shows die amino acid sequence (SEQ ID NO:213) derived from the coding sequence of SEQ ID NO:212 shown in Figure 77.
  • Figure 79 shows a nucleotide sequence (SEQ ID NO:220) of a native sequence PR0272 cDNA, wherein SEQ ID NO:220 is a clone designated herein as "UNQ239" and/or "DNA40620-1183".
  • Figure 80 shows die amino acid sequence (SEQ ID NO:221) derived from the coding sequence of SEQ ID NO:
  • Figure 81 shows a nucleotide sequence (SEQ ID N0.226) of a native sequence PR0294 cDNA, wherein SEQ ID NO:226 is a clone designated herein as "UNQ257” and/or "DNA40604-1187".
  • Figure 82 shows the amino acid sequence (SEQ ID NO:227) derived from the coding sequence of SEQ ID NO:226 shown in Figure 81.
  • Figure 83 shows a nucleotide sequence (SEQ ID NO:235) of a native sequence PR0295 cDNA, wherein SEQ ID NO:235 is a clone designated herein as "UNQ258" and/or "DNA38268-1188".
  • Figure 84 shows die amino acid sequence (SEQ ID NO:236) derived from the coding sequence of SEQ ID NO:235 shown in Figure 83.
  • Figures 85A-B show a nucleotide sequence (SEQ ID NO:244) of a native sequence PR0293 cDNA, wherein SEQ ID NO:244 is a clone designated herein as "UNQ256" and/or "DNA37151-1193" .
  • Figure 86 shows the amino acid sequence (SEQ ID NO:245) derived from the coding sequence of SEQ ID NO:244 shown in Figures 85A-B.
  • Figures 89 A-B show a nucleotide sequence (SEQ ID NO:249) of a native sequence PR0247 cDNA, wherein SEQ ID NO:249 is a clone designated herein as "UNQ221 " and/or "DNA35673-1201 " .
  • Figure 88 shows die amino acid sequence (SEQ ID NO:250) derived from the coding sequence of SEQ ID NO:249 shown in Figure 87.
  • Figure 89 shows a nucleotide sequence (SEQ ID NO:254) of a native sequence PRO302 cDNA, wherein
  • SEQ ID NO:254 is a clone designated herein as "UNQ265" and/or "DNA40370-1217” .
  • Figure 90 shows die ammo acid sequence (SEQ ID NO:255) derived from the coding sequence of SEQ ID NO:254 shown in Figure 89.
  • Figure 91 shows a nucleotide sequence (SEQ ID NO:256) of a native sequence PRO303 cDNA, wherein SEQ ID NO:256 is a clone designated herein as "UNQ266" and/or "DNA42551-1217” .
  • Figure 92 shows die amino acid sequence (SEQ ID NO:257) derived from the coding sequence of SEQ ID NO:256 shown in Figure 91.
  • Figure 93 shows a nucleotide sequence (SEQ ID NO:258) of a native sequence PRO304 cDNA, wherein SEQ ID NO:258 is a clone designated herein as "UNQ267" and/or "DNA39520-1217” .
  • Figure 94 shows me amino acid sequence (SEQ ID NO:259) derived from the coding sequence of SEQ ID NO:
  • Figure 95 shows a nucleotide sequence (SEQ ID NO:260) of a native sequence PRO307 cDNA, wherein SEQ ID NO:260 is a clone designated herein as "UNQ270" and/or "DNA41225-1217” .
  • Figure 96 shows die amino acid sequence (SEQ ID NO:261) derived from the coding sequence of SEQ ID NO :260 shown in Figure 95.
  • Figure 97 shows a nucleotide sequence (SEQ ID NO:262) of a native sequence PR0343 cDNA, wherein SEQ ID NO:262 is a clone designated herein as "UNQ302" and/or "DNA43318-1217” .
  • Figure 98 shows die amino acid sequence (SEQ ID NO:263) derived from the coding sequence of SEQ ID NO:262 shown in Figure 97.
  • Figure 99 shows a nucleotide sequence (SEQ ID NO:284) of a native sequence PR0328 cDNA, wherein
  • SEQ ID N0.284 is a clone designated herein as "UNQ289” and/or “DNA40587-1231 ".
  • Figure 100 shows the amino acid sequence (SEQ ID NO:285) derived from the coding sequence of SEQ ID NO:284 shown in Figure 99.
  • Figures 101A-B show a nucleotide sequence (SEQ ID NO:289) of a native sequence PR0335 cDNA, wherein SEQ ID NO:289 is a clone designated herein as "UNQ287" and/or "DNA41388-1234" .
  • Figure 102 shows die amino acid sequence (SEQ ID NO:290) derived from the coding sequence of SEQ ID NO:289 shown in Figures 103 A-B.
  • Figure 103 shows a nucleotide sequence (SEQ ID NO:291) of a native sequence PR0331 cDNA, wherein SEQ ID NO:291 is a clone designated herein as "UNQ292" and/or "DNA40981-1234" .
  • Figure 104 shows me ammo acid sequence (SEQ ID NO:292) derived from the coding sequence of SEQ ID NO:291 shown in Figure 103.
  • Figures 105A-B show a nucleotide sequence (SEQ ID NO:293) of a native sequence PR0326 cDNA, wherein SEQ ID NO:293 is a clone designated herein as "UNQ287" and/or "DNA37140-1234" .
  • Figure 106 shows die ammo acid sequence (SEQ ID NO:294) derived from the coding sequence of SEQ ID NO:293 shown in Figures 105A-B.
  • Figures 107 A-B show a nucleotide sequence (SEQ ID NO:309) of a native sequence PR0332 cDNA, wherein SEQ ID NO:309 is a clone designated herein as "UNQ293" or "DNA40982-1235” .
  • Figure 108 shows the amino acid sequence (SEQ ID NO:310) derived from the coding sequence of SEQ ID NO:
  • Figure 109 shows a nucleotide sequence (SEQ ID NO:314) of a native sequence PR0334 cDNA, wherein SEQ ID NO:314 is a clone designated herein as "UNQ295" or "DNA41379-1236" .
  • Figure 110 shows the ammo acid sequence (SEQ ID NO:315) derived from the coding sequence of SEQ ID NO:314 shown in Figure 109.
  • Figure 111 shows a nucleotide sequence (SEQ ID NO:319) of a native sequence PR0346 cDNA, wherein SEQ ID NO:319 is a clone designated herein as "UNQ305" or "DNA44167-1243".
  • Figure 112 shows the amino acid sequence (SEQ ID NO:320) derived from the coding sequence of SEQ ID NO:319 shown in Figure 111.
  • Figure 113 shows a nucleotide sequence (SEQ ID NO:324) of a native sequence PR0268 cDNA, wherein
  • SEQ ID NO:324 is a clone designated herein as "UNQ235" or "DNA39427-1179” .
  • Figure 114 shows the amino acid sequence (SEQ ID NO:325) derived from the coding sequence of SEQ ID NO: 324 shown in Figure 113.
  • Figure 115 shows a nucleotide sequence (SEQ ID NO:331) of a native sequence PRO330 cDNA, wherein SEQ ID NO:331 is a clone designated herein as "UNQ290" or "DNA40603-1232" .
  • Figure 116 shows die amino acid sequence (SEQ ID NO:332) derived from the coding sequence of SEQ ID NO:331 shown in Figure 115.
  • Figure 117 shows a nucleotide sequence (SEQ ID NO:338) of a native sequence PR0339 cDNA, wherein SEQ ID NO:338 is a clone designated herein as "UNQ229" or "DNA43466-1225” .
  • Figure 118 shows die amino acid sequence (SEQ ID NO:339) derived from the coding sequence of SEQ ID NO:
  • Figure 119 shows a nucleotide sequence (SEQ ID NO:340) of a native sequence PRO310 cDNA, wherein SEQ ID NO:340 is a clone designated herein as "UNQ273" or "DNA43046-1225".
  • Figure 120 shows the amino acid sequence (SEQ ID NO:341) derived from the coding sequence of SEQ ID NO:340 shown in Figure 119.
  • Figure 121 shows a nucleotide sequence (SEQ ID NO:376) of a native sequence PR0244 cDNA, wherein SEQ ID NO:376 is a clone designated herein as "UNQ218" or “DNA35668-1171 " .
  • Figure 122 shows die amino acid sequence (SEQ ID NO:377) derived from the coding sequence of SEQ ID NO:376 shown in Figure 121.
  • PRO polypeptide and "PRO” as used herein and when immediately followed by a numerical designation refer to various polypeptides, wherein the complete designation (i.e., PRO/number) refers to specific polypeptide sequences as described herein.
  • PRO/number polypeptide and “PRO/number” as used herein encompass native sequence polypeptides and polypeptide variants (which are further defined herein).
  • the PRO polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • a “native sequence PRO polypeptide” comprises a polypeptide having die same amino acid sequence as die corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence PRO polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific PRO polypeptide (e.g. , an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally- occurring aUeUc variants of the polypeptide.
  • the native sequence PR0211 is a mature or full-length native sequence PR0211 polypeptide comprising ammo acids 1 to 353 of Figure 2 (SEQ ID NO:2)
  • the native sequence PR0217 is a mature or full-length native sequence PR0217 polypeptide comprising amino acids 1 to 379 of Figure 4 (SEQ ID NO:4)
  • the native sequence PRO230 is a mature or fuU-lengfh native sequence PRO230 polypeptide comprising amino acids 1 to 467 of Figure 6 (SEQ ID NO: 12)
  • the native sequence PR0232 polypeptide is a mature or fuU-lenglh native sequence PR0232 polypeptide comprising amino acids 1 to 114 of Figure 9 (SEQ ID NO: 18)
  • the native sequence PR0187 is a mature or full-length native sequence PR0187 comprising amino acids 1 to 205 of Figure 11 (SEQ ID NO:23)
  • the native sequence PR0265 polypeptide is a mature or fuU-length native sequence PR0265 polypeptide comprising amino acids 1 to
  • the native-sequence PR0317 is a full-lengtii native-pre-sequence PR0317 comprising amino acids 1 to 366 of Fig. 42 (SEQ ID NO: 114) or a mature native-sequence PR0317 comprising amino acids 19 to 366 of Fig. 42 (SEQ ID NO: 114), die native sequence PRO301 is a mature or full- length native sequence PRO301 comprising ammo acids 1 to 299 of Fig.
  • die native sequence PR0224 polypeptide is a matore or full-length native sequence PR0224 polypeptide comprising amino acids 1 to 282 of Figure 46 (SEQ ID NO: 127), the native sequence PR0222 polypeptide is a matore or full-length native sequence PR0222 polypeptide comprising amino acids 1 to 490 of Figure 48 (SEQ ID NO: 132), the native sequence PR0234 is a mature or full-length native sequence novel lectin comprising amino acids 1 to 382 of Fig.
  • die native sequence PR0231 polypeptide is a mature or full-length native sequence PR0231 polypeptide comprising amino acids 1 to 428 of Figure 52 (SEQ ID NO: 142), the native sequence PR0229 polypeptide is a mature or fuU-length native sequence PR0229 polypeptide comprising amino acids 1 to 347 of Figure 54 (SEQ ID NO:148), the native sequence PR0238 polypeptide is a mature or full-length native sequence PR0238 polypeptide comprising amino acids 1 to 310 of Figure 56 (SEQ ID NO: 153), the native sequence PR0233 polypeptide is a mature or fuU-lengdi native sequence PR0233 polypeptide comprising ammo acids 1 to 300 of Figure 58 (SEQ ID NO: 159), die native sequence PR0223 polypeptide is a matore or full-length native sequence PR0223 polypeptide comprising amino acids 1 to 476 of Figure 60 (SEQ ID NO: 164), the native sequence PR0235 polypeptide is
  • the native sequence PR0334 polypeptide is a mature or full-length native sequence PR0334 polypeptide comprising ammo acids 1 to 509 of Figure 110 (SEQ ID NO:315)
  • the native sequence PR0346 is a mature or full-length native sequence PR0346 comprising amino acids 19 to 339 of Fig.
  • the native sequence PR0268 polypeptide is a matore or fuU-length native sequence PR0268 polypeptide comprising amino acids 1 to 280 of Figure 114 (SEQ ID NO:325) or the native sequence PR0268 polypeptide is an extracellular domain of the full-length PR0268 protein, wherein the putative transmembrane domain of the full-length PR0268 protein is encoded by nucleotides beginning at nucleotide 559 as shown in Figure 113, the native sequence PRO330 polypeptide is a mature or Ml-lengui native sequence PRO330 polypeptide comprising amino acids 1 to 533 of Figure 116 (SEQ ID NO:332), die native sequence PR0339 polypeptide is a mature or full-length native sequence
  • PRO polypeptide variants include, for instance, PRO polypeptides wherein one or more amino acid residues are added, or deleted, at die N- or C-terminus of the full-length native amino acid sequence.
  • a PRO polypeptide variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the amino acid sequence of die full-length native amino acid sequence as disclosed herein.
  • PR0317 variants or "PR0317 sequence variants” as defined herein mean biologically active PR0317s as defined below havmg less than 100% sequence identity with the PR0317 isolated from recombinant cell culture or from mammalian fetal kidney tissue having the deduced sequence described in Figure 42.
  • a biologicaUy active PR0317 variant will have an amino acid sequence havmg at least about 70% amino acid sequence identity with the PR0317 of Figure 42, preferably at least about 75%, more preferably at least about 80%, still more preferably at least about 85 % , even more preferably at least about 90 % , and most preferably at least about 95 % (i.e.
  • PR0317 fragments have a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid residues, preferably about 10-150 residues, that is identical to die sequence of die PR0317 shown in Figure 42.
  • Other preferred PR0317 fragments mclude tiiose produced as a result of chemical or enzymatic hydrolysis or digestion of he purified PR0317.
  • a “chimeric PR0317” is a polypeptide comprising full-lengtii PR0317 or one or more fragments thereof fused or bonded to a second protein or one or more fragments thereof.
  • the chimera will typically share at least one biological property in common with PR0317.
