Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• This invention relates to newly identified polynucleotides, polypeptides encoded by these polynucleotides, antibodies that bind these polypeptides, uses of such polynucleotides, polypeptides, and antibodies, and their production.
• sorting signals are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
• One type of sorting signal called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER).
• ER endoplasmic reticulum
• both groups of proteins can be further directed to another organelle called the Golgi apparatus.
• the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.
• Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein.
• vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space - a process called, exocytosis: Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered.
• proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a "linker" holding the protein to the membrane.
• the present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
• isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
• isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
• a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
• the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
• polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
• the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5 ' . or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25> 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• a "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC.
• the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted ' protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
• a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
• the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
• a representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.
• the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
• the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
• a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC.
• “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C; Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions.
• Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
• washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
• blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
• the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• polynucleotide which hybridizes only to polyA ⁇ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
• the polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
• the polypeptide of the present invention can be composed of amino acids joined to each other by peptide "bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
• the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
• polypeptides may be branched , for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, -and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
• SEQ ID NO:X refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in . Table 1.
• a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.) Polynucleotides and Polypeptides of the Invention
• polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group consisting of:
• DSHPSPSVTS (SEQ ID NO: 51) and/or MLSRPRCGPRPKDWLLDASSRLSETRRDSRPKGGQPVSCFSAGSRVPAGVGV
• polypeptides encoding these polypeptides are also encompassed by the invention.
• fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 91%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement there of are encompassed by the invention.
• Antibodies that bind polypeptides of the invention are also encompassed by the invention.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the translation product of this gene shares sequence homology with metallocarboxypeptidase (see, e.g., Genbank Accession AAD15985; all references available through this accession are hereby incorporated by reference herein, for example, DNA Cell Biol. 18, 175-185 (1999)) which is thought to play a role in development.
• Carboxypeptidases are enzymes that remove amino acids off the carboxyl end of polypeptides. which is thought to be important in processing neuroendocrine peptides.
• the translation product of this clone is expected to share at least some biological activities with carboxypetidase proteins. Such activities are known in the art, some of which are described elsewhere herein.
• This gene is expressed primarily in the developing human fetus (e.g., whole fetus, fetal bone, and the pluripotent human embryonic carcinoma cell line NTERA2) and to a lesser extent in B-cells.
• Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: developmental disorders and immunological disorders.
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken from an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• tissue distribution of the expression of this gene and homology to a carboxypeptidase indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for the detection, diagnosis, and treatment of developmental disorders, particularly those affecting bone tissue.
• Expression within embryonic tissue and other cellular sources marked by proliferating cells suggests that this gene may play a role in the regulation of cellular division.
• the polynucleotides and or polypeptides corresponding to this gene may show utility in the diagnosis and treatment of cancer and other proliferative disorders.
• embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation.
• this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy.
• the expression of this gene in B-cells indicates it could be involved in the differentiation and activation of B cells, for example,
• the polynucleotides and/or polypeptides corresponding to this gene are useful for the treatment or detection of immune or hematopoietic disorders including arthritis, asthma, autoimmune diseases, immunodeficiency diseases, leukemia, and also in treatment of cancer patients with a depleted immune system.
• the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 ahd may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides.
• a-b comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3763 of SEQ ID NO:l 1, b is an integer of 15 to 3777, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:l 1, and where b is greater than or equal to a + 14.
• polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention.
• polypeptides of the invention comprise, or alternatively consist of the following amino acid sequence: IRHERARRATERSASSSMAGLRi ⁇ QPGCYCRTAAAVNLLLGVFQVLLPCCRP GGAQGQAIEPLPNVVELWQAEEGELLLPTQGDSEEGLEEPSQEQSFSDKLFSG KGLHFQPSVLDFGIQFLGHPVAKILHAYNPSRDSEVWNSVFAAAGHFHVPP VPCRVIPAMGKTSSELFSYLTEEGSI (SEQ ID NO: 53).
• hematopoietic cells and tissues e.g., fetal liver spleen.
• Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: infectious diseases and immune/hematopoietic disorders and diseases.
• polypeptides and antibodies directed to. these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken from an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• this gene in stimulated B-cells and dendritic cells suggests that the gene could be important for the treatment or detection of Immune or hematopoietic disorders including arthritis, leukemia, and also in treatment of cancer patients with a depleted immune system.
• Expression of this gene in cells of the immune system, particularly in germinal center B cells indicates that the polynucleotides and/or polypeptides corresponding to this gene, (and/or antibodies raised against those polypeptides) are useful to treat infectious diseases and disorders.
• Expression of this gene in germinal center B cells which are known to be activated B cells undergoing an immune response, isotype switching and somatically hypermutating.
• the polynucleotides and/or polypeptides corresponding to this gene may have clinical utility as an agent to bolster immune responses, by for example, inducing isotype switching or the production of higher affinity antibodies.
• the polynucleotides and/or polypeptides corresponding to this gene could also be used to detect, diagnose or treat patients with immune system disorders such as immunodeficiencies (e.g, CVID, SCID and X linked agammaglobulinemia) autoimmune diseases (e.g. lupus, Rheumatoid arthritis, Sjorgen's syndrome), allergies, and asthma.
• immunodeficiencies e.g, CVID, SCID and X linked agammaglobulinemia
• autoimmune diseases e.g. lupus, Rheumatoid arthritis, Sjorgen's syndrome
• allergies e.g. lupus, Rheumatoid arthritis, Sjorgen's syndrome
• the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. '
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 12 Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention.
• related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a.
• nucleotide sequence described by the general formula of a-b where a is any integer between 1 to 1699 of SEQ ID NO: 12, b is an integer of 15 to 1713, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a + 14.
• the nucleotide sequence of this gene shares sequence homology with human 17-beta-hydroxysteroid dehyrogenase IV (human 17beta-HSD TV) (See, e.g., Genbank Accession AF057740; all references available through this accession are hereby incorporated by reference herein), which is thought to be involved in inhibiting the stimulation of FSH secretion a multifunctional enzyme that is localized in the peroxisomes.
• the N-terminal part has dehydrogenase activity, the central part has hydratase activity, and the carboxy-terminal part is responsible for sterol transport. Mutations in the human 17beta-HSD IV cDNA can lead to a severe peroxisomal disorder. Based on the sequence similarity, the translation product of this clone is expected to share at least some biological activities with human 17beta-HSD TV proteins. Such activities are known in the art, some of which are described elsewhere herein.
• This gene is expressed primarily in ovarian cancer.
• Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: ovarian cancer.
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken irom an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for the diagnosis and treatment cancers, especially ovarian tumors.
• polynucleotides and polypeptides corresponding to this gene may have utility in monitoring, inhibiting or preventing tumor progression or metastases, or even promoting tumor regression.
• the expression in ovarian tissue indicates that the polynucleotides and/or polypeptides corresponding to this gene, (and/or antibodies raised against those polypeptides) are useful in the detection, diagnosis, and treatment of diseases and disorders of the ovary (e.g., oophoritis, polycystic ovary syndrome, ovarian cancer, and menstral disorders).
• the protein may also be used to determine unknown biological activities (as well as antibodies raised against those polypeptides), to raise antibodies, ' as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 921 of SEQ ID NO: 13, b is an integer of 15 to 935, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13, and where b is greater than or equal to a + 14.
• polypeptides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: ovarian cancer.
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken from an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for the diagnosis and treatment cancers, especially ovarian tumors.
• polynucleotides and polypeptides corresponding to this gene may have utility in monitoring, inhibiting or preventing tumor progression or metastases, or even promoting tumor regression.
• the expression in ovarian tissue indicates that the polynucleotides and/or polypeptides corresponding to this gene, (and/or antibodies raised against those polypeptides) are useful in the detection, diagnosis, and treatment of diseases and disorders of the ovary (e.g., oophoritis, polycystic ovary syndrome, ovarian cancer, and menstral disorders).
• the protein may also be used to determine unknown biological activities (as well as antibodies raised against those polypeptides), to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 610 of SEQ ID NO: 14, b is an integer of 15 to 624, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.
• the translation product of this gene has homology to citrate lyase (See e.g., GenBank Accession Number CAB02709; all references available through this accession are hereby incorporated by reference herein) which is involved in lipid metabolism. Based on the sequence similarity, the translation product of this clone is . expected to share at least some biological activities with citrate lyase proteins. Such activities are known in the art, some of which are described elsewhere herein.
• This gene is expressed primarily in the digestive system (e.g., colon intestine, liver, colon), human embryo, and in cells of the immune system (e.g., macrophages, primary dendritic cells, fetal liver spleen).
• the digestive system e.g., colon intestine, liver, colon
• the immune system e.g., macrophages, primary dendritic cells, fetal liver spleen.
• Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: diseases and disorders of the digestive system (e.g., ulcerative colitis) and the immune system.
• diseases and conditions which include but are not limited to: diseases and disorders of the digestive system (e.g., ulcerative colitis) and the immune system.
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken from an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• tissue distribution of the expression of this gene indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for detection, diagnosis and/or treatment of diseases and disorders of the digestive system such as ulcerative colitis, cancers of the digestive system - especially colon cancer, Crohn's disease, divertivcular disease, irritable bowel syndrome, celiac sprue and digestive disorders associated with the maladsorption of nutrients across the gut lining or secretion of substances (enzymes, mucus) into the gut.
• the expression of this gene primarily in the embryo, suggests a key role in embryo development and could be used in the treatment and or detection of embryonic development defects.
• this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other pro liferative disorders.
• embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation.
• this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy.
• Expression of this gene product in antigen presenting cells such as B cells and dendritic cells indicates that it plays a role in mediating responses to infection and controlling immunological responses, such as those that occur during immune surveillance.
• tissue distribution indicates that the polypeptides or polynucleotides are useful for treatment, prophylaxis, and diagnosis of immune and autoimmune diseases, such as lupus, transplant rejection, allergic reactions, arthritis, asthma, immunodeficiency diseases, leukemia, and AIDS.
• the polypeptides or polynucleotides of the present invention are also useful in the treatment, prophylaxis, and detection of thymus disorders, such as Graves Disease, lymphocytic thyroiditis, hyperthyroidism, and hypothyroidism.
• the expression observed predominantly in dendritic cells also indicates that the polynucleotides or polypeptides are important in treating and/or detecting graft versus host reaction, graft versus host disease, transplant rejection, myelogenous leukemia, bone marrow fibrosis, and myeloproliferative disease.
• the polypeptides or polynucleotides are also useful to enhance or protect proliferation, differentiation, and functional activation of hematopoietic progenitor cells (e.g., developing T cells), useful in treating cancer patients undergoing chemotherapy or patients undergoing bone marrow transplantation.
• polypeptides corresponding to this gene may also be useful in the treatment or prevention of infectious diseases, for example by boosting the immune response to an antigen.
• the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 15 Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1268 of SEQ ID NO: 15, b is an integer of 15 to 1282, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a + 14.
• polypeptides of the invention comprise, or alternatively consist of the following amino acid sequence selected from the group consisting of:
• polypeptides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: diseases and disorders of the immune system and bones (such as bone cancer, osteoporosis, osteopetrosis, and arthritis).
• diseases and conditions which include but are not limited to: diseases and disorders of the immune system and bones (such as bone cancer, osteoporosis, osteopetrosis, and arthritis).
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue or cell types e.g., cancerous and wounded tissues
• bodily fluids e.g., serum, plasma, urine, synovial fluid and spinal fluid
• another tissue or sample taken from an individual having such a disorder relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.
• tissue distribution of the expression of this gene in bone marrow, a major site of hematopoiesis suggests that the polynucleotides and/or polypeptides corresponding to this gene (and/or antibodies raised against those polypeptides) are useful for the treatment or detection of immune or hematopoietic disorders (such as arthritis, asthma, immunodeficiency diseases, leukemia, and anemia).
• immune or hematopoietic disorders such as arthritis, asthma, immunodeficiency diseases, leukemia, and anemia.
• polypeptides and/or polypeptides corresponding to this gene are useful in the detection, diagnosis and treatment of diseases and disorders of the bone including but not limited to: Ewing's sarcoma, osteoporosis, osteoarthritis, achoondroplasia, osteogenesis imperfecta, rickets, osteomalacia, gigantism, bone cysts, bone cancer, Paget's disease, and rheumatoid arthritis.
• tissue distribution of this gene in cancers of the lung and breast indicate that the gene may also be important in diagnosis or treatment of cancers and other proliferative disorders.
• the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 16 amino acid sequences
• amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2128 of SEQ ID NO:16, b is an integer of 15 to 2142, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 16, and where b is greater than or equal to a
• polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention.
• polypeptides of the invention comprise, or alternatively consist of the following amino acid sequence: TRPPRAAGPGVPEPRAAPLPLLAMAPPPACRSPMSPPPPLLLLLLLSLALLGAR ARAEPA
• GSAVPAQSRPCVDCHAFEFMQRALQDLRKTACSLDARTETLLLQAERRALC ACWPAGH SEQ ID NO: 55.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• fragments and variants of these polypeptides (such as, for example, fragments as. described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement there of are encompassed by the invention.
• Antibodies that bind polypeptides of the invention are also encompassed by the invention.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• This gene is expressed primarily in tissues and cells of the reproductive system (e.g. testis, ovarian cancer, endometrial stromal cells), and in the colon.
• reproductive system e.g. testis, ovarian cancer, endometrial stromal cells
• Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: diseases and disorders of the gastrointestinal system (such as colon cancer and diverticulitis) and reproductive system (such as ovarian cancer).
• diseases and conditions which include but are not limited to: diseases and disorders of the gastrointestinal system (such as colon cancer and diverticulitis) and reproductive system (such as ovarian cancer).
• polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
• tissue distribution of the expression of this gene indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis or treatment of tumor progression, in particular in carcinomas of the ovary and endometrium.
• this gene product is useful in the treatment of infertility, impotence, oophoritis, polycystic ovary syndrome, ovarian cancer, endometriosis, and menstral disorders.
• Expression of this gene in the colon indicates that the polynucleotides and/or polypeptides corresponding to this gene (and/or antibodies raised against those polypeptides) are useful in the detection, diagnosis and treatment of diseases and disorders of the colon including ulcerative colitis and colon cancer.
• the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
• Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 451 of SEQ ID NO:17, b is an integer of 15 to 465, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a + 14.
• Table 1 summarizes the information corresponding to each "Gene No.” described above.
• the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous ("overlapping") sequences obtained from the "cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones.
• the overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
• the cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones corresponding to the same gene. "Vector” refers to the type of vector contained in the cDNA Clone ID.
• Total NT Seq refers to the total number of nucleotides in the contig identified by "Gene No.”
• the deposited clone may contain all or most of these sequences, reflected, by the nucleotide position indicated as “5' NT of Clone Seq.” and the "3 ' NT of Clone Seq.” of SEQ ID NO:X.
• the nucleotide position of SEQ ID . NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon.”
• the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep.”
• the translated amino acid sequence beginning with the methionine, is identified as "AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques.
• the polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
• SEQ ID NO:Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep.”
• the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion.”
• the amino acid position of SEQ ID NO:Y of the last amino acid in the open reading frame is identified as "Last AA of ORF.”
• SEQ JD NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the-art and described further below.
• SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention.
• polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1. Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors.
• the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
• the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence.
• the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9%) identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
• the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1.
• the nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits.
• amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
• the present invention also relates to the genes corresponding to SEQ ID NO: 1
• the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
• allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue. '
• Table 2 provides predicted epitopes contained in certain embodiments of the invention and polynucleotide sequences that may be disclaimed according to certain embodiments of the invention.
• the first column refers to each "Gene #” described above in Table 1.
• the second column provides the sequence identifier, "NT SEQ ID NO:X”, for polynucleotide sequences disclosed in Table 1.
• the third column provides the sequence identifier, "AA SEQ ID NO:Y”, for polypeptide sequences disclosed in Table 1.
• the fourth column provides a unique integer "ntA” where "ntA” is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X
• the fifth column provides a unique integer "ntB” where "nfB” is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both ntA and ntB correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where ntB is greater than or equal to a + 14.
• This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 2 as "Predicted epitopes". Polypeptides of the invention may possess one, two, three, four, five or more antigenic epitopes comprising residues described in Table 2. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
• Table 3 summarizes the expression profile of polynucleotides corresponding to the clones disclosed in Table 1.
• the first column provides a unique clone identifier, "Clone ID”, for a cDNA clone related to each contig sequence disclosed in Table 1.
• Column 2 "Library Code(s)” shows the expression profile of tissue and/or cell line libraries which express the polynucleotides of the invention.
• Each Library Code in column 2 represents a tissue/cell source identifier code corresponding to the Library Code and Library description provided in Table 5. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested.
• Table 4 provides a key to the Library Code disclosed in Table 3.
• Column 1 provides the Library Code disclosed in Table 3, column 2.
• Column 2 provides a description of the tissue or cell source from which the corresponding library was derived.
• Library codes corresponding to diseased Tissues are indicated in column 3 with the word "disease”.
• polypeptides of the invention can be prepared in any suitable manner.
• Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
• polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
• a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
• Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.
• the present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and or a cDNA contained in ATCC deposit Z.
• the present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z.
• Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.
• the present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ LD NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone.
• Polynucleotides encoding the mature forms are also encompassed by the invention.
• proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
• cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
• the present invention provides secreted polypeptides having a sequence shown in SEQ JD NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
• SEQ JD NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
• cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
• the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
• the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
• the predicted signal sequence will be capable of directing the secreted protein to the ER.
• the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below).
• a mammalian cell e.g., COS cells, as desribed below.
• polynucleotide and Polypeptide Variants The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.
• the present invention also encompasses variants of the polypeptide sequence disclosed in SEQ LD NO:Y and/or encoded by a deposited clone.