  • the second protein will typically be a cytokine, growth factor, or hormone such as a neurotrophic or angiogenic factor such as GDNF or VEGF, or another member of the TGF- superfamily such as EBAF-1.
  • Another exemplary preferred PR0317 chimera is a "domain chimera” that consists of the N-terminal residues substituted witii one or more, but not all, of the residues of the human EBAF-1.
  • the PR0317 chimera would have individual or blocks of residues from the human EBAF-1 sequence added or substituted into the PR0317 sequence. For example, one or more of those segments of EBAF-1 that are not homologous could be substituted into die corresponding segments of PR0317. It is contemplated tiiat this "PR0317-EBAF-1 domain chimera" will have an agonist biological activity.
  • Percent (%) amino acid sequence identity with respect to the PRO polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with d e amino acid residues m me specific PRO polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve die maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of dete ⁇ riining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megahgn (DNASTAR) software. The preferred software alignment program is BLAST.
  • Percent (%) nucleic acid sequence identity with respect to PRO-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical witii the nucleotides in me PRO nucleic acid sequence of interest, after aligning die sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using pubUcly available computer software such as BLAST, ALIGN or MegaUgn (DNASTAR) software. Those skiUed in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences bemg compared.
  • Isolated when used to describe the various polypeptides disclosed herein, means polypeptide tiiat has been identified and separated and/or recovered from a component of its natoral environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may mclude enzymes, hormones, and otiier proteinaceous or non-proteinaceous solutes.
  • die polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non- reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes polypeptide in situ witiiin recombinant ceUs, since at least one component of the PRO polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • An "isolated" PRO polypeptide nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natoral source of the PRO polypeptide nucleic acid.
  • An isolated PRO polypeptide nucleic acid molecule is other than in the form or setting in which it is found in nature.
  • Isolated PRO polypeptide nucleic acid molecules therefore are distinguished from the specific PRO polypeptide nucleic acid molecule as it exists in natoral cells.
  • an isolated PRO polypeptide nucleic acid molecule mcludes PRO polypeptide nucleic acid molecules contained in cells tiiat ordinarily express the PRO polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natoral ceUs.
  • Southern analysis or “Southern blotting” is a method by which me presence of DNA sequences in a restriction endonuclease digest of DNA or a DNA-containing composition is confirmed by hybridization to a known, labeled oligonucleotide or DNA fragment.
  • Southern analysis typically involves electrophoretic separation of DNA digests on agarose gels, denaturation of the DNA after electrophoretic separation, and transfer of the DNA to nitroceUulose, nylon, or another suitable membrane support for analysis with a radiolabeled, biotinylated, or enzyme- labeled probe as described in sections 9.37-9.52 of Sambrook et al. , Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
  • RNA sequences tiiat hybridize to a known probe such as an oUgonucleotide, DNA fragment, cDNA or fragment thereof, or RNA fragment.
  • the probe is labeled with a radioisotope such as 32 P, or by biotinylation, or with an enzyme.
  • the RNA to be analyzed is usually electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe, using standard techniques weU known in the art such as those described in sections 7.39-7.52 of Sambrook et al., supra.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic ceUs are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that die DNA sequences bemg linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • antibody is used in the broadest sense and specifically covers single anti-PRO polypeptide monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-PRO polypeptide antibody compositions with polyepitopic specificity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • Activity refers to form(s) of PRO polypeptide which retain the biologic and/or immunologic activities of the specific native or naturally-occurring PRO polypeptide.
  • the activity of a PR0332 polypeptide preferably involves the regulation of extracellular matrix, cartilage, or bone function.
  • PR0317-associated disorder refers to a pathological condition or disease wherein PR0317 is over- or underexpressed.
  • disorders include diseases of die female genital tract or of the endometrium of a mammal, including hyperplasia, endometritis, endometriosis, wherein the patient is at risk for infertility due to endometrial factor, endometrioma, and endometrial cancer, especially those diseases involving abnormal bleeding such as a gynecological disease.
  • diseases involving angiogenesis wherem the angiogenesis results in a patiiological condition, such as cancer involving solid tumors (the therapy for the disorder would result in decreased vascularization and a decline in growth and metastasis of a variety of tumors).
  • the angiogenesis may be beneficial, such as for ischemia, especially coronary ischemia.
  • these disorders include tiiose found hi patients whose hearts are functioning but who have a blocked blood supply due to atiierosclerotic coronary artery disease, and those with a functioning but underperfused heart, including patients witii coronary arterial disease who are not optimal candidates for angioplasty and coronary artery by-pass surgery.
  • the disorders also include diseases involving the kidney or originating from the kidney tissue, such as polycystic kidney disease and chronic and acute renal failure.
  • Treatment refers to both tiierapeutic treatment and prophylactic or preventative measures. Those in need of treatment include tiiose already witii the disorder as well as those prone to have the disorder of those in which the disorder is to be prevented.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, and die like.
  • the mammal herein is a human.
  • Carriers as used herem include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed tiiereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologicaUy acceptable carriers include buffers such as phosphate, citrate, and otiier 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 polyvinylpyrroUdone; ammo acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and otiier 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 TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and otiier organic acids
  • antioxidants including ascorbic acid
  • agonist is used to refer to peptide and non-peptide analogs of the native PRO polypeptides
  • native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or mature PRO polypeptide
  • antibodies specifically binding such native PRO polypeptides provided that they retain at least one biological activity of a native PRO polypeptide.
  • the agonists of the present invention retain the quaUtative binding recognition properties and receptor activation properties of the native PRO polypeptide.
  • antagonists are used to refer to a molecule inhibiting a biological activity of a native PRO polypeptide of die present invention wherein native PRO polypeptide refers to pro-PRO polypeptide, pre-PRO polypeptide, prepro-PRO polypeptide, or matore PRO polypeptide.
  • the antagonists herein inhibit the binding of a native PRO polypeptide of die present invention.
  • Preferred antagonists essentially completely block the binding of a native PR0317 polypeptide to a PR0317 polypeptide receptor to which it otherwise binds.
  • Such receptors may include the Type I and Type II, and possibly Type III receptors identified for the TGF- superfamily. Kolodziejczyk and Hall, supra.
  • a PRO polypeptide "antagonist” is a molecule which prevents, or interferes with, a PRO antagonist effector function (e.g. a molecule which prevents or interferes with bmdmg and/or activation of a PRO polypeptide receptor by PRO polypeptide). Such molecules can be screened for their ability to competitively inhibit PRO polypeptide receptor activation by monitoring binding of native PRO polypeptide in die presence and absence of die test antagonist molecule, for example. Examples of PR0317 polypeptide antagonists include neutralizing antibodies against F-2. An antagonist of the mvention also encompasses an antisense polynucleotide against the PRO polypeptide gene, which antisense polynucleotide blocks transcription or translation of the PRO polypeptide gene, thereby inhibiting its expression and biological activity.
  • “Stringent conditions” means (1) employing low ionic strength and high temperature for washing, for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide wtih 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrroUdone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide wtih 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrroUdone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C.
  • Another example is use of 50% formamide, 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 ⁇ g/ml), 0.1 % SDS, and 10% dextran sulfate at 42 °C, with washes at 42°C in 0.2 x SSC and 0.1 % SDS.
  • Yet another example is hybridization using a buffer of 10% dextran sulfate, 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.
  • Modely stringent conditions are described in Sambrook et al., supra, and include the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength, and %SDS) less stringent than described above.
  • An example of moderately stringent conditions is a condition such as overnight incubation at 37 C C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 °C.
  • the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. , as necessary to accommodate factors such as probe length and die like.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present appUcation as PR0211 and PR0217.
  • Applicants have identified and isolated cDNA encoding PR0211 and PR0217 polypeptides, as disclosed in further detail in the Examples below.
  • BLAST FastA format
  • the cDNA sequence DNA32292-1131 ( Figure 1, SEQ ID NO:l) has 36% identify and a Blast score of 209 with PAC6_RAT and 31 % identify and a Blast score of 206 with Fibulin- 1, isoform c precursor.
  • the cDNA sequence DNA33094-1131 ( Figure 3, SEQ ID NO:3) has 36% identity and a Blast score of 336 with eastern newt tenascin, and 37% identity and a Blast score of 331 with human tenascin-X precursor. Accordingly, it is presently believed that PR0211 and PR0217 polypeptides disclosed in the present application are newly identified members of the EGF-like family and possesses properties typical of the EGF-like protein family.
  • PRO230 Full-length polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO230.
  • Applicants have identified and isolated cDNA encoding a PRO230 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence aUgnment computer programs Applicants found that a cDNA sequence encodmg full- length native sequence PRO230 has 48% amino acid identity with the rabbit tubulointerstitial nephritis antigen precursor.
  • PRO230 polypeptide disclosed in the present application is a newly identified member of the tubulointerstitial nephritis antigen family and possesses die ability to be recognized by human autoantibodies in certain forms of tubulointerstitial nephritis.
  • PR0232 nucleotide sequences encoding polypeptides referred to in the present application as PR0232.
  • Applicants have identified and isolated cDNA encoding a PR0232 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence aUgnment computer programs Applicants found that a portion of the full-length native sequence PR0232 (shown in Figure 9 and SEQ ID NO: 18) has 35% sequence identity with a stem cell surface antigen from GaUus gaUus. Accordingly, it is presently believed that the PR0232 polypeptide disclosed in the present appUcation may be a newly identified stem cell antigen.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0187.
  • Applicants have identified and isolated cDNA encoding a PRO 187 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that a fuU-length native sequence PRO 187 (shown in Figure 15) has 74% amino acid sequence identity and BLAST score of 310 with various androgen-induced growth factors and FGF-8.
  • PRO 187 polypeptide disclosed in the present application is a newly identified member of the FGF-8 protein family and may possess identify activity or property typical of the FGF-8-like protein family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0265.
  • PR0265 polypeptides referred to in the present application as PR0265.
  • Applicants have identified and isolated cDNA encoding a PR0265 polypeptide, as disclosed in further detail in the Examples below.
  • programs such as BLAST and FastA sequence alignment computer programs, Applicants found that various portions of the PR0265 polypeptide have significant homology with the fibromodulin protein and fibromodulin precursor protein.
  • the DNA encoding die PR0265 polypeptide has significant homology with platelet glycoprotein V, a member of the leucine rich related protein famtiy involved in skin and wound repair.
  • tiiat PR0265 polypeptide disclosed in the present application is a newly identified member of the leucme rich repeat family and possesses protein protein binding capabilities, as well as be involved in skin and wound repair as typical of this family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0219.
  • PR0219 polypeptides referred to in the present application as PR0219.
  • Applicants have identified and isolated cDNA encoding a PR0219 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0219 polypeptide have significant homology with the mouse and human matrilin-2 precursor polypeptides. Accordingly, it is presently believed that PR0219 polypeptide disclosed in the present application is related to the matrilin-2 precursor polypeptide.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0246.
  • Applicants have identified and isolated cDNA encoding a PR0246 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence aUgnment computer programs Applicants found tiiat a portion of the PR0246 polypeptide has significant homology with die human ceU surface protein HCAR. Accordingly, it is presently believed that PR0246 polypeptide disclosed in die present appUcation may be a newly identified membrane-bound virus receptor or tumor cell-specific antigen.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0228.
  • PR0228 polypeptides referred to in the present application as PR0228.
  • Applicants have identified and isolated cDNA encoding a PR0228 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0228 polypeptide have significant homology with the EMRl protein.
  • the DNA encodmg the PR0228 polypeptide has significant homology with latrophilin, macrophage-restricted cell surface glycoprotein, B0457.1 and leucocyte antigen CD97 precursor.
  • PR0228 polypeptide disclosed in the present application is a newly identified member of the seven transmembrane superfamily and possesses characteristics and functional properties typical of this family.
  • PR0228 is a new member of the subgroup within this family to which CD97 and EMRl belong.
  • PR0533 nucleotide sequences encoding polypeptides referred to in the present application as PR0533.
  • Applicants have identified and isolated cDNA encoding a PR0533 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that a fuU-lengdi native sequence PR0533 (shown in Figure 22 and SEQ ID NO:59) has a Blast score of 509 and 53 % amino acid sequence identity with fibroblast growth factor (FGF). Accordingly, it is presently believed that PR0533 disclosed in the present application is a newly identified member of the fibroblast growth factor family and may possess activity typical of such polypeptides.
  • FGF fibroblast growth factor
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0245.
  • Applicants have identified and isolated cDNA encoding a PR0245 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence aUgnment computer programs Applicants found tiiat a portion of the amino acid sequence of the PR0245 polypeptide has 60% amino acid identity with the human c-myb protein. Accordingly, it is presently believed tiiat die PR0245 polypeptide disclosed in die present appUcation may be a newly identified member of the transmembrane protein tyrosine kinase family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO220, PR0221 and PR0227.
  • PRO220 has identified and isolated cDNAs encoding a PRO220, PR0221 and PR0227 polypeptide, respectively, as disclosed in further detaU in the Examples below.
  • PRO220 has amino acid identity with the ammo acid sequence of a leucine rich protein wherein the identity is 87 % .
  • PRO220 additionaUy has amino acid identity witii the neuronal leucine rich protein wherein the identity is 55 % .
  • PR0221 has amino acid identity with the SLIT protein precursor, wherein different portions of these two proteins have the respective percent identities of 39% , 38%, 34%, 31 %, and 30%.
  • PR0227 has amino acid identity with the amino acid sequence of platelet glycoprotein V precursor. The same results were obtained for human glycoprotein V. Different portions of these two proteins show the following percent identities of 30% , 28%, 28%, 31 %, 35%, 39% and 27% .
  • PRO220, PR0221 and PR0227 polypeptides disclosed in the present application are newly identified members of the leucine rich repeat protein superfamily and that each possesses protein-protein binding capabUities typical of the leucine rich repeat protein superfamily. It is also believed tiiat they have capabilities similar to those of SLIT, the leucine rich repeat protein and human glycoprotein V.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0258.
  • PR0258 polypeptides referred to in the present application as PR0258.