• “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
• the present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 91%, 98% or 99% identical to, for example, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence contained in a deposited cDNA clone or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO: Y, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in a deposited clone, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein).
• a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 91%, 98% or 99% identical to, for example, the nucleotide
• Polynucleotides which hybridize to these nucleic acid molecules under stringent •hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• the present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%o, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).
• nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
• nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• the query sequence may be an entire sequence shown inTable 1, the ORF (open reading frame), or any fragment specified as described herein.
• nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs.
• a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)).
• a sequence alignment the query and subject sequences are both DNA sequences.
• An RNA sequence can be compared by converting U's to T's.
• the result of said global sequence alignment is in percent identity.
• the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity.
• the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention.
• the final percent identity would be 90%.
• a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/ aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
• polypeptide having an amino acid sequence at least, for example, 95%> identical to a query amino acid sequence of the present invention
• the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
• alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• whether any particular polypeptide is at least 80%, 85%,
• the result of said global sequence alignment is in percent identity.
• the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention.
• the 10 unpaired residues represent 10%) of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from, the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%). h another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
• the variants may contain alterations in the coding regions, non-coding regions, or both.
• polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
• Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
• variants in which 5-10, 1-5, or 1-2 aniino acids are substituted, deleted, or added in any combination are also preferred.
• Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
• Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
• one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
• Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein.
• Gayle and coworkers J.
• the invention further includes polypeptide variants which show substantial biological activity.
• variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
• guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change. The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function.
• the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
• tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Tip, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
• variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification or (v) fusion of the polypeptide with another compound, such as albumin (including, but not limited to, recombinant albumin (see, e.g., U.S.
• a further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions.
• a peptide or polypeptide it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
• the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
• the present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.
• a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, o.r encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:Y.
• the nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
• a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID ⁇ NO:X.
• nucleotide fragments include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.
• polynucleotide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto, or the cDNA contained
• these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone.
• Protein (polypeptide) fragments maybe "freestanding,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
• Representative examples of polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region.
• polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length.
• polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1- 60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
• polypeptide and polynucleotide fragments characterized by structural of functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
• Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention.
• polynucleotides encoding these domains are also contemplated.
• polypeptide fragments are biologically active fragments.
• Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
• the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity.
• a polypeptide demonstrating a "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein.
• Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or Iigand for a polypeptide of the invention.
• polypeptides of the invention and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
• various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
• competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al, 1995, Microbiol. Rev. 59:94-123.
• physiological correlates of binding of a polypeptide of the invention to its substrates can be assayed.
• assays described herein may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo).
• Other methods will be known to the skilled artisan and are within the scope of the invention.
• the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ JD NO:X), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as dete ⁇ nined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about.15 to about 30 amino acids.
• Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
• Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
• Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
• Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
• Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art.
• immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
• the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
• a carrier protein such as an albumin
• immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
• Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• polypeptides of the present invention can be fused to heterologous polypeptide sequences.
• polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
• polypeptides and/or antibodies of the present invention may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
• polypeptides and/or antibodies of the present invention are fused with the mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety.
• polypeptides and/or antibodies of the present invention are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S.
• Patent 5,766,883 herein incorporated by reference in its entirety.
• Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) ' may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).
• polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
• Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al, Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813).
• antigens e.g., insulin
• FcRn binding partner such as IgG or Fc fragments
• IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide.
• an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
• alteration of polynucleotides corresponding to SEQ JD NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
• polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ JD NO. ⁇ , and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody- antigen binding).
• TCR T-cell antigen receptors
• Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• the immunoglobulin molecules of the invention are IgGl.
• the immunoglobulin molecules of the invention are IgG4.
• the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
• the antibodies of the invention may be from any animal origin including birds and mammals.
• the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
• the antibodies of the present invention may be monospecific, bispecific, • trispecific or of greater multispecificity- Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
• a heterologous epitope such as a heterologous polypeptide or solid support material.
• Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-teiminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
• Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
• Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.
• Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
• Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%), at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%> identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
• antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof.
• Antibodies that do not bind polypeptides with less than 95%, less ⁇ than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
• the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
• antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 "3 M, 5 X 10 "4 M, 10 "4 M, 5 X 10 "5 M, 10 ⁇ 5 M, 5 X 10 "6 M, 10 '6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 "8 M, 5 X 10 '9 M, 10 "9 M, 5 X 10- 10 M, 10 '10 M, 5 X 10 "H M, 10 "n M, 5 X 10 "12 M, 10"12 M, 5 X 10 '13 M, 10 '13 M, 5 X 10 '14 M, 10 "14 M, 5 X 10 "15 M, or 10 "15 M.
• the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
• the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
• Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
• antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
• the invention features both receptor-specific antibodies and ligand-specific antibodies.
• the invention also features receptor-specific antibodies which do not prevent Iigand binding but prevent receptor activation.
• Receptor activation i.e., signaling
• receptor activation can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• antibodies are provided that inhibit Iigand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
• the invention also features receptor-specific antibodies which both prevent Iigand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound Iigand.
• receptor-specific antibodies which both prevent Iigand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound Iigand.
• neutralizing antibodies which bind the Iigand and prevent binding of the Iigand to the receptor, as well as antibodies which bind the Iigand, thereby preventing receptor activation, but do not prevent the Iigand from binding the receptor.
• antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
• the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
• the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161 (4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al, J.
• Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
• the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
• the antibodies of the present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
• antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuchdes, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
• the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular Iigand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicarnycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• the antibodies of the present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
• a polypeptide of the invention can be administered to various host animals. including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
• adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
• the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated.
• the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
• Hybridomas are selected and cloned by limited dilution.
• the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
• Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
• the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
• Antibody fragments which recognize specific epitopes may be generated by known techniques.
• Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab. fragments) or pepsin (to produce F(ab')2 fragments).
• F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
• the antibodies of the present invention can also be generated using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
• phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
• Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
• Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage . gene III or gene VIII protein.
• Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol. Methods . 184:177-186 (1995); " Kettleborough et al., Eur. J. Immunol.
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
• Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
• Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and a framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
• Human antibodies are particularly desirable for therapeutic treatment of human patients.
• Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,881 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
• the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
• the mouse heavy and light chain immunoglobulin genes may be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
• the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
• Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
• a selected non-human monoclonal antibody e.g., a mouse antibody
• antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
• antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a Iigand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its Iigand.
• Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide Iigand.
• anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
• the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
• the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ JD NO. ⁇ .
• the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
• a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be
• nucleotide sequence and corresponding amino acid sequence of the. antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non- human antibody, as described supra.
• the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
• the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
• Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
• Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
• Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
• the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
• an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
• a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
• the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
• Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
• the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
• a variety of host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 : 1791 ( 1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
• the virus grows in Spodopterafrugiperda cells.
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• an AcNPV promoter for example the polyhedrin promoter
• a number of viral-based expression systems may be utilized.
• the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
• This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region ofthe viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame ofthe desired coding sequence to ensure translation ofthe entire insert.
• exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
• the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
• a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function ofthe protein.
• Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.
• eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used.
• Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D
• normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• stable expression is preferred.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
• adenine phosphoribosyltransferase genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci.
• the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture ofhost cell will increase the number of copies ofthe marker gene. Since the amplified region is associated with the antibody gene, production ofthe antibody will also increase (Grouse et al, Mol. Cell. Biol. 3:257 (1983)).
• the host cell may be co-transfected with two expression vectors ofthe invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• an antibody molecule ofthe invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• differential solubility e.g., differential solubility
• the antibodies ofthe present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids ofthe polypeptide) ofthe present invention to generate fusion proteins.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• the antibodies may be specific for antigens other ' than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids ofthe polypeptide) ofthe present invention.
• antibodies may be used to target the polypeptides ofthe present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides ofthe present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., . Harbor et al, supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.
• the present invention further includes compositions comprising the polypeptides ofthe present invention fused or conjugated to antibody domains other , than the variable regions.
• the polypeptides ofthe present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions.
• Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
• Methods for fusing or conjugating the polypeptides ofthe present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
• polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification.
• One reported example describes chimeric proteins consisting ofthe first two domains ofthe human CD4-polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
• polypeptides ofthe present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5
• Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
• the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
• the marker amino acid sequence is a hexa-histidine peptide,. such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• apQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311
• hexa- histidine provides for convenient purification ofthe fusion protein.
• Other peptide tags useful for purification include, but are not limited to,. the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
• the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
• an example of a luminescent material includes luminol
• examples of bioluminescent materials include luciferase, luciferin, and aequorin
• suitable radioactive material include 1251, 1311, l llln or99Tc.
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213BL
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fiuorouracil decarbazine), alkylating agents (e.g., mechlorethan ine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.
• the conjugates ofthe invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No.
• a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
• biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-1 interleukin-1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification ofthe target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc.
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• the antibodies ofthe invention maybe utilized for immunophenotyping of cell lines and biological samples.
• the translation product ofthe gene ofthe present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types.
• Monoclonal antibodies directed against a specific epitope, or combination of epitopes will allow for the screening of cellular populations expressing the marker.
• Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S.
• MRD minimal residual disease
• GVHD Graft-versus-Host Disease
• the antibodies ofthe invention may be assayed for immunospecific binding by any method known in the art.
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
• immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIP A buffer (1%.
• protein phosphatase and/or protease inhibitors e.g., EDTA, PMSFi, aprotinin, sodium vanadate
• the ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
• One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding ofthe antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
• immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
• Western blot analysis generally comprises preparing protein samples, electrophoresis ofthe protein samples in a polyacrylamide gel (e.g., 8%>- 20% SDS- PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3%> BSA or nonfat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence ofthe antigen.
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen.
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a second antibody conjugated to a detectable compound may be added following the addition ofthe antigen of interest to the coated well.
• ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
• the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection ofthe antibody bound to the labeled antigen.
• labeled antigen e.g., 3H or 125I
• the affinity ofthe antibody of interest for a particular antigen and the binding off -rates can be determined from the data by scatchard plot analysis.
• Competition with a second antibody can also be determined using radioimmunoassays.
• the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
• the present invention is further directed to antibody-based therapies which involve administering antibodies ofthe invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more ofthe disclosed diseases, disorders, or conditions.
• Therapeutic compounds ofthe invention include, but are not limited to, antibodies ofthe invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies ofthe invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
• the antibodies ofthe invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide ofthe invention, including, but not lirnited to, any one or more ofthe diseases, disorders, or conditions described herein.
• the treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary ofthe ways in which the antibodies ofthe present invention may be used therapeutically includes binding polynucleotides or polypeptides ofthe present invention locally or systemically in the body or by direct cytotoxicity ofthe antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• lymphokines or hematopoietic growth factors such as, e.g., IL-2, IL-3 and IL-7
• the antibodies of the ⁇ nvention maybe administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).
• treatments e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents.
• administration of products of a species origin or species reactivity in the case of antibodies
• human antibodies, fragments derivatives, analogs, or nucleic acids are administered ⁇ to a human patient for therapy or prophylaxis.
• polypeptides or polynucleotides of the present invention It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, ofthe present invention.
• Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides ofthe invention, including fragments thereof.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 " 4 M, 10 “4 M, 5 X 10 "5 M, 10 “5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 "7 M, 10 “7 M, 5 X 10 “8 M, 10 '8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 "10 M, 10 ⁇ 10 M, 5 X 10 "11 M, 10 "n M, 5 X 10 "12 M, 10 “12 M, 5 X lO -13 M, lO - 13 M, 5 X 10 "!4 M, 10 "14 M, 5 X 10 "15 M, and 10 "15 M.
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide ofthe invention, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect.
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at. a desired site in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad.
• the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, ofthe antibody.
• nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
• This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
• microparticle bombardment e.g., a gene gun; Biolistic, Dupont
• coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a Iigand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc.
• nucleic acid-ligand complexes can be formed in which the Iigand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
• the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
• viral vectors that contains nucleic acid sequences encoding an antibody ofthe invention are used.
• a retroviral vector can be used (see Miller et al, Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery ofthe gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al, Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644- 651 (1994); Kiem et al, Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
• adenovirus vectors are used.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo ofthe resulting recombinant cell.
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol.
• the technique should provide for the stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• Recombinant blood cells e.g., hematopoietic stem or progenitor cells
• Recombinant blood cells are preferably administered intravenously.
• the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass " any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood ⁇ fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71 :973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61 :771 (1986)).
• the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression ofthe nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription.
• the compounds or pharmaceutical compositions ofthe invention are preferably tested in " vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
• the effect ofthe compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition ofthe invention, preferably an antibody ofthe invention.
• the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below. .
• a compound ofthe invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor- mediated endocytosis (see, e.g., Wu and Wu, J. BioL Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
• Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
• the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• a protein including an antibody, ofthe invention
• care must be taken to use materials to which the protein does not absorb.
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et . al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
• the compound or composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al, Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al, Science 228:190 (1985); During et al, Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)).
• a controlled release system can be placed in proximity ofthe therapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
• the present invention also provides pharmaceutical compositions.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
• Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site ofthe injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• the compounds ofthe invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• the amount ofthe compound ofthe invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques.
• in vitro assays may optionally be employed . to help identify optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness ofthe disease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg ofthe patient's body weight.
• the dosage administered to a patient is between 0.1 mg/kg and 20 nig/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg ofthe patient's body weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of antibodies ofthe invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) ofthe antibodies by modifications such as, for example, lipidation.
• the invention also provides a pharmaceutical pack or kit comprising one or more'containers filled with one or more ofthe ingredients ofthe pharmaceutical compositions ofthe invention.
• Optionally associated with such cbntainer(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide ofthe invention.
• the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression ofthe polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
• the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression ofthe polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression ofthe polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development ofthe disease, or may provide a means for detecting the disease prior
• Antibodies ofthe invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096 (1987)).
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression ofthe polypeptide of interest.
• Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system
• the size ofthe subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m ⁇ c.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of
• the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or
• time interval following administration is 5 to 20 days or 5 to 10 days.
• monitoring ofthe disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• Presence ofthe labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• kits that can be used in the above methods.
• a kit comprises an antibody ofthe invention, preferably a purified antibody, in one or more containers.
• the kits ofthe present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
• the kits ofthe present invention further comprise a control antibody which does not react with the polypeptide of interest.
• kits ofthe present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest.
• Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen ofthe kit may also be attached to a solid support.
• the detecting means ofthe above-described kit includes a solid support to which said polypeptide antigen is attached.
• a kit may also include a non-attached reporter-labeled anti-human antibody.
• binding ofthe antibody to the polypeptide antigen can be detected by binding ofthe said reporter-labeled antibody.
• the invention includes a diagnostic kit for use in screening serum containing antigens ofthe polypeptide ofthe invention.
• the . diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
• the antibody is attached to a solid support.
• the antibody may be a monoclonal antibody.
• the detecting means ofthe kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods ofthe present invention.
• the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
• the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
• the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
• the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment ofthe protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
• sfreptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• the invention provides an assay system or kit for carrying out this diagnostic method.
• the kit generally includes a support with surface- bound ⁇ recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
• any polypeptide ofthe present invention can be used to generate fusion proteins.
• the polypeptide ofthe present invention when fused to a second protein, can be used as an antigenic tag.
• Antibodies raised against the polypeptide ofthe present invention can be used to indirectly detect the second protein by binding to the polypeptide.
• secreted proteins target cellular locations based on trafficking signals, the polypeptides ofthe present invention can be used as targeting molecules once fused to other proteins.
• domains that can be fused to polypeptides ofthe present invention include not only heterologous signal sequences, but also other heterologous functional regions.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• fusion proteins may also be engineered to improve characteristics ofthe polypeptide ofthe present invention. For instance, a region of additional amino acids, particularly charged amino acids, maybe added to the N-terminus ofthe polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation ofthe polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
• polypeptides of the present invention can be combined with parts ofthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• the Fc part in a fusion, protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• EP-A 0232 262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins, such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J.
• the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification ofthe fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification ofthe fusion protein.
• HA hemagglutinin protein
• the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
• the vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion ofthe transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end ofthe polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, 293, and Bowes melanoma cells
• plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE- 9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, ⁇ RIT5 available from Pharmacia Biotech, Inc.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ.pGAPZ, pGAPZalph, P PIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, CA).
• a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
• HPLC high performance liquid chromatography
• Polypeptides ofthe present invention can also be recovered from: products purified from natural, sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
• a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
• the polypeptides ofthe present invention may be glycosylated or may be non- glycosylated.
• polypeptides ofthe invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
• the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of . the amino acid to which the N-terminal methionine is covalently linked.
• the yeast Pichia pastoris is used to express the polypeptide ofthe present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
• a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O .
• This reaction is catalyzed by the enzyme alcohol oxidase.
• Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 . Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one ofthe two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% ofthe total soluble protein in Pichia pastoris.
• a heterologous coding sequence such as, for example, a polynucleotide ofthe present invention, under the transcriptional regulation of all or part ofthe AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
• the plasmid vector pPIC9K is used to express DNA encoding a polypeptide ofthe invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998.
• This expression vector allows expression and secretion of a protein ofthe invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
• PHO alkaline phosphatase
• yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, ⁇ PIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
• high-level expression of a heterologous coding sequence such as, for example, a polynucleotide ofthe present invention
• a heterologous coding sequence such as, for example, a polynucleotide ofthe present invention
• an expression vector such as, for example, pGAPZ or pGAPZalpha
• the invention also encompasses primary, secondary, and immortalized host cells, of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence); and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenous polynucleotides.
• endogenous genetic material e.g., coding sequence
• genetic material e.g., heterologous polynucleotide sequences
• heterologous control regions e.g., promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• heterologous control regions e.g., promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• polypeptides ofthe invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al, Nature, 310:105-111 (1984)).