  • Applicants have identified and isolated cDNA encoding a PR0258 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0258 polypeptide have significant homology with the CRTAM and poliovirus receptors. Accordingly, it is presently believed that PR0258 polypeptide disclosed in the present appUcation is a newly identified member of the Ig superfamily and possesses virus receptor capabilities or regulates immune function as typical of this family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0266.
  • PR0266 polypeptides referred to in the present application as PR0266.
  • Applicants have identified and isolated cDNA encoding a PR0266 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0266 polypeptide have significant homology with the SLIT protein from Drosophilia. Accordingly, it is presently believed that PR0266 polypeptide disclosed in the present application is a newly identified member of the leucine rich repeat family and possesses ligand-ligand bmdmg activity and neuronal development typical of this family.
  • SLIT has been shown to be useful in the stody and treatment of Alzheimer's disease, supra, and thus, PR0266 may have involvement in the stody and cure of this disease.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0269.
  • Applicants have identified and isolated cDNA encoding a PR0269 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found tiiat tiie amino acid sequence encoded by nucleotides 314 to 1783 of the ftiU-lengtii native sequence PR0269 (shown in Figure 35 and SEQ ID NO:95) has significant homology to human urinary thrombomodulin and various thrombomodulin analogues respectively, to which it was aligned. Accordingly, it is presentiy beUeved tiiat PR0269 polypeptide disclosed in the present application is a newly identified member of the thrombomodulin family. 15. Full-length PRQ287 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0287.
  • PR0287 polypeptides referred to in the present application as PR0287.
  • Applicants have identified and isolated cDNA encoding a PR0287 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0287 polypeptide have significant homology with the type 1 procoUagen C-proteinase enhancer protein precursor and type 1 procoUagen C- proteinase enhancer protein. Accordingly, it is presently believed that PR0287 polypeptide disclosed in the present application is a newly identified member of the C-proteinase enhancer protein family.
  • PR0214 Full-length PRQ214 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encodmg polypeptides referred to in the present application as PR0214.
  • Applicants Uave identified and isolated cDNA encoding a PR0214 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that a full-length native sequence PR0214 polypeptide (shown in Figure 40 and SEQ ID NO: 109) has 49% amino acid sequence identity with HT protein, a known member ofthe EGF-famtiy.
  • the comparison resulted in a BEAST score of 920, with 150 matching nucleotides. Accordingly, it is presentiy beUeved that die PR0214 polypeptide disclosed in the present application is a newly identified member of the famUy comprising EGF domains and may possess activities or properties typical of the EGF-domain containing family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0317.
  • cDNA encoding a PR0317 polypeptide has been identified and isolated, as disclosed in further detail in the Examples below.
  • BLASTTM and Fas ATM sequence aUgnment computer programs it was found that a full-length native-sequence PR0317 (shown in Figure 42 and SEQ ID NO: 114) has 92% amino acid sequence identity with EBAF-1. Further, it is closely aligned with many other members of the TGF- superfamily.
  • PR0317 disclosed in the present appUcation is a newly identified member of die TGF- superfamily and may possess properties that are therapeutically useful in conditions of uterine bleeding, etc.
  • PR0317 may be useful in diagnosing or treating abnormal bleeding mvolved in gynecological diseases, for example, to avoid or lessen the need for a hysterectomy.
  • PR0317 may also be useful as an agent that affects angiogenesis in general, so PR0317 may be useful in anti-tumor indications, or conversely, in treating coronary ischemic conditions.
  • PR0317 has shown up in several tissues as well, but it does look to have a greater concentration in uterus. Hence, PR0317 may have a broader use by the body than EBAF-1. It is contemplated that, at least for some indications, PR0317 may have opposite effects from EBAF-1. 18. Full-length PRO301 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO301.
  • Applicants have identified and isolated cDNA encoding a PRO301 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that a fuU-length native sequence PRO301 (shown in Figure 44 and SEQ ID NO:119) has a Blast score of 246 corresponding to 30% amino acid sequence identity with human A33 antigen precursor.
  • tiiat PRO301 disclosed in the present application is a newly identified member of the A33 antigen protein family and may be expressed in human neoplastic diseases such as colorectal cancer.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0224.
  • Applicants have identified and isolated cDNA encodmg a PR0224 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and Fas A sequence alignment computer programs Applicants found that full-length native PR0224 ( Figure 46, SEQ ID NO: 127) has amino acid identity with apolipoprotein E receptor 2906 from homo sapiens.
  • the aUgnments of different portions of these two polypeptides show amino acid identities of 37%, 36%, 30%, 44%, 44% and 28% respectively.
  • FuU-length native PR0224 (Figure 46, SEQ ID NO:127) also has ammo acid identity with very low-density lipoprotein receptor precursor from gall. The alignments of different portions of these two polypeptides show amino acid identities of 38%, 37%, 42%, 33%, and 37% respectively. Additionally, fuU-length native PR0224 (Figure 46, SEQ ID NO: 127) has amino acid identity with the chicken oocyte receptor P95 from
  • GaUus gaUus The alignments of different portions of these two polypeptides show amino acid identities of 38%
  • PR0224 polypeptide disclosed in the present application is a newly identified member of the low density lipoprotein receptor famUy and possesses the structural characteristics required to have the functional abitity to recognize and endocy tose low density lipoproteins typical of the low density
  • the present invention provides newly identified and isolated nucleotide sequences encodmg polypeptides referred to in the present application as PR0222.
  • Applicants have identified and isolated cDNA encoding a PR0222 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and Fas A sequence aUgnment computer programs Applicants found that a sequence encoding fuU-length native sequence PR0222 (shown in Figure 48 and SEQ ID NO: 132) has 25-26% amino acid identity with mouse complement factor h precursor, has 27-29% amino acid identity with complement receptor, has 2547% amino acid identity with mouse complement C3b receptor type 2 long form precursor, has 40% amino acid identity with human hypothetical protein kiaa0247.
  • PR0222 polypeptide disclosed in the present application is a newly identified member of the complement receptor famtiy and possesses activity typical of the complement receptor family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in tiie present application as PR0234.
  • Applicants have identified and isolated cDNA encoding a PR0234 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST FasA-format sequence aUgnment computer programs
  • AppUcants found tiiat a cDNA sequence encoding full-lengtii native sequence PR0234 has 31 % identity and Blast score of 134 with E-selectin precursor.
  • tiiat tUe PR0234 polypeptides disclosed in tiie present application are newly identified members of the lectin/selectin family and possess activity typical of the lectin/selectin family.
  • PR0231 Full-length polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0231.
  • Applicants have identified and isolated cDNA encoding a PR0231 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and Fas A sequence alignment computer programs Applicants found that the full-length native sequence PR0231 polypeptide (shown in Figure 52 and SEQ ID NO: 142) has 30 % and 31 % amino acid identity with human and rat prostatic acid phosphatase precursor proteins, respectively. Accordingly, it is presently believed tiiat the PR0231 polypeptide disclosed in the present application may be a newly identified member of the acid phosphatase protein family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0229.
  • PR0229 polypeptides referred to in the present application as PR0229.
  • Applicants Uave identified and isolated cDNA encoding a PR0229 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and Fas A sequence alignment computer programs Applicants found that various portions of the PR0229 polypeptide have significant homology with antigen wcl.l, M130 antigen, T cell surface glycoprotein CD6 and CD6. It also is related to Sp-alpha.
  • PR0229 polypeptide disclosed in the present application is a newly identified member of the famtiy containing scavenger receptor homology, a sequence motif found in a number of proteins involved in immune function and thus possesses immune function and /or segments which resist degradation, typical of this family.
  • PR0238 polypeptide disclosed in the present application is a newly identified member of the reductase family and possesses reducing activity typical of the reductase family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0233.
  • Applicants have identified and isolated cDNA encoding a PR0233 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found tiiat various portions of the PR0233 polypeptide have significant homology with the reductase protein.
  • Applicants have also found that the DNA encoding the PR0233 polypeptide has significant homology with proteins from Caenorhabditis elegans.
  • PR0233 polypeptide disclosed in the present application is a newly identified member of the reductase family and possesses the ability to effect the redox state of the cell typical of tiie reductase family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0223.
  • PR0223 polypeptides referred to in the present application as PR0223.
  • Applicants Uave identified and isolated cDNA encoding a PR0223 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that the PR0223 polypeptide has significant homology with various serine carboxypeptidase polypeptides. Accordingly, it is presently believed that PR0223 polypeptide disclosed in the present application is a newly identified serine carboxypeptidase.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0235.
  • PR0235 polypeptides referred to in the present application as PR0235.
  • Applicants Uave identified and isolated cDNA encoding a PR0235 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0235 polypeptide have significant homology witii the various plexin protems. Accordingly, it is presently believed that PR0235 polypeptide disclosed in the present application is a newly identified member of the plexin family and possesses cell adhesion properties typical of the plexin famtiy.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides refe ⁇ ed to in the present appUcation as PR0236 and PR0262.
  • Applicants have identified and isolated cDNA encoding PR0236 and PR0262 polypeptides, as disclosed in further detail in the Examples below.
  • PR0236 and PR0262 polypeptides have significant homology with various ⁇ -galactosidase and ⁇ -galactosidase precursor polypeptides. Accordingly, it is presentiy beUeved that the PR0236 and PR0262 polypeptides disclosed in the present application are newly identified ⁇ -galactosidase homologs.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0239.
  • Applicants have identified and isolated cDNA encoding a PR0239 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0239 polypeptide have significant homology with densin proteins. Accordingly, it is presently believed that PR0239 polypeptide disclosed in the present appUcation is a newly identified member of the densin family and possesses cell adhesion and the ability to effect synaptic processes as is typical of the densin family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0257.
  • PR0257 polypeptides referred to in the present application as PR0257.
  • Applicants Uave identified and isolated cDNA encoding a PR0257 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0257 polypeptide have significant homology with the ebnerin precursor and ebnerin protein. Accordingly, it is presently believed that PR0257 polypeptide disclosed in die present application is a newly identified protein member which is related to the ebnerin protein.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO260.
  • Applicants have identified and isolated cDNA encoding a PRO260 polypeptide, as disclosed in further detail in the Examples below.
  • programs such as BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PRO260 polypeptide have significant homology with the alpha-1-fucosidase precursor. Accordingly, it is presently believed that PRO260 polypeptide disclosed in the present application is a newly identified member of the fucosidase family and possesses enzymatic activity related to fucose residues typical of the fucosidase family.
  • the present invention provides newly identified and isolated nucleotide sequences encodmg polypeptides referred to in the present application as PR0263.
  • PR0263 nucleotide sequences encodmg polypeptides referred to in the present application as PR0263.
  • Applicants have identified and isolated cDNA encoding a PR0263 polypeptide, as disclosed in further detati in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0263 polypeptide have significant homology with the CD44 antigen and related proteins.
  • PR0263 polypeptide disclosed in the present application is a newly identified member of the CD44 antigen family and possesses at least one of the properties associated with these antigens, i.e., cancer and HIV marker, cell-cell or cell- matrix interactions, regulating cell traffic, lymph node homing, transmission of growth signals, and presentation of chemokines and growth facors to traveling cells.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO270.
  • Applicants have identified and isolated cDNA encoding a PRO270 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found tiiat that various portions of the PRO270 polypeptide have significant homology with various thioredoxin proteins. Accordingly, it is presently believed that PRO270 polypeptide disclosed in the present appUcation is a newly identified member of the thioredoxin family and possesses the ability to effect reduction-oxidation (redox) state typical of the thioredoxin family.
  • redox reduction-oxidation
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0271.
  • Applicants have identified and isolated cDNA encoding a PR0271 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that the PR0271 polypeptide has significant homology with various link protems and precursors thereof. Accordingly, it is presentiy believed that PR0271 polypeptide disclosed in the present application is a newly identified link protein homolog.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0272.
  • Applicants have identified and isolated cDNA encoding a PR0272 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence aUgnment computer programs Applicants found that various portions of the PR0272 polypeptide have significant homology with the human reticulocalbin protein and its precursors.
  • the DNA encodmg the PR0272 polypeptide has significant homology with the mouse reticulocalbin precursor protein. Accordingly, it is presentiy beUeved that PR0272 polypeptide disclosed in the present appUcation is a newly identified member of the reticulocalbin family and possesses the ability to bind calcium typical of the reticulocalbin family.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0294.
  • PR0294 polypeptides referred to in the present application as PR0294.
  • Applicants have identified and isolated cDNA encoding a PR0294 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence aUgnment computer programs Applicants found that various portions of the PR0294 polypeptide have significant homology with the various portions of a number of collagen protems . Accordingly, it is presently believed that PR0294 polypeptide disclosed in the present application is a newly identified member of the collagen family. 37. Full-length PRQ295 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0295.
  • PR0295 polypeptides referred to in the present application as PR0295.
  • Applicants have identified and isolated cDNA encoding a PR0295 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found tiiat various portions of the PR0295 polypeptide have significant homology with integrin proteins. Accordingly, it is presently believed that PR0295 polypeptide disclosed in the present application is a newly identified member of the integrin family and possesses cell adhesion typical of the integrin family.
  • PR0293 Full-length PRQ293 Polypeptides
  • PR0293 nucleotide sequences encoding polypeptides referred to in the present application as PR0293.
  • Applicants have identified and isolated cDNA encoding a PR0293 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence aUgnment computer programs Applicants found tiiat portions of the PR0293 polypeptide have significant homology with the neuronal leucine rich repeat proteins 1 and 2, (NLRR-1 and NLRR-2), particularly NLRR-2. Accordingly, it is presentiy beUeved that PR0293 polypeptide disclosed in the present application is a newly identified member of the neuronal leucine rich repeat protein family and possesses ligand-ligand binding activity typical of the NRLL protein family.
  • PR0247 polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0247.
  • Applicants have identified and isolated cDNA encoding a PR0247 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0247 polypeptide have significant homology with densin.
  • Applicants have also found that the DNA encodmg the PR0247 polypeptide has significant homology with a number of other proteins, includmg KIAA0231. Accordingly, it is presently believed that PR0247 polypeptide disclosed in the present appUcation is a newly identified member of the leucine rich repeat family and possesses ligand binding abilities typical of this family.
  • PRO302. PRO303. PRO304. PRO307 and PRQ343 Polypeptides The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present appUcation as PRO302, PRO303, PRO304, PRO307 and PR0343.
  • Applicants have identified and isolated cDNA encoding PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides, as disclosed in further detati in the Examples below.
  • AppUcants found that various portions of the PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides have significant homology with various protease proteins. Accordingly, it is presently believed that the PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides disclosed in the present application are newly identified protease proteins.
  • 41 Full-length PRQ328 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0328.
  • PR0328 has identified and isolated cDNA encoding a PR0328 polypeptide, as disclosed in further detail in the Examples below.
  • GLIP human gUoblastoma protein
  • CRISP cysteine ricU secretory protein
  • PR0328 polypeptide disclosed in the present appUcation is a newly identified member of the GLIP or CRISP families and possesses transcriptional regulatory activity typical of the GLIP or CRISP families.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides refe ⁇ ed to in the present appUcation as PR0335, PR0331 or PR0326.
  • Applicants Uave identified and isolated cDNA encoding a PR0335, PR0331 or PR0326 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0335, PR0331 or PR0326 polypeptide have significant homology with LIG-1, ALS and in the case of PR0331, additionally, decorin.
  • PR0335, PR0331 and PR0326 polypeptides disclosed in the present appUcation are newly identified members of the leucme rich repeat superfamily, and particularly, are related to LIG-1 and possess the biological functions of this family as discussed and referenced herein.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0332.
  • Applicants have identified and isolated cDNA encoding PR0332 polypeptides, as disclosed in further detail in the Examples below.
  • AppUcants found tiiat a fuU-length native sequence PR0332 (shown in Figure 108 and SEQ ID NO:310) has about 3040% amino acid sequence identity with a series of known proteoglycan sequences, includmg, for example, fibromodulin and fibromodulin precursor sequences of various species (FMOD BOVIN, FMOD.CHICK, FMOD.RAT, FMOD.MOUSE, FMOD.HUMAN, P.R36773), osteomoduUn sequences (AB000114 . 1, AB007848.1), decorin sequences (CFU83141.1, OCU03394.1, P . R42266, P .
  • PR0332 disclosed in the present application is a new proteoglycan-type molecule, and may play a role in regulating extracellular matrix, cartilage, and/or bone function.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0334.
  • PR0334 polypeptides referred to in the present application as PR0334.
  • Applicants have identified and isolated cDNA encoding a PR0334 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PR0334 polypeptide have significant homology with fibulin and fibrillin. Accordingly, it is presently believed that PR0334 polypeptide disclosed in the present appUcation is a newly identified member of the epidermal growth factor family and possesses properties and activities typical of this family.
  • PR0346 Full-length PRQ346 Polypeptides
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0346.
  • Applicants have identified and isolated cDNA encoding a PR0346 polypeptide, as disclosed in further detail in the Examples below.
  • AppUcants found that a fuU-length native sequence PR0346 (shown in Figure 112 and SEQ ID NO:320) has 28 % amino acid sequence identity with carcinoembryonic antigen.
  • PR0346 disclosed in the present application is a newly identified member of the carcinoembryonic protein family and may be expressed in association witii neoplastic tissue disorders.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PR0268.
  • PR0268 polypeptides referred to in the present application as PR0268.
  • Applicants have identified and isolated cDNA encoding a PR0268 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence aUgnment computer programs Applicants found that portions of the PR0268 polypeptide have significant homology with the various protein disulfide isomerase proteins. Accordingly, it is presently believed that PR0268 polypeptide disclosed in the present application is a homolog of the protein disulfide isomerase p5 protein.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as PRO330.
  • Applicants have identified and isolated cDNA encoding a PRO330 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FasA sequence alignment computer programs Applicants found that various portions of the PRO330 polypeptide have significant homology with the murine prolyl 4-hydroxylase alpha-II subunit protein. Accordingly, it is presently believed that PRO330 polypeptide disclosed in the present application is a novel prolyl 4-hydroxylase subunit polypeptide.
  • the present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present appUcation as PR0339 and PRO310.
  • Applicants have identified and isolated cDNA encoding a PR0339 polypeptide, as disclosed in further detail in the Examples below.
  • Applicants have also identified and isolated cDNA encodmg a PRO310 polypeptide, as disclosed in further detail in the Examples below.
  • BLAST and FastA sequence alignment computer programs Applicants found that various portions of the PR0339 and PRO310 polypeptides have significant homology with small secreted proteins from C. elegans and are distantly related to fringe.
  • PR0339 also shows homology to collagen-like polymers.
  • the present invention provides newly identified and isolated nucleotide sequences encoding C-type lectins referred to in the present application as PR0244.
  • PR0244 C-type lectins
  • applicants Uave identified and isolated cDNA encoding PR0244 polypeptides, as disclosed in further detail in the Examples below.
  • AppUcants found that a fuU-length native sequence PR0244 (shown in Figure 122 and SEQ ID NO:377) has 43% amino acid sequence identity with the hepatic lectin gaUus gaUus (LECH- CHICK), and 42% amino acid sequence identity with an HIV gpl20 bmdmg C-type lectin (A46274).
  • PR0244 disclosed in the present application is a newly identified member of the C-lectin superfamUy and may play a role in immune function, apoptosis, or in the pathogenesis of atherosclerosis.
  • PR0244 may be useful in identifying tumor-associated epitopes.
  • PRO Polypeptide variants can be prepared.
  • PRO polypeptide variants can be prepared by introducing appropriate nucleotide changes into the PRO polypeptide DNA, or by synthesis of the desired PRO polypeptide.
  • ammo acid changes may alter post-translational processes of the PRO polypeptides, such as changing the number or position of glycosylation sites or .altering the membrane anchoring characteristics.
  • Variations in the native full-length sequence PRO polypeptides or in various domains of the PRO polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934.
  • Variations may be a substitution, deletion or Uisertion of one or more codons encoding the PRO polypeptide that results in a change in the ammo acid sequence of the PRO polypeptide as compared with the native sequence PRO polypeptide.
  • the variation is by substitotion of at least one amino acid with any other ammo acid in one or more of the domains of the PRO polypeptide.
  • Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the PRO polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of 1 to 5 amino acids. The variation allowed may be determined by systematically malting insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity in the in vitro assay described in the Examples below.
  • restriction selection mutagenesis [WeUs et al., PhUos. Trans. R. Soc. London SerA. 217:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the desired PRO polypeptide variant DNA.
  • Scanning ammo acid analysis can also be employed to identify one or more amino acids along a contiguous sequence.
  • preferred scanning amino acids are relatively small, neutral amino acids.
  • amino acids include alanine, glycine, serine, and cysteine.
  • Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant.
  • Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.); Chothia, Mol. Biol.. 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
  • Covalent modifications of PRO polypeptides are included within the scope of this mvention.
  • One type of covalent modification includes reacting targeted amino acid residues of the PRO polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PRO polypeptide.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking a PRO polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-PRO polypeptide antibodies, and vice-versa.
  • Commonly used crosslinking agents include, e.g.
  • l,l-bis(diazoacetyl)-2-pbenylethane, glutaraldehyde, N-hydroxysuccinimide esters for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, includmg disuccinimidyl esters sucU as 3,3'-dithiobis(succU ⁇ imidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as metiiyl-3-[(p-azidophenyl)dithio]propioimidate.
  • Another type of covalent modification of the PRO polypeptides included within the scope of this mvention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in a native sequence PRO polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence PRO polypeptide. Addition of glycosylation sites to the PRO polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitotion by, one or more serine or threonine residues to the native sequence PRO polypeptide (for O-linked glycosylation sites).
  • the PRO polypeptide amino acid sequence may optionaUy be altered through changes at the DNA level, particularly by mutating the DNA encoding the PRO polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the PRO polypeptide polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. BiocUem.. pp. 259-306 (1981). Removal of carbohydrate moieties present on the PRO polypeptide may be accomplished chemically or enzymatically or by mutational substitotion of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddm, et al., Arch.
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo- glycosidases as described by Thotakura et al., Meth. Enzvmol.. 138:350 (1987).
  • Another type of covalent modification of PRO polypeptides of the invention comprises linking the PRO polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyetiiylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
  • nonproteinaceous polymers e.g., polyetiiylene glycol, polypropylene glycol, or polyoxyalkylenes
  • the PRO polypeptides of the present invention may also be modified in a way to form a chimeric molecule comprising a PRO polypeptide fused to another, heterologous polypeptide or amino acid sequence.
  • a chimeric molecule comprises a fusion of the PRO polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the amino- or carboxyl- terminus of the PRO polypeptide. The presence of such epitope-tagged forms of the PRO polypeptide can be detected using an antibody against the tag polypeptide.
  • the epitope tag enables the PRO polypeptide to be readtiy purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • the chimeric molecule may comprise a fusion of the PRO polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
  • tag polypeptides and then respective antibodies are well known in the art. Examples include poly- histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5
  • Tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology. 6:1204-1210 (1988)]; the KT3 epitope peptide
  • Amino acid sequence variants of PR0317 are prepared by introducing appropriate nucleotide changes into the PR0317 DNA, or by in vitro synthesis of the desired PR0317 polypeptide.
  • Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence shown for human PR0317 in Figure 42. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post- translational processes of the PR0317, such as changing the number or position of glycosylation sites.
  • PR0317 is presumably encoded by multi-exon genes.
  • Alternative mRNA constructs which may be attributed to different mRNA splicing events following transcription, and which share large regions of identity with the cDNAs claimed herein, are considered to be within the scope of the present invention.
  • the location of the mutation site and the nature of the mutation will depend on the PR0317 characteristic(s) to be modified.
  • candidate PR0317 antagonists or agonists will be initially selected by locating sites that are identical or highly conserved among PR0317, EBAF-1, LEFTY, and otiier members of the TGF- superfamily.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • a useful method for identification of certain residues or regions of the PR0317 polypeptide that are preferred locations for mutagenesis is caUed "alanine scanning mutagenesis," as described by Cunningham and Wells,
  • a residue or group of target residues are identified (e.g. , charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitotion.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis is conducted at the target codon or region and the PR0317 variants produced are screened for the optimal combination of desired activity.
  • the location and nature of the mutation chosen will depend upon the PR0317 characteristic to be modified.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues, and typically are contiguous. Contiguous deletions ordinarily are made in even numbers of residues, but single or odd numbers of deletions are within the scope hereof.
  • Deletions may be introduced into regions of low homology among PR0317, EBAF-1 , and other members of the TGF- superfamily which share the most sequence identity to the human PR0317 amino acid sequence to modify the activity of PR0317. Deletions from PR0317 in areas of substantial homology with one of the receptor binding sites of other members of the TGF- superfamily will be more likely to modify the biological activity of PR0317 more significantly. The number of consecutive deletions will be selected so as to preserve the tertiary structore of PR0317 in the affected domain, e.g. , beta-pleated sheet or alpha helix.
  • Amino acid sequence insertions mclude amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • Intrasequence insertions i.e. , insertions within the mature PR0317 sequence
  • terminal insertions include mature PR0317 with an N-terminal methionyl residue, an artifact of the direct production of mature PR0317 in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to tiie N-terminus of the mature PR0317 molecule to facUitate the secretion of mature PR0317 from recombinant bosts.
  • signal sequences may be obtained from, and thus homologous to, the intended host cell species, but also may be from other members of the TGF- superfamily. Suitable sequences include STII or lpp for E. coli, alpha factor for yeast, and viral signals such as herpes gD or the native EBAF-1 sequence for mammalian cells.
  • insertional variants of the PR0317 molecule include the fusion to the N- or C-terminus of PR0317 of immunogenic polypeptides, e.g. , bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins havmg a long half-life such as immunoglobulin constant regions (or other immunoglobulin regions), albumin, or ferritin, as described in WO 89/02922 published
  • a third group of variants are amino acid substitotion variants. These variants have at least one amino acid residue in the PR0317 molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s) of PR0317 and sites where the amino acids found in the known analogues are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there is also a high degree of sequence identity at the selected site within various animal PR0317 species, or where the amino acids found in known members of the TGF- superfamily and novel PR0317 are substantially different in terms of side-chain bulk, charge, or hydrophobicity, but where there also is a high degree of sequence identity at the selected site within various animal analogues of such members (e.g. , among all the animal EBAF-1 molecules).
  • This analysis will highlight residues that may be involved in the modulation of endometrial tissue or angiogenesis, and therefore, variations at these sites may affect such activities.
  • Otiier sites of interest are those in which particular residues of the PR0317 obtained from various species are identical among all animal species of PR0317 and other members of the TGF- superfamily, this degree of conservation suggesting importance in achieving biological activity common to these cytokines. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.
  • Met fM leu
  • phe phe
  • ile leu
  • Substantial modifications in function or immunological identity of the PR0317 are accomplished by selecting substitutions that differ significantiy in their effect on maintaining (a) the structore of the polypeptide backbone in the area of the substitotion, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Natorally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitotions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitotion sites or, more preferably, into the remaining (non-conserved) sites.
  • protease cleavage sites tiiat are present in the molecule. These sites are identified by inspection of the encoded amino acid sequence, in the case of trypsin, e.g. , for an arginyl or lysinyl residue.
  • protease cleavage sites are identified, they are rendered inactive to proteolytic cleavage by substituting the targeted residue with another residue, preferably a basic residue such as glutamine or a hydrophiUc residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue.
  • any methionyl residues other than the starting methionyl residue of the signal sequence, or any residue located withm about three residues N- or C-terminal to each such methionyl residue, is substituted by another residue (preferably in accord with Table 1) or deleted.
  • another residue preferably in accord with Table 1
  • about 1-3 residues are inserted adjacent to such sites.
  • nucleic acid molecules encodmg amino acid sequence variants of PR0317 are prepared by a variety of methods known in the art. These methods mclude, but are not limited to, isolation from a natoral source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion, and insertion variants of PR0317 DNA. This technique is well known in the art as described by AdeUnan et al. , DNA.