• a polypeptide corresponding to a fragment of a polypeptide sequence ofthe invention can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro- amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (
• the invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular Iigand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation ofthe protein.
• the chemical moieties for derivitizatio ⁇ may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a . therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,0.00, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• the polyethylene glycol may have a branched structure.
• Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl Biochem. Biotechnol. 56:59-12 (1996); Vorobjev et al, Nucleosides Nucleotides 18:2145-2150 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
• polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains ofthe protein.
• attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG, to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• The' amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
• specific amino acid residues e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine
• amino acid residues e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof
• polyethylene glycol as an illustration ofthe present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terfninally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be ⁇ accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer is achieved.
• pegylation ofthe proteins ofthe invention may be accomplished by any number of means.
• polyethylene glycol may be attached to the protein either directly or by an intervening linker.
• Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. ofHematol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; . and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO CH 2 CF ).
• MPEG monmethoxy polyethylene glycol
• ClSO CH 2 CF tresylchloride
• polyethylene glycol is directly attached to amine groups ofthe protein.
• the invention includes protein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group. .
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
• U.S. Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG- succinimidylsuccinate, MPEG activated with l,l'-carbonyldiimidazole, MPEG- 2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG- succinate derivatives.
• the number of polyethylene glycol moieties attached to each protein ofthe invention may also vary.
• the pegylated proteins ofthe invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
• the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249- 304 (1992).
• the polypeptides ofthe invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers ofthe polypeptides ofthe invention, their preparation, and compositions (preferably, Therapeutics) containing them.
• the polypeptides ofthe invention are monomers, dimers, trimers or tetramers.
• the multimers ofthe invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers.
• the term homomer refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences.
• a homomer ofthe invention is a multimer containing only polypeptides having an identical amino acid sequence.
• a homomer ofthe invention is a multimer containing polypeptides having different amino acid sequences.
• the multimer ofthe invention is a homodimer ⁇ e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences).
• the homomeric multimer ofthe invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides ofthe invention.
• the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer ofthe invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers ofthe invention may be the result of hydrophobic, hydrophilic; ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers ofthe invention such as, for example, homodimers or homotrimers, are formed when polypeptides ofthe invention contact one another in solution.
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides ofthe invention contact antibodies to the polypeptides ofthe invention (including antibodies to the heterologous polypeptide sequence in a fusion protein ofthe invention) in solution.
• multimers ofthe invention are formed by covalent associations with and/or between the polypeptides ofthe invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein ofthe invention.
• covalent associations are between the heterologous sequence contained in a fusion protein ofthe invention (see, e.g., US Patent Number 5,478,925).
• the covalent associations are between the heterologous sequence contained in an Fc fusion protein ofthe invention (as described herein).
• covalent associations of fusion proteins ofthe invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
• two or more polypeptides ofthe invention are joined through peptide ' linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
• Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization ofthe proteins in which they are found.
• Leucine zippers were originally identified in several DNA-binding proteins. (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins.
• leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
• leucine zipper domains suitable for producing soluble multimeric proteins ofthe invention are those described in PCT application WO 94/10308, hereby incorporated by reference.
• Recombinant fusion proteins comprising a polypeptide ofthe invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
• Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.
• Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.
• One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al.
• proteins ofthe invention are associated by interactions between Flag® polypeptide sequence contained in- fusion proteins ofthe invention containing Flag® polypeptide seuqence.
• associations proteins ofthe invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins ofthe invention and anti- Flag® antibody.
• the multimers ofthe invention may be generated using chemical techniques known in the art.
• polypeptides desired to be contained in the multimers ofthe invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers ofthe invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polypeptides ofthe invention may -be routinely Un ⁇
• multimers ofthe invention may be generated using genetic engineering techniques known in the art.
• polypeptides contained in multimers ofthe invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polynucleotides coding for a homodimer ofthe invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides ofthe invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• the polynucleotides ofthe present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide ofthe present invention can be used as a chromosome marker.
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ JD NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ JD NO:X will yield an amplified fragment.
• somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sub localization ofthe polynucleotides can be achieved with panels of specific chromosome fragments.
• Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).
• FISH fluorescence in situ hybridization
• the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and or multiple chromosomes).
• the polynucleotides ofthe present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
• HAPPY mapping high range restriction mapping
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
• the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
• the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject.
• the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide ofthe present invention and a suitable container.
• the kit includes two polynucleotide probes defining an internal region of the polynucleotide ofthe present invention, where each probe has one strand containing a 31 'mer-end internal to the region.
• the probes may be useful as primers for polymerase chain reaction amplification.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide ofthe present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• measuring the expression level of polynucleotide ofthe present invention is intended qualitatively or quantitatively measuring or estimating the level ofthe polypeptide ofthe present invention or the level ofthe mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample).
• the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder.
• a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide ofthe present invention or mRNA. As indicated, biological samples in
• tissue biopsy is the preferred source.
• the method(s) provided above may preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support.
• the support may be a "gene chip” or a "biological chip” as described in US Patents 5,837,832, 5,874,219, and 5,856,174.
• a gene chip with polynucleotides ofthe present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e.
• the present invention encompasses polynucleotides ofthe present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art.
• PNA peptide nucleic acids
• the use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip.
• a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P.
• PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible.
• PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined ' with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15- mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis. The present invention is useful for detecting cancer in mammals. .
• pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism.
• c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated.
• a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA.
• Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, FL (1988).
• Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241 : 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding ofthe polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region ofthe gene involved in transcription (triple helix - see Lee et al., Nucl. Acids Res. 3:173 (1979); Cooney et al., Science 241 :456
• Polynucleotides ofthe present invention are also useful in gene therapy.
• One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
• the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
• Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.
• the polynucleotides are also useful for identifying individuals from minute biological samples.
• the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
• RFLP restriction fragment length polymorphism
• an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
• This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, orst ⁇ len, making positive identification. difficult.
• the polynucleotides ofthe present invention can be used as additional DNA markers for RFLP.
• the polynucleotides ofthe present invention can also be used as an alternative to RFLP, by determining the actual- base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique JD database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
• DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,urine,fecal matter, etc.
• body fluids e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,urine,fecal matter, etc.
• gene sequences amplified from polymorphic loci such as DQa class II HLA, gene, are used in forensic biology to identify individuals.
• polynucleotides ofthe present invention can be used as polymorphic markers for forensic purposes.
• reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
• Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences ofthe present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
• the polynucleotides ofthe present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
• a polypeptide ofthe present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
• protein expression in tissues can be studied with classical immunohistological methods.
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc)
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• proteins can also be detected in vivo by imaging.
• Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR.
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
• suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 1311, 112fr ⁇ , 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal.
• an appropriate detectable imaging moiety such as a radioisotope (for example, 1311, 112fr ⁇ , 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al., "Inimunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc.
• the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide ofthe present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
• a diagnostic method of a disorder which involves (a) assaying the expression of a polypeptide ofthe present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development ofthe disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
• a more definitive diagnosis of this type may allow health professionals to employ preventative measures
• polypeptides ofthe present invention can be used to treat, prevent, and/or diagnose disease.
• patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels ofthe polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to.
• the polypeptide e.g., insulin
• a different polypeptide e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins
• an oncogene or tumor supressor e.g., an oncogene or tumor supressor
• a receptor to reduce the activity of a membrane bound receptor by competing with it for free Iigand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement ofthe immune response to proliferative cells or tissues).
• free Iigand e.g., soluble TNF receptors used in reducing inflammation
• desired response e.g., blood vessel growth inhibition, enhancement ofthe immune response to proliferative cells or tissues.
• antibodies directed to a polypeptide ofthe present invention can also be used to treat, prevent, and/or diagnose disease.
• administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction ofthe polypeptide.
• administration of ah antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
• polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides ofthe present invention can be used to test the following biological activities. Gene Therapy Methods
• Another aspect ofthe present invention is to gene therapy methods for treatingor preventing disorders, diseases and conditions.
• the gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) 5. sequences into an animal to achieve expression of a polypeptide ofthe present
• This method requires a polynucleotide which codes for a polypeptide ofthe invention that operatively linked to a promoter and any other genetic elements necessary for the expression ofthe polypeptide by the target tissue.
• gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which 0 is herein incorporated by reference.
• cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide ofthe invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
• a polynucleotide DNA or RNA
• Such methods are well-known in the 5 art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J.
• the cells which are engineered are arterial cells.
• the arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
• the polynucleotide constructs can be 5 delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).
• the polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
• the polynucleotide ofthe invention is delivered as a naked 0 polynucleotide.
• naked polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like.
• the polynucleotides ofthe invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
• the polynucleotide vector constructs ofthe invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication.
• Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen.
• Other suitable vectors will be readily apparent to the skilled artisan.
• Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RS V) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters.
• the promoter also may be the native promoter for the polynucleotides ofthe invention.
• one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature ofthe polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production ofthe desired polypeptide for periods of up to six months.
• the polynucleotide construct ofthe invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
• Interstitial space ofthe tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone.
• the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
• an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight.
• the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
• this dosage will vary according to the tissue site of injection.
• the appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues.
• parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes ofthe nose.
• naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
• the naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.
• the constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
• the polynucleotide constructs ofthe invention are complexed in a liposome preparation.
• Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
• cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
• Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem.,
• Cationic liposomes are readily available.
• N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference).
• Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
• Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.
• DOTAP l,2-bis(oleoyloxy)-3-(trimethylammonio)propane liposomes.
• DOTMA liposomes Preparation of DOTMA liposomes is explained in the literature, see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference.
• anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.
• Such materials include phosphatidyl, choline, cholesterol, ' phosphatidyl ethanolamine, dioleoylphosphatidyl choiine (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), _ among others.
• DOPC dioleoylphosphatidyl choiine
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphoshatidyl ethanolamine
• DOPC dioleoylphosphatidyl choline
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphosphatidyl ethanolamine
• DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water.
• the sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC.
• negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
• Other methods are known and available to those of skill in the art.
• the liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs. being preferred.
• MLVs multilamellar vesicles
• SUVs small unilamellar vesicles
• LUVs large unilamellar vesicles
• the various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology , 101:512-527 (1983), which is herein incorporated by reference.
• MLVs containing nucleic acid can be prepared by depositing a thin film of phospho lipid on the walls of a glass tube and subsequently hydrating with a solution ofthe material to be encapsulated.
• SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes.
• the material to be entrapped is added to a suspension of preformed MLVs and then sonicated.
• liposomes containing cationic lipids the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA.
• the liposome and DNA form a very stable complex due to binding ofthe positively charged liposomes to the cationic DNA.
• SUVs find use with small nucleic acid fragments.
• LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca 2+ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc.
• the ratio of DNA to liposomes will be from about 10:1 to about 1:10.
• the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
• U.S. Patent NO: 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
• cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides ofthe invention.
• Retro viruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloprohferative Sarcoma Virus, and mammary tumor virus.
• the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
• packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT- 19-17-H2, RCRE, RCRTP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy , 1 :5-14 (1990), which is incorporated herein by reference in its entirety.
• the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO precipitation.
• the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
• the producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides ofthe invention.
• retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
• the transduced eukaryotic cells will express polypeptides ofthe invention.
• cells are engineered, ex vivo or in vivo, with polynucleotides ofthe invention contained in an adenovirus vector.
• Adenovirus can be manipulated such that it encodes and expresses 'polypeptides ofthe invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration ofthe viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)).
• adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha- 1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434 (1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
• Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al, Cell , 68:143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein incorporated by reference.
• the adenovirus vector Ad2 is useful and can be grown in human 293 cells.
• These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products ofthe genes deleted from the vector.
• Ad2 other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
• the adenoviruses used in the present invention are replication deficient.
• Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles.
• the resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells.
• Replication deficient adenoviruses may be deleted in one or more of all or a portion ofthe following genes: Ela, Elb, E3, E4, E2a, or LI through L5. .
• the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV).
• AAV adeno-associated virus
• AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol., 158:97 (1992)). It is also one ofthe few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
• an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration.
• the polynucleotide construct containing polynucleotides ofthe invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989).
• the recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
• helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
• packaging cells Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct ofthe invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.
• Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g.
• Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein.
• the targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence.
• the targeting sequence will be sufficiently near the 5' end ofthe desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
• the promoter and the targeting sequences can be amplified using PCR.
• the amplified promoter contains distinct restriction enzyme sites on the 5 ' and 3' ends.
• the 3 ' end of the first targeting sequence contains the same restriction enzyme site as the 5 ' end ofthe amplified promoter and the 5 ' end ofthe second targeting sequence contains the same restriction site as the 3 ' end ofthe amplified promoter.
• the amplified promoter and targeting sequences are digested and ligated together.
• the promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above.
• the P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
• the promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control ofthe promoter. The promoter then drives the expression ofthe endogenous sequence.
• polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins.
• Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
• the polynucleotide encoding a polypeptide ofthe invention contains a secretory signal sequence that facilitates secretion ofthe protein.
• the signal sequence is positioned in the coding region ofthe polynucleotide to be expressed towards or at the 5 ' end ofthe coding region.
• the signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected.
• the signal sequence may be chemically synthesized using methods known in the art. Any mode of administration of any ofthe above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect.
• biolistic injectors particle accelerators (i.e., "gene guns”
• gelfoam sponge depots other commercially available depot materials
• osmotic pumps e.g., Alza minipumps
• oral or suppositorial solid (tablet or pill) pharmaceutical formulations e.g., osmotic pumps
• decanting or topical applications during surgery e.g., direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of
• a preferred method of local administration is by direct injection.
• a recombinant molecule ofthe present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
• Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
• Another method of local administration is to contact a polynucleotide construct ofthe present invention in or around a surgical wound.
• a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
• compositions useful in systemic administration include recombinant molecules ofthe present invention complexed to a targeted delivery vehicle ofthe present invention.
• Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
• Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA , 189:11277-11281 (1992), which is incorporated herein by reference).
• Oral delivery can be performed by c ⁇ mplexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art.
• Topical delivery can be performed by mixing a polynucleotide construct ofthe present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
• a lipophilic reagent e.g., DMSO
• Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity ofthe substance, the age and weight ofthe animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
• Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly
• polynucleotides or polypeptides, or agonists or antagonists ofthe present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
• Polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with the following systems.
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions ofthe immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
• Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
• immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
• a polypeptide of the invention may be used to treat diseases and disorders ofthe immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide ofthe invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1, column 8 (Tissue Distribution Library Code).
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies.
• B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late- onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency),
• IgM, IgG and IgA deficiency with increased IgM antibody deficiency - with normal or elevated Igs
• Ig heavy chain deletions Ig heavy chain deletions
• CVI common variable immunodeficiency
• Ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides ofthe invention, and/or agonists or antagonists thereof.
• Examples of congenital immunodeficiencies in which T cell and or B cell , function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22qll.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T- lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
• SCID severe combined immunodefici
• DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides ofthe invention, or antagonists or agonists thereof.
• Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides ofthe invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6- phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including Cl, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma,
• the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders.
• Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in ah immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, maybe an effective therapy in preventing autoimmune disorders.
• Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention include, but are not limited to, one or more ofthe following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemo lytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura., purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellit
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions ofthe invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and or prognosed with the compositions ofthe invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility
• glomerulonephritis often characterized, e.g., by glomerular basement membrane antibodies or immune complexes
• bullous pemphigoid often characterized, e.g., by IgG and complement in basement membrane
• Sjogren's syndrome often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)
• diabetes mellitus often characterized, e.g., by cell-mediated and humoral islet cell antibodies
• adrenergic drug resistance including adrenergic drug resistance with asthma or cystic fibrosis
• Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions ofthe invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often
• the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies ofthe present invention.
• rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• idiopathic thrombocytopenia purpura is treated, prevented, and or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• IgA nephropathy is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a immunosuppressive agent(s).
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells.
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, . and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia.
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
• Allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof.
• these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
• polypeptides or polynucleotides ofthe invention may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions.
• allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to modulate IgE concentrations in vitro or in vivo.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions.
• polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists ofthe invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions.
• Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia- reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokme induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AJDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocardit
• tissue-specific inflammatory disorders including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthit
• polypeptides, antibodies, or polynucleotides ofthe invention, and or agonists or antagonists thereof are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease.
• Organ rejection occurs by host immune cell destruction ofthe transplanted tissue through an immune response.
• an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
• Polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
• polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
• polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
• Polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented.
• the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen.
• polypeptides, antibodies, polynucleotides and/or agomsts or antagonists ofthe present invention are used as an adjuvant to enhance tumor-specific immune responses.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an adjuvant to enhance anti- viral immune responses.
• Anti- viral immune responses that may be enhanced using the compositions ofthe invention as an adjuvant include virus and virus associated diseases or symptoms described herein or otherwise known in the art.
• the compositions ofthe invention are used as an adjuvant to enhance an immime response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B).
• compositions ofthe invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
• a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses.
• Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions ofthe invention as an adjuvant include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art.
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
• compositions ofthe invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, and Borrelia burgdorferi.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an adjuvant to enhance anti-parasitic immune responses.
• Anti-parasitic immune responses that may be enhanced using the compositions ofthe invention as an adjuvant include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art.
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to a parasite.
• the compositions ofthe invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides ofthe invention.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
• an animal e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human
• an animal e.g., mouse
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a stimulator of B cell responsiveness to pathogens.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an activator of T cells.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent to induce higher affinity antibodies.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent to increase serum immunoglobulin concentrations.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent to accelerate recovery of immunocompromised individuals.
• polypeptides, antibodies, polynucleotides and or agonists or antagonists ofthe present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation).
• compositions ofthe invention may be administered prior to, concomitant with, and/or after transplantation.