  • PR0317 DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the smgle-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of PR0317. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and wUl code for the selected alteration in the PR0317 DNA.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the OUgonucleotides are readily synthesized using techniques known in the art such as that described by
  • the DNA template can be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13mpl8 and M13mpl9 vectors are suitable), or those vectors that contain a smgle-stranded phage origin of replication as described by Viera et al. Meth. Enzymol.. 153: 3 (1987). Thus, the DNA that is to be mutated may be inserted into one of these vectors to generate single-stranded template. Production of the single-stranded template is described in Sections 4.214.41 of Sambrook et al , supra.
  • single-stranded DNA template may be generated by denaturing double-stranded plasmid (or other) DNA using standard techniques.
  • the oligonucleotide is hybridized to the single-stranded template under suitable hybridization conditions.
  • a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of die template using the oligonucleotide as a primer for synthesis.
  • a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of PR0317, and the other strand (the original template) encodes the native, unaltered sequence of PR0317.
  • This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101.
  • the cells are grown, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabeled with W P to identify the bacterial colonies that contain the mutated DNA.
  • the mutated region is then removed and placed in an appropriate vector for protein production, generaUy an expression vector of the type typically employed for transformation of an appropriate host.
  • the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s).
  • the modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from the Amersham
  • This mixture is added to the template-oligonucleotide complex.
  • DNA polymerase Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated.
  • this new strand of DNA wUl contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.
  • the template strand can be digested with ⁇ r ⁇ lll nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded.
  • a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of aU four deoxyribonucleotide tripUosphates, ATP, and DNA ligase.
  • This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.
  • DNA encoding PR0317 mutants with more than one amino acid to be substituted may be generated in one of several ways . If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitotions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
  • a separate oligonucleotide is generated for each ammo acid to be substituted.
  • the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitotions.
  • the alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
  • the first round is as described for the single mutants: wUd-type DNA is used for the template, an oligonucleotide encodmg tiie first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated.
  • the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
  • this template already contains one or more mutations.
  • the oligonucleotide encoding the additional desired amino acid substitotion(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
  • This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
  • PCR mutagenesis is also suitable for making amino acid variants of PR0317. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA.
  • the PCR technique generaUy refers to the following procedure (see Erlich, PCR Technology.
  • primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA.
  • the sequence of the second primer is located withm 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced.
  • PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.
  • the starting material is the plasmid (or other vector) comprising the PR0317 DNA to be mutated.
  • the codon(s) in the PR0317 DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis metiiod to introduce them at appropriate locations in the PR0317 DNA.
  • the plasmid is cut at these sites to linearize it.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the destied mutation(s) is synthesized using standard procedures. The two strands are synfliesized separately and then hybridized together using standard techniques.
  • This double-stranded oUgonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated PR0317 DNA sequence.
  • Covalent modifications of PR0317 are also included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of the PR0317 with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PR0317.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking PR0317 to a water-insoluble support matrix or surface for use in the metiiod for purifying anti-PR0317 antibodies, and vice-versa.
  • crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, mcludmg disuccininiidyl esters such as 3,3'-dithiobis- (succinimidylpropionate), bifunctional maleimides sucU as bis-N-maleimido-l,8-octane, and agents such as methyl-3- ((p-azidophenyl)dithio)propioimidate .
  • Another type of covalent modification of the PR0317 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native-sequence PRO polypeptide, and/or adding one or more glycosylation sites that are not present in the native-sequence PRO polypeptide.
  • the deduced amino acid sequence of PR0317 shown in Figure 42 (SEQ ID NO: 114) has one predicted N-linked glycosylation site at residue 160.
  • Addition of glycosylation sites to the PR0317 polypeptide may be accomplished by altering the amino acid sequence.
  • the alteration may be made, for example, by the addition of, or substitotion by, one or more serine or threonine residues to the native-sequence PR0317 (for O-linked glycosylation sites).
  • the PR0317 amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encodmg the PR0317 polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Removal of carbohydrate moieties present on the PR0317 polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding ammo acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Ha muddin, et al. , Arch. Biochem. Biophvs.. 259:52 (1987) and by Edge et al. , Anal. Biochem.. 118:131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo- glycosidases as described by Thotakura et al. , Meth. Enzymol.. 138:350 (1987).
  • PR0317 polypeptide comprises linking the PR0317 polypeptide to one of a variety of nonproteinaceous polymers, e.g. , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • nonproteinaceous polymers e.g. , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • the PR0317 of the present invention may also be modified in a way to form a cnimeric molecule comprising PR0317 fused to another, heterologous polypeptide or amino acid sequence.
  • a chimeric molecule comprises a fusion of the PR0317 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generaUy placed at the amino- or carboxyl- terminus of the PR0317. The presence of such epitope-tagged forms of the PR0317 can be detected using an antibody against the tag polypeptide.
  • the epitope tag enables the PR0317 to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • the chimeric molecule may comprise a fusion of the PR0317 with an immunoglobulin or a particular region of an immunoglobulin.
  • such a fusion could be to the Fc region of an IgG molecule .
  • tag polypeptides and their respective antibodies are well known in tiie art. Examples include poly- histidine (poly-his) or poly-histidine-glycine (poly-Uis-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et al. , Mol. Cell. Biol.. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies thereto (Evan et al. Molecular and Cellular Biology. 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al , Protein Engineering.
  • poly-his poly-his
  • poly-Uis-gly poly-Uis-gly
  • tag polypeptides include the Flag-peptide (Hopp et al , Bio/Technology. 6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al. Science. 255:192-194 (1992)); an "-tubutin 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)).
  • PRO polypeptide sequence or portions thereof, may be produced by direct peptide synthesis using solid- phase techniques [see, e.g., Stewart et al., Solid-PUase Peptide Synthesis. W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation.
  • Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's instructions.
  • Various portions of the desired PRO polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length PRO polypeptide.
  • DNA encoding PRO polypeptides may be obtained from a cDNA library prepared from tissue believed to possess the desired PRO polypeptide mRNA and to express it at a detectable level. Accordingly, human PRO polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The PRO polypeptide-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.
  • Probes such as antibodies to the desired PRO polypeptide or oligonucleotides of at least about 20-80 bases
  • Screening the cDNA or genomic library witii the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
  • An alternative means to isolate the gene encodmg the desired PRO polypeptide is to use PCR methodology [Sambrook et al., supra: Dieffenbach et al., PCR PrimerA Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].
  • the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.
  • the oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32 P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al , supra
  • Sequences identified m such library screemng methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or otiier private sequence databases
  • Sequence identity (at either the ammo acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as BLAST, ALIGN, DNAstar, and INHERIT which employ various algorithms to measure homology
  • Nucleic acid havmg protein coding sequence may be obtained by screemng selected cDNA or genomic libraries usmg the deduced amino acid sequence disclosed herem for the first time, and, if necessary, usmg conventional primer extension procedures as descnbed m Sambrook et al , supra, to detect precursors and processmg intermediates of mRNA that may not have been reverse-transcribed mto cDNA
  • Host cells are transfected or transformed with expression or cloning vectors described herem for PRO polypeptide production and cultured in conventional nutrient media modified as appropriate for mducmg promoters, selecting transformants, or amplifying the genes encodmg the desired sequences
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation
  • principles, protocols, and practical techmques for maximizing the productivity of cell cultures can be found m Mammalian Cell Biotechnology a Practical Approach. M Butler, ed (IRL Press, 1991) and Sambrook et al , supra
  • Suitable host cells for clomng or expressing the DNA in the vectors herem include prokaryote, yeast, or higher eukaryote cells
  • Suitable prokaryotes m include but are not limited to eubacte ⁇ a, such as Gram-negative or Gram-positive organisms, for example, Enterobacte ⁇ aceae such as E coli
  • E coli strains are publicly available, such as E coli K12 stram MM294 (ATCC 31,446), E coli X1776 (ATCC 31,537), E coli stram W3110 (ATCC 27,325) and K5 772 (ATCC 53,635)
  • Other suitable prokaryotic host cells m include Enterobacte ⁇ aceae such as Eschenchia, e g , E coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e g , Salmonella typhimurtum, Serratia, e.g.
  • E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC 53,635). These examples are illustrative rather than limiting.
  • Stram W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations .
  • the host cell secretes minimal amounts of proteolytic enzymes.
  • strain W3110 may be modified to effect a genetic mutation in the genes encodmg proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1A2, which has the complete genotype tonA ; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E.
  • E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)I69 degP ompTka ;
  • E. coli W3110 strain 37D6 which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvGka ;
  • E. coli W3110 strain 40B4 which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and anE. coli strain havmg mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for PRO polypeptide-encoding vectors.
  • Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.
  • Others include Schizosaccharomyces pombe (Beach and Nurse, Nature. 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al , Bio/Technology.
  • K. lactis MW98-8C, CBS683, CBS4574; Louvencourt et al , L Bacteriol.. 737 [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/Technologv. 8: 135 (1990)
  • K . thermotolerans K.
  • Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al , Biochem. Biophvs. Res. Commun.. 112: 284-289 [1983]; Tilburn et al. , Gene. 26: 205-221 [1983]; Yelton et al. , Proc Natl. Acad. Sci. USA.
  • Methylotropic yeasts are suitable herein and mclude, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs. 269 (1982).
  • Suitable host cells for the expression of glycosylated PRO polypeptides are derived from multicellular organisms.
  • invertebrate cells mclude insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.
  • useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol.. 26:59 (1977)); Chinese hamster ovary ceUs ⁇ DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
  • mice settoti ceUs TM4, Mather, Biol. Reprod.. 23:243-251 (1980)
  • human lung cells W138, ATCC CCL 75
  • human liver cells Hep G2, HB 8065
  • mouse mammary tumor MMT 060562, ATCC CCL51. The selection of the appropriate host cell is deemed to be within the skill in the art.
  • the nucleic acid encodmg a desired PRO polypeptide may be inserted into a repUcable vector for cloning (amplification of the DNA) or for expression.
  • a repUcable vector for cloning (amplification of the DNA) or for expression.
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • the PRO polypeptide of interest may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the PRO polypeptide DNA that is inserted into the vector.
  • the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
  • the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (includmg Saccharomyces and Kluyveromyces ⁇ -factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP
  • mammaUan signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of repUcation from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammatian cells.
  • Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. , ampicillin, neomycin, methotrexate, or tetracycUne, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g. , the gene encodmg D-alanine racemase for Bacilli.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the PRO polypeptide nucleic acid, such as DHFR or thymidine kinase.
  • An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc Natl. Acad. Sci. USA. 77:4216 (1980).
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature. 282:39 (1979); Kingsman et al., Gene. 7:141 (1979); Tschemper et al., Gene.
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the abtiity to grow in tryptophan, for example, ATCC No. 44076 or PEP4- 1 [Jones, Genetics. 85:12 (1977)].
  • Expression and cloning vectors usually contain a promoter operably linked to the PRO polypeptide nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase and lactose promoter systems [Chang et al. , Nature. 275:615 (1978); Goeddel et al., Nature. 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res.. 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA. 80:21-25 (1983)]. Promoters for use in bacterial systems also wtil contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encodmg the desired PRO polypeptide.
  • S.D. Shine-Dalgarno
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionetii, glyceraldehyde-3-phos ⁇ hate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
  • PRO polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus
  • Enhancers are cis-acting elements of DNA, usuaUy about from 10 to 300 bp, that act on a promoter to increase its transcription.
  • Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic ceU virus.
  • Examples include the SV40 enhancer on the late side of the repUcation origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the PRO polypeptide coding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms
  • sequences are commonly avatiable from the 5' and, occasionally 3' , untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding PRO polypeptides.
  • StiU other methods, vectors, and host cells suitable for adaptation to the synthesis of PRO polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature. 222:620-625 (1981); Mantei et al., Nature. 281 :4046 (1979); EP 117,060; and EP 117,058.
  • Gene ampUfication and or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc Natl. Acad. Sci. USA. 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • the antibodies in torn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • Gene expression may be measured by immunological methods, such as immunohistochemical staining of ceUs or tissue sections and assay of ceU culture or body fluids, to quantitate directly the expression of gene product.
  • Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence PRO polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to a PRO polypeptide DNA and encodmg a specific antibody epitope.
  • PRO polypeptides may be recovered from culture medium or from host cell lysates . If membrane- bound, it can be released from the membrane usmg a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of PRO polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • PRO polypeptides may be desired to purify PRO polypeptides from recombinant ceU proteins or polypeptides.
  • the following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope- tagged forms of the PRO polypeptide.
  • Nucleotide sequences (or their complement) encodmg the PRO polypeptides of the present invention have various appUcations in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA.
  • PRO polypeptide-encoding nucleic acid will also be useful for the preparation of PRO polypeptides by the recombinant techniques described herein.
  • the full-length native sequence PRO polypeptide-encoding nucleic acid or portions thereof may be used as hybridization probes for a cDNA library to isolate the full-length PRO polypeptide gene or to isolate still other genes (for instance, those encodmg naturally-occurring variants of the PRO polypeptide or PRO polypeptides from other species) which have a desired sequence identity to the PRO polypeptide nucleic acid sequences.
  • the length of the probes will be about 20 to about 50 bases.
  • the hybridization probes may be derived from the nucleotide sequence of any of the DNA molecules disclosed herein or from genomic sequences including promoters, enhancer elements and nitrons of native sequence PRO polypeptide encoding DNA.
  • a screening method will comprise isolating the coding region of the PRO polypeptide gene usmg the known DNA sequence to synthesize a selected probe of about 40 bases.
  • Hybridization probes may be labeled by a variety of labels, includmg radionucleotides such as 2 P or 35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the specific PRO polypeptide gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.
  • ESTs disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
  • the probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related PRO polypeptide sequences.
  • Nucleotide sequences encodmg a PRO polypeptide can also be used to construct hybridization probes for mapping the gene which encodes that PRO polypeptide and for the genetic analysis of individuals with genetic disorders .
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
  • the PRO polypeptide can be used in assays to identify its ligands. Simtiarly, inhibitors of the receptor/Ugand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Screening assays can be designed to find lead compounds that mimic the biological activity of a native PRO polypeptide or a Ugand for the PRO polypeptide. Such screening assays will mclude assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds.
  • the assays can be performed in a variety of formats, including protein-protein bmdmg assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.
  • Nucleic acids which encode a PRO polypeptide or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in torn, are useful in the development and screening of therapeutically useful reagents.
  • a transgenic animal e.g., a mouse or rat
  • a transgene is a DNA which is integrated mto the genome of a cell from which a transgenic animal develops.
  • cDNA encodmg a PRO polypeptide of mterest can be used to clone genomic DNA encoding the PRO polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding the PRO polypeptide.
  • Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009.
  • particular cells would be targeted for PRO polypeptide transgene incorporation with tissue-specific enhancers.
  • Transgenic animals that mclude a copy of a transgene encoding a PRO polypeptide introduced into tiie germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding the PRO polypeptide.
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
  • non-human homologues of PRO polypeptides can be used to construct a PRO polypeptide
  • cDNA encodmg a PRO polypeptide can be used to clone genomic DNA encodmg tiie PRO polypeptide in accordance with established techmques.
  • a portion of the genomic DNA encodmg a PRO polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, CeU. 51:503 (1987) for a description of homologous recombination vectors].
  • the vector is introduced mto an embryonic stem ceU line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell. 69:915 (1992)].
  • the selected cells are then injected mto a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152].
  • a chimeric embryo can tiien be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their abiUty to defend against certain pathological conditions and for their development of pathological conditions due to absence of the PRO polypeptide.
  • therapeutic indications mclude disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (e.g., enterocoUtis, ZolUnger-Ellison syndrome, gastrointestmal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation (e.g., psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.
  • enterocoUtis e.g., enterocoUtis, ZolUnger-Ellison syndrome, gastrointestmal ulceration and congenital microvillus atrophy
  • skin diseases associated with abnormal keratinocyte differentiation e.g., psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.
  • PR0232 polypeptide and nucleic acid encoding it possess sequence homology to a cell surface stem ceU antigen and its encoding nucleic acid
  • probes based upon the PR0232 nucleotide sequence may be employed to identify other novel stem cell surface antigen proteins.
  • Soluble forms of tiie PR0232 polypeptide may be employed as antagonists of membrane bound PR0232 activity both in vitro and in vivo.
  • PR0232 polypeptides may be employed in screening assays designed to identify agonists or antagonists of the native PR0232 polypeptide, wherein such assays may take the form of any conventional cell-type or biochemical binding assay.
  • the PR0232 polypeptide may serve as a molecular marker for the tissues in which the polypeptide is specifically expressed.
  • FGF-8 has been implicated in cellular differentiation and embryogenesis, includmg the patterning which appears during limb formation. FGF-8 and the
  • PRO 187 molecules of the invention therefore are likely to have potent effects on cell growth and development.
  • Diseases which relate to cellular growth and differentiation are therefore suitable targets for therapeutics based on functionality similar to FGF-8.
  • diseases related to growth or survival of nerve cells including Parkinson's disease, Alzheimer's disease, ALS, neuropathies.
  • disease related to uncontrolled cell growth e.g., cancer, would also be expected therapeutic targets.
  • PR0265 polypeptides disclosed herem other methods for use with PR0265 are described in U.S. Patent 5,654,270 to Ruoslahti et al.
  • PR0265 can be used in comparison with the fibromodulin disclosed therein to compare its effects on reducing dermal scarring and other properties of the fibromodulin described therein including where it is located and with what it binds and does not.
  • PR0219 polypeptides of the present invention which play a regulatory role in the blood coagulation cascade may be employed in vivo for therapeutic purposes as well as for in vitro purposes. Those of ordinary skill in the art will well know how to employ PR0219 polypeptides for such uses.
  • PR0246 polypeptides of the present invention which serve as cell surface receptors for one or more viruses will find other uses. For example, extracellular domains derived from these PR0246 polypeptides may be employed therapeuticaUy in vivo for lessening the effects of viral infection. Those PR0246 polypeptides which serves as tumor specific antigens may be exploited as therapeutic targets for anti-tumor drugs, and the like. Those of ordinary skill in the art will well know how to employ PR0246 polypeptides for such uses.
  • Assays in which connective growth factor and other growth factors are usually used should be performed with PR0261.
  • An assay to determine whether TGF beta induces PR0261, indicating a role in cancer is performed as known in the art. Wound repair and tissue growth assays are also performed with PR0261. The results are applied accordingly.
  • PR0228 polypeptides should be used in assays in which EMRl , CD97 and latroph ⁇ in would be used in to determine their relative activities. The results can be applied accordingly. For example, a competitive binding assay with PR0228 and CD97 can be performed with the ligand for CD97, CD55.
  • Native PR0533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequence. Residues 3 to 216 have a Blast score of 509, corresponding to 53% homology to fibroblast growth factor. At the nucleotide level, DNA47412, the EST from which PCR oligos were generated to isolate the full length DNA49435-1219, has been observed to map to llpl5. Sequence homology to the llpl5 locus would indicate that PR0533 may have utility in the treatment of Usher Syndrome or Atrophia areata.
  • fibroblast growth factors can act upon cells in both a mitogenic and non-mitogenic manner. These factors are mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived ceUs, inducing granulosa ceUs, adrenal cortical ceUs, chrondrocytes, myoblasts, corneal and vascular endothelial cells
  • vascular smooth muscle cells vascular smooth muscle cells
  • lens retina and prostatic epithelial cells
  • oligodendrocytes vascular smooth muscle cells
  • astrocytes vascular smooth muscle cells
  • chrondocytes vascular smooth muscle cells
  • myoblasts and osteoblasts.
  • Non-mitogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (chemotaxis), initiation of new blood vessel formulation (angiogenesis), modulation of nerve regeneration and survival (neurotrophism), modulation of endocrine functions, and stimulation or suppression of specific cellular protein expression, extraceUular matrix production and cell survival. Baird, A. & Bohlen, P., Handbook of Exp. Phrmacol. 25(1): 369418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healmg, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors, have been suggested to minimize myocardium damage in heart disease and surgery (U.S. P. 4,378,437).
  • PR0245 polypeptide and nucleic acid encodmg it possess sequence homology to a transmembrane protein tyrosine kinase protein and its encoding nucleic acid
  • probes based upon the PR0245 nucleotide sequence may be employed to identify other novel transmembrane tyrosine kinase proteins.
  • Soluble forms of the PR0245 polypeptide may be employed as antagonists of membrane bound PR0245 activity both in vitro and in vivo.
  • PR0245 polypeptides may be employed in screening assays designed to identify agonists or antagonists of the native PR0245 polypeptide, wherein such assays may take the form of any conventional cell-type or biochemical binding assay.
  • the PR0245 polypeptide may serve as a molecular marker for the tissues in which the polypeptide is specifically expressed.
  • PRO220, PR0221 and PR0227 all have leucine rich repeats. Additionally, PRO220 and PR0221 have homology to SLIT and leucme rich repeat protein. Therefore, these proteins are useful in assays described in the Uterature, supra, wherein the SLIT and leucine rich repeat protein are used.
  • PR0227 can be used in an assay to determine the affect of PR0227 on neurodegenerative disease. AdditionaUy, PR0227 has homology to human glycoprotein V. In the case of PR0227, this polypeptide is used in an assay to determine its affect on bleeding, clotting, tissue repair and scarring.
  • the PR0266 polypeptide can be used in assays to determine if it has a role in neurodegenerative diseases or their reversal.
  • PR0269 polypeptides and portions thereof which effect the activity of thrombin may also be useful for in vivo therapeutic purposes, as weU as for various in vitro applications.
  • PR0269 polypeptides and portions thereof may have therapeutic use as an antithrombotic agent with reduced risk for hemorrhage as compared with heparin.
  • Peptides havmg homology to thrombomodulin are particularly desirable.
  • PR0287 polypeptides and portions thereof which effect the activity of bone morphogenic protein "BMPl'VprocoUagen C-proteinase (PCP) may also be useful for in vivo therapeutic purposes, as weU as for various in vitro applications.
  • PR0287 polypeptides and portions thereof may have therapeutic applications in wound healmg and tissue repair.
  • Peptides having homology to procoUagen C-proteinase enhancer protein and its precursor may also be used to induce bone and/or cartilage formation and are therefore of particular interest to the scientific and medical communities.
  • Therapeutic indications for PR0214 polypeptides include disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (e.g., enterocolitis,
  • ZolUnger-EUison syndrome gastrointestinal ulceration and congenital microvillus atrophy
  • skin diseases associated with abnormal keratinocyte differentiation e.g., psoriasis, epithelial cancers sucU as lung squamous cell carcinoma, epidermoid carcinoma of die vulva and gliomas.
  • mice deficient in members of the TGF- superfamily are reported in Matzuk, Trends in Endocrinol. and Metabol.. 6: 120-127 (1995).
  • the PR0317 polypeptide, as well as PR0317-specific antibodies, inhibitors, agonists, receptors, or their analogs, herein are useful in treating PR0317-associated disorders. Hence, for example, they may be employed in modulating endometrial bleeding angiogenesis, and may also have an effect on kidney tissue. Endometrial bleeding can occur in gynecological diseases such as endometrial cancer as abnormal bleeding. Thus, the compositions herein may find use in diagnosing and treating abnormal bleeding conditions in the endometrium, as by reducing or eUminating the need for a hysterectomy.
  • the molecules herein may also find use in angiogenesis applications such as anti-tumor indications for which the antibody against vascular endothelial growth factor is used, or, conversely, ischemic indications for which vascular endothelial growth factor is employed.
  • Bioactive compositions comprising PR0317 or agonists or antagonists thereof may be administered in a suitable therapeutic dose determined by any of several methodologies mcludmg clinical studies on mammalian species to determine maximal tolerable dose and on normal human subjects to determine safe dose. Additionally, the bioactive agent may be complexed with a variety of well established compounds or compositions which enhance stabiUty or pharmacological properties such as half-life. It is contemplated that the therapeutic, bioactive composition may be deUvered by intravenous infusion into the bloodstream or any other effective means which could be used for treating problems of the kidney, uterus, endometrium, blood vessels, or related tissue, e.g., in the heart or genital tract.
  • Dosages and administration of PR0317, PR0317 agonist, or PR0317 antagonist in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. See, for example, Mordenti and Rescigno, Pharmaceutical Research. 2: 17-25 (1992); Morenti et al. , Pharmaceutical Research. 8:1351-1359 (1991); and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development. Yacobi et al. (eds) (Pergamon Press: NY, 1989), pp. 42-96.
  • An effective amount of PR0317, PR0317 agonist, or PR0317 antagonist to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the mammal. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • a typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day.
  • the clinician wtil administer PR0317, PR0317 agonist, or PR0317 antagonist, until a dosage is reached that achieves the desired effect for treatment of the above mentioned disorders.
  • PR0317 or an PR0317 agonist or PR0317 antagonist may be administered alone or in combination with another to achieve the desired pharmacological effect.
  • PR0317 itself, or agonists or antagonists of PR0317 can provide different effects when administered therapeutically.
  • Such compounds for treatment will be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although the pH may vary according to the characteristics of the PR0317, agonist, or antagonist being formulated and the condition to be treated. Characteristics of the treatment compounds include solubility of the molecule, half-life, and antigenicity/immunogenicity; these and other characteristics may aid in defining an effective carrier.
  • PR0317 or PR0317 agonists or PR0317 antagonists may be delivered by known routes of administration mcludmg but not limited to topical creams and gels; transmucosal spray and aerosol, transdermal patch and bandage; injectable, intravenous, and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes.
  • routes of administration mcludmg but not limited to topical creams and gels; transmucosal spray and aerosol, transdermal patch and bandage; injectable, intravenous, and lavage formulations; and orally administered liquids and pills, particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration will be determined by the attending physician and will vary according to each specific situation.
  • Such determinations of admimstration are made by considering multiple variables such as the condition to be treated, the type of mammal to be treated, the compound to be administered, and the pharmacokinetic profile of the particular treatment compound. Additional factors which may be taken into account mclude disease state (e.g. severity) of the patient, age, weight, gender, diet, time of administration, drug combination, reaction sensitivities, and tolerance/response to therapy. Long-acting treatment compound formulations (such as liposomally encapsulated PR0317 or PEGylated PR0317 or PR0317 polymeric microspheres, such as polylactic acid-based microspheres) might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular treatment compound.
  • diseases state e.g. severity
  • Long-acting treatment compound formulations such as liposomally encapsulated PR0317 or PEGylated PR0317 or PR0317 polymeric microspheres, such as polylactic acid-based microspheres
  • Normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day, preferably about 1 ⁇ g/kg/day to 10 mg/kg/day, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the Uteratore; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting the uterus, for example, may necessitate delivery in a manner different from that to another organ or tissue, such as cardiac tissue.
  • microencapsulation of PR0317 is contemplated.
  • Microencapsulation of recombinant proteins for sustained release has been successfuUy performed with human growth hormone (rhGH), interferon- (rhIFN- ), interleukin-2, and MN rgpl20.
  • rhGH human growth hormone
  • rhIFN- interferon-
  • MN rgpl20 MN rgpl20.
  • sustained-release formulations of these proteins were developed usmg poly-lactic-coglycolic acid
  • PLGA polymer due to its biocompatibility and wide range of biodegradable properties.
  • the degradation products of PLGA, lactic and glycolic acids, can be cleared quickly within the human body.
  • the degradability of this 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. Ctiasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.
  • PR0317 20% w/w PR0317 with the lowest possible initial burst ( ⁇ 20%) is necessary.
  • the encapsulated protem to be released should maintain its integrity and stability over the desired release period. It is contemplated that conditions or diseases of the uterus, endometrial tissue, or other genital tissues or cardiac tissues may precipitate damage that is treatable with PR0317 or PR0317 agonist where PR0317 expression is reduced in the diseased state; or with antibodies to PR0317 or other PR0317 antagonists where the expression of
  • PR0317 is increased in the diseased state. These conditions or diseases may be specifically diagnosed by the probing tests discussed above for physiologic and pathologic problems which affect the function of the organ.
  • the PR0317, PR0317 agonist, or PR0317 antagonist may be administered to a mammal with another biologically active agent, either separately or in the same formulation to treat a common indication for which they are appropriate.
  • PR0317 can be administered together with EBAF-1 for those indications on which they demonstrate the same quaUtative biological effects.
  • EBAF-1 may be administered together with an antagonist to PR0317, such as an anti-PR0317 antibody.
  • PR0317 may be administered together with VEGF for coronary ischemia where such indication is warranted, or with an anti-VEGF for cancer as warranted, or, conversely, an antagonist to PR0317 may be administered with VEGF for coronary ischemia or with anti-VEGF to treat cancer as warranted. These administrations would be in effective amounts for treating such disorders.
  • Native PRO301 (SEQ ID NO: 119) has a Blast score of 246 and 30% homology at residues 24 to 282 of Figure 44 with A33_HUMAN, an A33 antigen precursor.
  • A33 antigen precursor as explained in the Background is a tumor-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders.
  • Native PRO301 (SEQ ID NO: 119) and A33_HUMAN also show a Blast score of 245 and 30% homology at residues 21 to 282 of Fig. 44 witii A33_HUMAN, the variation dependent upon how spaces are inserted into the compared sequences.
  • Native PRO301 also has a Blast score of 165 and 29% homology at residues 60 to 255 of Fig. 44 with HS46KDA 1, a human coxsackie and adenovirus receptor protein, also known as cell surface protein HCAR. This region of PRO301 also shows a similar Blast score and homology with
  • PR0234 polypeptides of the invention includes treatments associated with leukocyte homing or the interaction between leukocytes and the endothelium during an inflammatory response. Examples include asthma, rheumatoid arthritis, psoriasis and multiple sclerosis
  • PR0231 polypeptide and nucleic acid encodmg it possess sequence homology to a putative acid phosphatase and its encoding nucleic acid
  • probes based upon the PR0231 nucleotide sequence may be employed to identify other novel phosphatase proteins
  • Soluble forms of the PR0231 polypeptide may be employed as antagonists of membrane bound PR0231 activity both in vitro and in vivo
  • PR0231 polypeptides may be employed m screemng assays designed to identify agomsts or antagonists of the native PR0231 polypeptide, wherem such assays may take the form of any conventional ceU-type or biochemical bmdmg assay
  • the PR0231 polypeptide may serve as a molecular marker for the tissues m which the polypeptide is specifically expressed
  • PR0229 polypeptides can be fused with peptides of interest to determine whether the fusion peptide has an increased half-life over the peptide of interest
  • the PR0229 polypeptides can be used accordmgly to mcrease the half-Ufe of polypeptides of mterest Portions of PR0229 which cause the increase m half-life are an embodunent of the mvention herein
  • PR0238 can be used m assays which measure its ability to reduce substrates, mcludmg oxygen and Aceyl- CoA, and particularly, measure PR0238's ability to produce oxygen free radicals This is done by usmg assays which have been previously described PR0238 can further be used to assay for candidates which block, reduce or reverse its reducmg abilities This is done by performing side by side assays where candidates are added m one assay havmg PR0238 and a substrate to reduce, and not added in another assay, bemg the same but for the lack of the presence of the candidate
  • PR0233 polypeptides and portions thereof which have homology to reductase may also be useful for in vivo therapeutic purposes, as well as for various other applications
  • the identification of novel reductase proteins and related molecules may be relevant to a number of human disorders such as inflammatory disease, organ failure, atherosclerosis, cardiac injury, infertility, birth defects, premature agmg, AIDS, cancer, diabetic complications and mutations m general
  • oxygen free radicals and antioxidants appear to play important roles in a number of disease processes
  • the identification of new reductase protems and reductase-like molecules is of special importance m that such proteins may serve as potential therapeutics for a variety of different human disorders
  • Such polypeptides may also play important roles m biotechnological and medical research, as well as various industrial applications
  • PR0223 polypeptides of the present mvention which exhibit serine carboxypeptidease activity may be employed in vivo for therapeutic purposes as weU as for in vitro purposes
  • PR0235 polypeptides and portions thereof which may be mvolved in cell adhesion are also useful for in vivo therapeutic purposes, as weU as for vanous in vitro appUcations in addition, PR0235 polypeptides and portions thereof may have therapeutic applications m disease states which mvolve cell adhesion Given the physiological importance of ceU adhesion mechamsms in vivo, efforts are currently being under taken to identify new, native proteins which are mvolved in ceU adhesion Therefore, peptides havmg homology to plexin are of particular interest to the scientific and medical communities
  • the PR0236 and PR0262 polypeptides disclosed herein are homologous to vanous known ⁇ - galactosidase proteins
  • the PR0236 and PR0262 polypeptides disclosed herem will find use m conjugates of monoclonal antibodies and the polypeptide for specific killing of tumor cells by generation of active drug from a galactosylated prodrug (e.g., the generation of 5-fluorouridine from the prodrug ⁇ -D-galactosyl-5-fluorouridine).
  • the PR0236 and PR0262 polypeptides disclosed herein may also find various uses both in vivo and in vitro, wherein those uses will be similar or identical to uses for which ⁇ -galactosidase proteins are now employed.
  • PR0239 polypeptides and portions thereof which have homology to densin may also be useful for in vivo therapeutic purposes, as well as for vanous in vitro applications
  • PR0239 polypeptides and portions thereof may have therapeutic applications m disease states which involve synaptic mechanisms, regeneration or cell adhesion
  • peptides havmg homology to densin are of particular mterest to the scientific and medical communities
  • PRO260 polypeptides described herein can be used in assays to determine their relation to fucosidase.
  • the PRO260 polypeptides can be used in assays in determining their ability to remove fucose or other sugar residues from proteoglycans.
  • the PRO260 polypeptides can be assayed to determine if they have any functional or locational simUarities as fucosidase.
  • the PRO260 polypeptides can then be used to regulate the systems in which they are integral.
  • PR0263 can be used in assays wherein CD44 antigen is generally used to determine PR0263 activity relative to that of CD44. The results can be used accordingly.
  • PRO270 polypeptides and portions thereof which effect reduction-oxidation (redox) state may also be useful for in vivo therapeutic purposes, as well as for vanous in vitro applications More specifically, PRO270 polypeptides may affect the expression of a large vanety of genes thought to be involved m the pathogenesis of AIDS, cancer, atherosclerosis, diabetic complications and m pathological conditions mvolvmg oxidative stress such as stroke and inflammation In addition, PRO270 polypeptides and portions thereof may affect the expression of a genes which have a role m apoptosis Therefore, peptides havmg homology to thioredoxin are particularly desirable to the scientific and medical communities
  • PR0272 polypeptides and portions thereof which possess the ability to bind calcium may also have numerous in vivo therapeutic uses, as weU as vanous in vitro applications Therefore, peptides havmg homology to reticulocalbin are particularly desirable Those with ordinary skill in the art will know how to employ PR0272 polypeptides and portions thereof for such purposes.
  • PR0294 polypeptides and portions thereof which have homology to collagen may also be useful for in vivo therapeutic purposes, as well as for vanous other applications.
  • the identification of novel collagens and collage-like molecules may have relevance to a number of human disorders. Thus, the identification of new collagens and coUage-Uke molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders.
  • polypeptides may also play important roles in biotechnological and medical research as weU as various industrial applications. Given the large number of uses for collagen, there is substantial interest in polypeptides with homology to the collagen molecule. PR0295 polypeptides and portions thereof which have homology to integrin may also be useful for in vivo therapeutic purposes, as well as for various other applications.
  • the identification of novel integrins and integrin-like molecules may have relevance to a number of human disorders such as modulating the binding or activity of cells of the immune system. Thus, the identification of new integrins and integrin-like molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders.
  • Such polypeptides may also play important roles in biotechnological and medical research as well as various industrial applications. As a result, there is particular scientific and medical interest in new molecules, such as PR0295.
  • the peptide can be used in all applications that the known NLRR-1 and NLRR-2 polypeptides are used. The activity can be compared between these peptides and thus applied accordingly.
  • the PR0247 polypeptides described herein can be used in assays in which densin is used to determine the activity of PR0247 relative to densin or these other proteins. The results can be used accordingly in diagnostics and/or therapeutic applications with PR0247.
  • PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides of the present invention which possess protease activity may be employed both in vivo for therapeutic purposes and in vitro.
  • PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides of the present invention may be employed both in vivo for therapeutic purposes and in vitro.
  • Those of ordinary skill in the art wUl weU know how to employ the PRO302, PRO303, PRO304, PRO307 and PR0343 polypeptides of the present invention for such purposes.
  • PR0328 polypeptides and portions thereof which have homology to GLIP and CRISP may also be useful for in vivo therapeutic purposes, as well as for various other applications.
  • the identification of novel GLIP and CRISP-like molecules may have relevance to a number of human disorders which mvolve transcriptional regulation or are over expressed in human tumors
  • the identification of new GLIP and CRISP-like molecules is of special importance in that such proteins may serve as potential therapeutics for a variety of different human disorders.
  • Such polypeptides may also play important roles in biotechnological and medical research as well as in various industrial applications. As a result, there is particular scientific and medical interest in new molecules, such as PR0328.
  • PR0335, PR0331 or PR0326 uses in competitive assays with LIG-1, ALS and decorin to determine their relative activities. The results can be used accordingly.
  • PR0335, PR0331 or PR0326 can also be used in assays where LIG-1 would be used to determine if the same effects are incurred.
  • PR0332 contains GAG repeat (GKEK) at ammo acid positions 625-628 in Fig. 108 (SEQ ID NO:310). Slippage in such repeats can be associated with human disease. Accordingly, PR0332 can use useful for the treatment of such disease conditions by gene therapy, i.e. by introduction of a gene containing the correct GKEK sequence motif.
  • GKEK GAG repeat
  • PR0334 uses include use in assays in which fibrilUn or fibulin would be used to determine the relative activity of PR0334 to fibrilUn or fibulin.
  • PR0334 can be used in assays which require the mechamsms imparted by epidermal growth factor repeats.
  • Native PR0346 (SEQ ID NO:320) has a Blast score of 230, conesponding to 27% homology between amino acid residues 21 to 343 with residues 35 to 1040 CGM6_HUMAN, a carcinoembryonic antigen cgm ⁇ precursor.
  • This homology region includes nearly all but 2 N-terminal extracellular domain residues, includmg an immunoglobulin superfamily homology at residues 148 to 339 of PR0346 in addition to several transmembrane residues (340-343).
  • Carcinoembryonic antigen precursor as explained in the Background is a tumor-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders.
  • Native PR0346 SEQ ID NO:320
  • P_W06874 a human carcinoembryonic antigen CEA-d have a Blast score of 224 and homology of 28% between residues 2 to 343 and 67 to 342, respectively. This homology includes the entire extraceUular domain residues of native PR0346, minus the initiator methionine (residues 2 to 18) as well as several transmembrane residues (340-343).
  • PR0268 polypeptides which have protein disulfide isomerase activity will be useful for many applications where protein disulfide isomerase activity is desirable including, for example, for use in promoting proper disulfide bond formation in recombinantly produced proteins so as to increase the yield of correctly folded protein.
  • protein disulfide isomerase activity is desirable including, for example, for use in promoting proper disulfide bond formation in recombinantly produced proteins so as to increase the yield of correctly folded protein.
  • PR0268 polypeptides will be useful for many applications where protein disulfide isomerase activity is desirable including, for example, for use in promoting proper disulfide bond formation in recombinantly produced proteins so as to increase the yield of correctly folded protein.
  • PRO330 polypeptides of the present invention which possess biological activity related to that of the prolyl 4-hydroxylase alpha subunit protein may be employed both in vivo for therapeutic purposes and in vitro. Those of ordinary skill in the art will well know Uow to employ tiie PRO330 polypeptides of tiie present invention for such purposes.
  • Anti-PRO Polypeptide Antibodies The present invention further provides anti-PRO polypeptide antibodies.
  • Exemplary antibodies mclude polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
  • the anti-PRO polypeptide antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant wtil be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may mclude the PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins mclude but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the anti-PRO polypeptide antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma mediods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to ehcit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the PRO polypeptide of interest or a fusion protein thereof.
  • GeneraUy eidier peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized ceU line usmg a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103].
  • Immortalized cell lines are usuaUy transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma ceU lines are employed.
  • TUe hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will mclude hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol , 122:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc, New York, (1987) pp. 51-63].
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the PRO polypeptide of interest.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by Unmunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. , 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra! .
  • Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
  • the hybridoma ceUs may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encodmg the monoclonal antibodies of the mvention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specificaUy to genes encoding the heavy and Ught chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host ceUs.
  • the DNA also may be modified, for example, by substitoting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobuUn polypeptide.
  • non-immunoglobulm polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen- combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • the anti-PRO polypeptide antibodies of the invention may further comprise humanized antibodies or human antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typicaUy that of a human immunoglobulin [Jones et al, Nature, 221: 522-525 (1986); Riechmann et al, Nature, 222:323-329 (1988); and Presta, Curr. Op. Struct. Biol , 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • Humanization can be essentiaUy performed foUowing the method of Winter and co-workers [Jones et al, Nature, 321 : 522-525 (1986); Riechmann et al, Nature, 222:323-327 (1988); Verhoeyen et al, Science, 222:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the PRO polypeptide, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structore. The purification of the conect molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, EMBO J.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy- chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy- chain constant region
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
  • the antibodies may be prepared in vitro usmg known methods in synthetic protein chemistry, includmg those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose mclude iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • anti-PRO polypeptide antibodies of the invention have various utilities.
  • anti-PRO polypeptide antibodies may be used in diagnostic assays for a PRO polypeptide, e.g. , detecting its expression in specific cells, tissues, or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques. CRC Press, Inc. (1987) pp. 147-158].
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 1 C, 32 P, 35 S, o 25 I, a fluorescent or chemilumUiescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • a radioisotope such as 3 H, 1 C, 32 P, 35 S, o 25 I
  • a fluorescent or chemilumUiescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • Any method known in the art for conjugating the antibody to the detectable moiety may be employed, includmg those methods described by Hunter et al, Nature, 144:945 (1962);
  • Anti-PRO polypeptide antibodies also are useful for the affinity purification of PRO polypeptide from recombinant cell culture or natoral sources.
  • the antibodies against the PRO polypeptide are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the PRO polypeptide to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the PRO polypeptide, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the PRO polypeptide from the antibody.
  • therapeutic indications mclude disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (e.g., enterocoUtis, ZolUnger-EUison syndrome, gastrointestinal ulceration and congenital microvillus atrophy), skin diseases associated with abnormal keratinocyte differentiation (e.g., psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.
  • disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions e.g., enterocoUtis, ZolUnger-EUison syndrome, gastrointestinal ulceration and congenital microvillus atrophy
  • skin diseases associated with abnormal keratinocyte differentiation e.g., psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.
  • FGF-8 has been implicated in cellular differentiation and embryogenesis, includmg the patterning which appears during limb formation. FGF-8 and the PRO 187 molecules of the invention therefore are likely to have potent effects on ceU growth and development. Diseases which relate to ceUular growth and differentiation are therefore suitable targets for therapeutics based on functionaUty similar to FGF-8. For example, diseases related to growth or survival of nerve cells mcludmg Parkinson's disease, Alzheimer's disease, ALS, neuropathies. Additionally, disease related to uncontrolled cell growth, e.g., cancer, would also be expected therapeutic targets.
  • Native PR0533 is a 216 amino acid polypeptide of which residues 1-22 are the signal sequence. Residues
  • 3 to 216 have a Blast score of 509, corresponding to 53 % homology to fibroblast growth factor.
  • DNA47412 the EST from which PCR oligos were generated to isolate the full length DNA49435-1219, has been observed to map to llpl5. Sequence homology to the llpl5 locus would indicate that PR0533 may have utility in the treatment of Usher Syndrome or Atrophia areata.
  • fibroblast growth factors can act upon cells in both a mitogenic and non-mitogenic manner.
  • Non-mitogenic actions of fibroblast growth factors include promotion of cell migration into a wound area (chemotaxis), initiation of new blood vessel formulation (angiogenesis), modulation of nerve regeneration and survival (neurotrophism), modulation of endocrine functions, and stimulation or suppression of specific ceUular protein expression, extraceUular matrix production and cell survival. Baird, A. & Bohlen, P., Handbook of Exp. Phrmacol. 25(1): 369418 (1990). These properties provide a basis for using fibroblast growth factors in therapeutic approaches to accelerate wound healmg, nerve repair, collateral blood vessel formation, and the like. For example, fibroblast growth factors, have been suggested to minimize myocardium damage in heart disease and surgery (U.S. P. 4,378,437). Antibodies to these factors can be generated to modulate such effects.
  • Therapeutic indications for PR0214 polypeptides include disorders associated with the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal lesions (e.g., enterocolitis,
  • Zollinger-Eltison syndrome gastrointestinal ulceration and congenital microvillus atrophy
  • skin diseases associated with abnormal keratinocyte differentiation e.g., psoriasis, epithelial cancers such as lung squamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.
  • Anti-PR0317 antibodies find use in anti-tumor indications if they are angiostatic, or in coronary ischemic indications if they are angiogenic
  • Native PRO301 (SEQ ID NO:119) has a Blast score of 246 and 30% homology at residues 24 to 282 of Fig. 44 with A33_HUMAN, an A33 antigen precursor.
  • A33 antigen precursor as explained in the Background is a tumor-specific antigen, and as such, is a recognized marker and therapeutic target for the diagnosis and treatment of colon cancer. The expression of tumor-specific antigens is often associated with the progression of neoplastic tissue disorders.
  • Native PRO301 (SEQ ID NO: 119) and A33 HUMAN also show a Blast score of 245 and 30% homology at residues 21 to 282 of Fig. 44 with A33 HUMAN, the variation dependent upon how spaces are inserted mto the compared sequences.
  • Native PRO301 also has a Blast score of 165 and 29% homology at residues 60 to 255 of Fig. 44 with HS46KDA_1, a human coxsackie and adenovirus receptor protein, also known as ceU surface protem HCAR. This region of PRO301 also shows a similar Blast score and homology with HSU90716_1. Expression of such proteins is usually associated with viral infection and therapeutics for the prevention of such infection may be accordingly conceived. Accordingly, antibodies to the above identified antigens and receptors have therapeutic potential as diagnostic and treatment techniques.
  • PR0234 polypeptides of die invention mcludes treatments associated with leukocyte homing or the interaction between leukocytes and the endothelium during an inflammatory response. Examples include asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.
  • Cancer-associated or specific antigens permit the creation of tumor or cancer specific monoclonal antibodies (mAbs) which are specific to such tumor antigens.
  • mAbs monoclonal antibodies
  • Such mAbs which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease.
  • mAbs cancer specific monoclonal antibodies
  • Such mAbs which can distinguish between normal and cancerous cells are useful in the diagnosis, prognosis and treatment of the disease.
  • Particular antigens are known to be associated with neoplastic diseases, such as colorectal and breast cancer. Since colon cancer is a widespread disease, early diagnosis and treatment is an important medical goal.
  • Diagnosis and treatment of cancer can be implemented usmg monoclonal antibodies (mAbs) specific therefore having fluorescent, nuclear magnetic or radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs can be used for treatment in situ with minimal patient description.
  • EXAMPLE 1 Extracellular Domain Homology Screening to Identify Novel Polypeptides and cDNA Encodmg Therefor
  • the extraceUular domain (ECD) sequences (mcludmg the secretion signal sequence, if any) from about 950 known secreted proteins from the Swiss-Prot pubUc database were used to search EST databases.
  • the EST databases included public databases (e.g., Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQTM, Incyte Pharmaceuticals, Palo Alto, CA).
  • oligonucleotides were then synthesized and used to identify by PCR a cDNA library that contained the sequence of interest and for use as probes to isolate a clone of the full-length coding sequence for a PRO polypeptide.
  • Forward (.f) and reverse (.r) PCR primers generally range from 20 to 30 nucleotides and are often designed to give a PCR product of about 100-1000 bp in length.
  • the probe (.p) sequences are typically 40-55 bp in length.
  • additional oligonucleotides are synthesized when the consensus sequence is greater than about l-1.5kbp.
  • DNA from the libraries was screened by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology, with the PCR primer pair. A positive library was then used to isolate clones encoding the gene of interest usmg the probe oligonucleotide and one of the primer pairs.
  • the cDNA libraries used to isolate the cDNA clones were constructed by standard methods using commerciaUy avaUable reagents such as those from Invitrogen, San Diego, CA.
  • the cDNA was primed with oligo dT containing a Notl site, linked with blunt to Sail hemikinased adaptors, cleaved with Notl, sized appropriately by gel electrophoresis, and cloned in a defined orientation into a suitable cloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes et al., Science. 253:1278-1280 (1991)) in the unique Xhol and Notl sites.
  • a suitable cloning vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the Sfil site; see, Holmes

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Abstract

La présente invention concerne des polypeptides et des molécules d'acides nucléiques codant ces polypeptides. L'invention concerne également des vecteurs et des cellules hôtes comprenant ces séquences d'acides nucléiques. L'invention concerne ensuite des molécules de polypeptides chimériques où les polypeptides de l'invention sont fusionnés à des séquences de polypeptides hétérologues. L'invention concerne aussi des anticorps qui se lient aux polypeptides de l'invention. L'invention concerne enfin des procédés de production des polypeptides de l'invention.
EP98946090A 1997-09-17 1998-09-16 Polypeptides secretes et transmembranaires et acides nucleiques les codant Withdrawn EP1027434A2 (fr)

Priority Applications (12)

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DK01127794T DK1207168T3 (da) 1997-09-17 1998-09-16 Polypeptider og nucleinsyrer kodende derfor
EP04014066A EP1464654A3 (fr) 1997-10-28 1998-09-16 Protéines sécrétées et transmembranaire et acides nucléiques les codant
DK01127792T DK1205489T3 (da) 1997-11-21 1998-09-16 Polypeptider og nucleinsyrer kodende derfor
ES05024504T ES2333880T3 (es) 1997-09-17 1998-09-16 Polipeptidos y acidos nucleicos que los codifican.
EP01127794A EP1207168B1 (fr) 1997-09-17 1998-09-16 Polypeptide et acide nucleotidique le codant
DK01127795T DK1217006T3 (da) 1997-10-28 1998-09-16 Polypeptider og nucleinsyrer kodende for samme
EP01127792A EP1205489B1 (fr) 1997-11-21 1998-09-16 Polypeptide et polynucléotide le codant
EP01127791A EP1205546B1 (fr) 1997-10-24 1998-09-16 Polypeptide et acide nucléique le codant
DK01127791T DK1205546T3 (da) 1997-10-24 1998-09-16 Polypeptider og nucleinsyrer kodende derfor
EP01127795A EP1217006B1 (fr) 1997-10-28 1998-09-16 Polypeptides et acides nucléiques le codant
EP01127793A EP1205550B1 (fr) 1997-09-18 1998-09-16 Polypeptide et acide nucléique le codant
EP05024504A EP1659131B1 (fr) 1997-09-17 1998-09-16 Polypeptides et acides nucléiques les codant

Applications Claiming Priority (86)

Application Number Priority Date Filing Date Title
US5912297P 1997-09-17 1997-09-17
US5911397P 1997-09-17 1997-09-17
US5912197P 1997-09-17 1997-09-17
US5911797P 1997-09-17 1997-09-17
US5911597P 1997-09-17 1997-09-17
US5911997P 1997-09-17 1997-09-17
US5918497P 1997-09-17 1997-09-17
US59113P 1997-09-17
US59121P 1997-09-17
US59122P 1997-09-17
US59115P 1997-09-17
US59184P 1997-09-17
US59117P 1997-09-17
US59119P 1997-09-17
US5926397P 1997-09-18 1997-09-18
US5926697P 1997-09-18 1997-09-18
US59266P 1997-09-18
US59263P 1997-09-18
US6212597P 1997-10-15 1997-10-15
US62125P 1997-10-15
US6228797P 1997-10-17 1997-10-17
US6228597P 1997-10-17 1997-10-17
US62287P 1997-10-17
US62285P 1997-10-17
US6348697P 1997-10-21 1997-10-21
US63486P 1997-10-21
US6312897P 1997-10-24 1997-10-24
US6312797P 1997-10-24 1997-10-24
US6281497P 1997-10-24 1997-10-24
US6304597P 1997-10-24 1997-10-24
US6312097P 1997-10-24 1997-10-24
US6281697P 1997-10-24 1997-10-24
US6312197P 1997-10-24 1997-10-24
US62814P 1997-10-24
US63127P 1997-10-24
US63121P 1997-10-24
US63045P 1997-10-24
US63128P 1997-10-24
US63120P 1997-10-24
US62816P 1997-10-24
US6332797P 1997-10-27 1997-10-27
US6332997P 1997-10-27 1997-10-27
US63329P 1997-10-27
US63327P 1997-10-27
US6354497P 1997-10-28 1997-10-28
US6355097P 1997-10-28 1997-10-28
US6356497P 1997-10-28 1997-10-28
US6354297P 1997-10-28 1997-10-28
US6354997P 1997-10-28 1997-10-28
US6354197P 1997-10-28 1997-10-28
US63541P 1997-10-28
US63564P 1997-10-28
US63550P 1997-10-28
US63542P 1997-10-28
US63549P 1997-10-28
US63544P 1997-10-28
US6343597P 1997-10-29 1997-10-29
US6373297P 1997-10-29 1997-10-29
US6373597P 1997-10-29 1997-10-29
US6373497P 1997-10-29 1997-10-29
US6421597P 1997-10-29 1997-10-29
US6370497P 1997-10-29 1997-10-29
US6373897P 1997-10-29 1997-10-29
US63734P 1997-10-29
US63704P 1997-10-29
US63735P 1997-10-29
US64215P 1997-10-29
US63738P 1997-10-29
US63732P 1997-10-29
US63435P 1997-10-29
US64103P 1997-10-31
US63870P 1997-10-31
US64248P 1997-11-03
US64809P 1997-11-07
US65186P 1997-11-12
US65846P 1997-11-17
US65693P 1997-11-18
US66364P 1997-11-21
US66120P 1997-11-21
US66453P 1997-11-24
US66770P 1997-11-24
US66511P 1997-11-24
US66466P 1997-11-24
US66772P 1997-11-24
US66840P 1997-11-25
PCT/US1998/019330 WO1999014328A2 (fr) 1997-09-17 1998-09-16 Polypeptides secretes et transmembranaires et acides nucleiques les codant

Related Child Applications (7)

Application Number Title Priority Date Filing Date
EP04014066A Division EP1464654A3 (fr) 1997-10-28 1998-09-16 Protéines sécrétées et transmembranaire et acides nucléiques les codant
EP01127791A Division EP1205546B1 (fr) 1997-10-24 1998-09-16 Polypeptide et acide nucléique le codant
EP01127795A Division EP1217006B1 (fr) 1997-10-28 1998-09-16 Polypeptides et acides nucléiques le codant
EP01127794A Division EP1207168B1 (fr) 1997-09-17 1998-09-16 Polypeptide et acide nucleotidique le codant
EP01127793A Division EP1205550B1 (fr) 1997-09-18 1998-09-16 Polypeptide et acide nucléique le codant
EP05024504A Division EP1659131B1 (fr) 1997-09-17 1998-09-16 Polypeptides et acides nucléiques les codant
EP01127792A Division EP1205489B1 (fr) 1997-11-21 1998-09-16 Polypeptide et polynucléotide le codant

Publications (1)

Publication Number Publication Date
EP1027434A2 true EP1027434A2 (fr) 2000-08-16

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EP98946090A Withdrawn EP1027434A2 (fr) 1997-09-17 1998-09-16 Polypeptides secretes et transmembranaires et acides nucleiques les codant

Country Status (1)

Country Link
EP (1) EP1027434A2 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9914328A3 *

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