• compositions ofthe invention are administered after transplantation, prior to the beginning of recovery of T-cell populations.
• compositions ofthe invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
• polypeptides, antibodies, polynucleotides and or agonists or antagonists ofthe present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function.
• Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency.
• Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo.
• said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used in the pretreatment of bone marrow samples prior to transplant.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno- incompetence/immunodeficiency such as observed among SCID patients.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
• polypeptides, antibodies, polynucleotides and or agonists or antagonists ofthe present invention are used as a means of regulating secreted cytokines that are elicited by polypeptides ofthe invention.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used in one or more ofthe applications decribed herein, as they may apply to veterinary medicine.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a means of blocking various aspects of immune responses to foreign agents or self.
• diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a therapy for chronic hypergammaglobulinemia evident in- such 'diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
• MGUS monoclonal gammopathy of undetermined significance
• Waldenstrom's disease Waldenstrom's disease
• related idiopathic monoclonal gammopathies and plasmacytomas.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used to enhance or inhibit complement mediated cell lysis.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may be employed to treat adult respiratory distress syndrome (ARDS).
• ARDS adult respiratory distress syndrome
• polypeptides, antibodies, polynucleotides and/or agomsts or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
• polynucleotides or polypeptides, and/or agonists thereof are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction.
• polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections ofthe joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii.
• blood-borne infections e.g., sepsis, meningitis, septic arthritis, and/or
• diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with polynucleotides or polypeptides, and/or agonists ofthe present invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease ("CVJD”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.
• CVJD common variable immunodeficiency disease
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue- related cancers or neoplasms.
• cancers or neoplasms that may be prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non- Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled "Hyperproliferative Disorders" elsewhere herein.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
• compositions ofthe invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
• Antagonists ofthe invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms ofthe polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9).
• Agonists ofthe invention include, for example, binding or stimulatory antibodies, and soluble forms ofthe polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9).
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741).
• polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention are useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention in an organ system listed above.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity.
• hemostatic or thrombolytic activity polynucleotides or polypeptides, and/or agonists or antagonists ofthe present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
• blood coagulation diseases, disorders, and/or conditions e.g., afibrinogenemia, factor deficiencies, hemophilia
• blood platelet diseases, disorders, and/or conditions e.g., thrombocytopenia
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention include, but are not lirmted to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
• extrcorporeal devices e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines.
• a polypeptide ofthe invention may be used to prevent, diagnose, prognose, and/or treat diseases and disorders ofthe blood and/or blood forming organs associated with the tissue(s) in which the polypeptide ofthe invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1, column 8 (Tissue Distribution Library Code).
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to modulate hematopoietic activity (the formation of blood cells).
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutiophils, mast cells, macrophages) and platelets.
• blood cells such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutiophils, mast cells, macrophages) and platelets.
• myeloid cells e.g., basophils, eosinophils, neutiophils, mast cells, macrophages
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutiophils, mast cells, macrophages) and platelets.
• blood cells such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutiophils, mast cells, macrophages) and platelets.
• the ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to prevent, treat, or diagnose blood dyscrasia.
• Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis).
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias.
• Anemias that may be treated prevented or diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria).
• iron deficiency anemia e.g., hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, mega
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs.
• rhe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagomsts of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating thalassemias, including, but not limited to major and minor forms of alpha- thalassemia and beta-thalassemia.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary
• thrombocytopenia e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic pur
• Hemorhhagic Telangiectsia also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
• the effect ofthe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention on the clotting time of blood may be monitored using any ofthe clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee- White Clotting time.
• PTT whole blood partial thromboplastin time
• aPTT activated partial thromboplastin time
• ACT activated clotting time
• Lee- White Clotting time the Lee- White Clotting time.
• the polynucleotides, polypeptides, antibodies, and or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, . preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis ofthe liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti- * inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells.
• Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer.
• Leokocytoses include but are not limited to, eosinophilia, and accumulations of macrophages.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.
• Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia.
• Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and or treating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott- Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).
• lymphocytopenias decreased numbers of B and/or T lymphocytes
• drug treatments e.g., drug treatment with corticosteroids, cancer
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease..
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophihc syndrome, eosinophilia-myalgia syndrome, and Hand- Schuller-Christian disease.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides. »
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macro globulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/or diagnosing myeloprohferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.
• myeloprohferative disorders including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful as a treatment prior to surgery, to increase blood cell production.
• the polynucleotides, polypeptides, antibodies, and or agonists or antagonists ofthe present invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutiophils, eosionophils and macrophages.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful as an agent to increase cytokine production.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.
• polynucleotides or polypeptides, or agonists or antagonists ofthe present invention can be used to treat or detect hyperproliferative disorders, including neoplasms.
• Polynucleotides or polypeptides, or agonists or • antagonists ofthe present invention may inhibit the proliferation ofthe disorder through direct or indirect interactions.
• Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
• hyperproliferative disorders can be treated.
• This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
• hyperproliferative disorders that can be treated or detected by polynucleotides or polypeptides, or agonists or antagonists ofthe present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
• neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen,
• hyperproliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists ofthe present invention.
• hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
• Lymphocytic Leukemia Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AJDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer ofthe Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer,
• Pheochromocytoma Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer ofthe Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal
• polynucleotides or polypeptides, or agonists or antagonists ofthe present invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
• Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp.
• Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
• Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions ofthe invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, angiofoUicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia ofthe breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, f ⁇ bromuscular hyperplasia, focal epithelial hyperp
• Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell.
• Metaplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
• Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
• Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
• Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
• Dysplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions ofthe invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia
• compositions ofthe invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
• benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia
• leukoplakia keratoses
• Bowen's disease keratoses
• Farmer's Skin Farmer's Skin
• solar cheilitis solar keratosis
• a polypeptide of the invention may be used to diagnose and/or prognose disorders associated with the tissue(s) in which the polypeptide ofthe invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1, column 8 (Tissue Distribution Library Code).
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention conjugated to a toxin or a radioactive isotope, as described herein may be used to treat cancers and neoplasms, including, but not limited to those described herein.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention conjugated to a toxin or a radioactive isotope, as described herein may be used to treat acute myelogenous leukemia.
• polynucleotides, polypeptides, and or agonists or antagomsts of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis.
• diseases associated with increased cell survival or the inhibition of apoptosis include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's
• polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
• Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
• Diseases associated with increased apoptosis that, could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.
• neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis
• ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
• liver injury e.g., hepatitis related liver injury, ischemia/rep erfusion injury, cholestosis (bile duct injury) and liver cancer
• toxin- induced liver disease such as that caused by alcohol
• septic shock cachexia and anorexia.
• Hyperproliferative diseases and/or disorders that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
• neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvi
• hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention.
• hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,. Sezary Syndrome, Waldenstron's macro globulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
• Another preferred embodiment utilizes polynucleotides ofthe present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
• the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide ofthe present invention, wherein said polynucleotide represses said expression.
• polynucleotides ofthe present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides.
• the DNA construct encoding the poynucleotides ofthe present invention is. inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference).
• the viral vector is defective and will not transform non-proliferating cells, only proliferating cells.
• the polynucleotides ofthe present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression ofthe encoded protein product.
• an external stimulus i.e. magnetic, specific small molecule, chemical, or drug administration, etc.
• the beneficial therapeutic affect ofthe present mvention maybe expressly modulated (i.e. to increase, decrease, or inhibit expression ofthe present invention) based upon said external stimulus.
• Polynucleotides ofthe present invention may be useful in repressing expression of oncogenic genes or antigens.
• repressing expression ofthe oncogenic genes is intended the suppression ofthe transcription ofthe gene, the degradation ofthe gene transcript (pre-message RNA), the inhibition of splicing, the destruction ofthe messenger RNA, the prevention ofthe post-translational modifications ofthe protein, the destruction ofthe protein, or the inhibition ofthe normal function ofthe protein.
• polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification.
• the polynucleotide ofthe present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A.
• vaccinia virus system Chokrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art.
• vaccinia virus system Chokrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art.
• retrovirus or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack ofthe retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides ofthe present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
• the polynucleotides ofthe present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site.
• the polynucleotides ofthe present invention may also be administered to disease sites at the time of surgical intervention.
• cell proliferative disease any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
• any amount ofthe polynucleotides ofthe present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one ofthe polynucleotide of the present invention simultaneously to the same site.
• biologically inhibiting is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth ofthe cells.
• the biologically inhibitory dose may be determined by assessing the effects ofthe polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
• the present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more ofthe described disorders.
• Methods for producing anti-polypeptides and anti-polynucleotide ' antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein.
• Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary ofthe ways in which the antibodies ofthe present invention may be used therapeutically includes binding polynucleotides or polypeptides ofthe present invention locally or systemically in the body or by direct cytotoxicity ofthe antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• CDC complement
• ADCC effector cells
• Such treatment comprises administering a single or multiple doses ofthe antibody, or a fragment, derivative, or a conjugate thereof.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example., which serve to increase the number or activity of effector cells which interact with the antibodies.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 " °M, 5X10 "7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 "9 M, 10 "9 M, 5X10- 10 M, 10 "10 M, 5X10 " ⁇ M, 10 " ⁇ M, 5X10 "12 M, 10 '12 M, 5X10 "13 M, 10 "13 M, 5X10 " 14 M, 10 "14 M, 5X10 "15 M, and 10 "15 M.
• polypeptides ofthe present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein.
• said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor- specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference).
• Antibodies directed to polypeptides or polynucleotides ofthe present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).
• Polypeptides including protein fusions, ofthe present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis.
• Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death- domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing Iigand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference).
• TNF tumor necrosis factor
• TRAMP TNF-receptor-related apoptosis-mediated protein
• TRAIL TNF-related apoptosis-in
• said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(l-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact.
• Polypeptides, including protein fusions to, or fragments thereof, ofthe present invention are useful in inhibiting the metastasis of proliferative cells or tissues.
• Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231 : 125-41 , which is hereby incorporated by reference).
• Such thereapeutic affects ofthe present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
• the mvention provides a method of delivering compositions containing the polypeptides ofthe invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide ofthe present invention.
• compositions containing the polypeptides ofthe invention e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs
• Polypeptides or polypeptide antibodes ofthe invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
• Polypeptides, protein fusions to, or fragments thereof, ofthe present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides ofthe present invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
• proteins known to enhance the immune response e.g. chemokines
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention may be used to treat, prevent, diagnose, and/or prognose disorders ofthe renal system.
• Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions ofthe invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders ofthe kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
• Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions ofthe invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases ofthe kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoprohferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic
• Alport's syndrome nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders ofthe kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).
• SLE systemic lupus erythematosus
• compositions ofthe invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders ofthe kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers ofthe kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
• Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, ⁇ osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
• Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
• cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
• Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
• Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, .
• heart disease such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture
• heart valve diseases myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
• Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle- branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson- White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
• Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentiy tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, . congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• coronary disease such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge- Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
• Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
• Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.
• Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
• Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art.
• Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
• Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders ofthe respiratory system.
• Diseases and disorders ofthe respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer ofthe nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer ofthe nasophary
• Pseudomas spp. Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).
• Additional diseases and disorders ofthe respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycdsis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.
• bronchiolitis poliomyelitis
• croup respiratory syncytial viral infection
• mumps erythema infectiosum (fifth disease)
• roseola infantum erythema infectiosum
• Pneumocystis carinii pneumocystis pneumonia
• atypical pneumonias e.g., Mycoplasma and Chlamydia spp.
• opportunistic infection pneumonia nos ⁇ comial pneumonia, chemical pneumonitis, and aspiration pneumonia
• pleural disorders e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)
• obstructive airway diseases e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis
• occupational lung diseases e.g., silicosis, black lung (coal workers' .
• pneumoconiosis e.g., asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses
• Infiltrative Lung Disease e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schuller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelect
• angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Umegulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
• a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al, Biotech. P:630-634 (1991); Folkman et al, N. Engl J. Med., 333:1151-1163 (1995); Auerbach et al, J. Microvasc. Res. 2°:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J.
• the present invention provides for treatment of diseases or disorders associated with neovascularization by administration ofthe polynucleotides and/or polypeptides ofthe invention, as well as agomsts or antagonists ofthe present invention.
• Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists ofthe invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
• the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist ofthe invention.
• a polynucleotide, polypeptide, antagonist and/or agonist ofthe invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor.
• Cancers which may be treated with polynucleotides, polypeptides, antagonists and or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias.
• polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
• polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration.
• Polynucleotides, polypeptides, antagonists and or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter.
• the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
• Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis.
• disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometi ⁇ osis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures
• methods for treating hypertrophic scars and keloids comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist ofthe invention to a hypertrophic scar or keloid.
• polynucleotides, polypeptides, antagonists and/or agonists ofthe invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions.
• This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 " days after the initial injury), but before hypertrophic scar or keloid development.
• the present invention also provides methods for treating neovascular diseases ofthe eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retro lental fihroplasia and macular degeneration.
• neovascular diseases ofthe eye including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retro lental fihroplasia and macular degeneration.
• Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides ofthe present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al, Am. J. Ophthal 85:104- 710 (1978) and Gartner et al, Surv. Ophthal 22:291-312 (1978).
• neovascular diseases ofthe eye such as corneal neovascularization (including corneal graft neovascularization)
• corneal neovascularization including corneal graft neovascularization
• a compound as described above
• the cornea is a tissue which normally lacks blood vessels.
• capillaries may extend into the cornea from the pericorneal vascular plexus ofthe limbus.
• the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates.
• corneal neovascularization e.g., corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
• corneal infections e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis
• immunological processes e.g., graft rejection and Stevens-Johnson's syndrome
• alkali burns trauma, inflammation (of any cause)
• toxic and nutritional deficiency states e.g., as a complication of wearing contact lenses.
• the invention may be prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form.
• the solution or suspension may be prepared in its pure form and administered several times daily.
• anti-angiogenic compositions prepared as described above, may also be administered directly to the cornea.
• the anti-angiogenic composition is prepared with a muco- adhesive polymer which binds to cornea.
• the anti- angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy.
• Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
• the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance.
• the preferred site of injection may vary with the morphology ofthe individual lesion, but the goal ofthe administration would be to place the composition at the advancing front ofthe vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic comeal injection to "protect" the cornea from the advancing blood vessels.
• This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply.
• neovascular glaucoma comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma.
• the compound may be implanted by injection into the region ofthe anterior chamber angle.
• the compound may also be placed in any location such that the compound is continuously released into the aqueous humor.
• methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is. inhibited.
• proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration ofthe polynucleotide, polypeptide, antagonist and/or agonist in the retina.
• this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
• methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically, via intravitreous injection and/or via intraocular implants.
• disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• disorders and/or states which can be treated, prevented, diagnosed, and/or prognosed with the the polynucleotides, polypeptides, agonists and/or agonists ofthe invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hyper
• an amount ofthe compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method.
• Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
• Polynucleotides, polypeptides, agonists and/or agonists ofthe present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
• compositions in the form of, for example, a spray or film
• a compositions may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues.
• compositions e.g., in the form of a spray
• surgical meshes which have been coated with anti- angiogenic compositions ofthe present invention may be utilized in any procedure wherein a surgical mesh might be utilized.
• a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount ofthe anti- angiogenic factor.
• methods for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited.
• the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins ofthe tumor with the anti-angiogenic compound).
• the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration.
• the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
• polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors.
• anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
• polypeptides, polypeptides, agonists and/or agonists ofthe present invention may also be administered along with other anti-angiogenic factors.
• anti-angiogenic factors include: Anti-Invasive
• Lighter "d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes ofthe above-mentioned transition metal species include oxo transition metal complexes.
• vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes.
• Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate.
• Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
• tungsten and molybdenum complexes also include oxo complexes.
• Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes.
• Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid.
• Suitable tungsten oxides include tungsten (TV) oxide and tungsten (VI) oxide.
• Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes.
• Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates.
• Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid.
• Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate.
• Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
• anti-angiogenic factors include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res.
• SP- PG Sulphated Polysaccharide Peptidoglycan Complex
• the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate
• Staurosporine modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L- 3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.
• Bisantrene National Cancer Institute
• Lobenzarit disodium N-(2)-carboxyphenyl-4- chloroanthronilic acid disodium or "CCA”
• CCA chloroanthronilic acid disodium
• Thalidomide Angostatic steroid
• AGM-1470 carboxynaminolmidazole
• metalloproteinase inhibitors such as BB94.
• cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone- dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, ghoblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,
• polynucleotides, polypeptides, and/or antagonists ofthe invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
• Additional diseases or conditions associated with increased cell survival that could be treated or detected by polynucleotides or polypeptides, or agonists or antagonists ofthe present invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macro globulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcoma
• Diseases associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune- related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.
• neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotroph
• ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
• liver injury e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver- cancer
• toxin-induced liver disease such as that caused by alcohol
• septic shock cachexia and anorexia.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage ofthe dermis and epidermis, eye tissue wounds,.
• polynucleotides or polypeptides will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract.
• Polynucleotides or polypeptides, agonists or antagonists ofthe present invention may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes. Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to treat epidermo lysis bullosa, a defect in adherence ofthe epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could also be used to treat gastric and doudenal ulcers and help heal by scar formation ofthe mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
• Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, are diseases which result in destruction ofthe mucosal surface ofthe small or large intestine, respectively.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to promote the resurfacing ofthe mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease.
• Treatment with polynucleotides or polypeptides, agonists or antagonists ofthe present invention is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to treat diseases associate with the under expression.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to prevent and heal damage to the lungs due to various pathological states.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage.
• emphysema which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists ofthe present invention.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
• liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used treat or prevent the onset of diabetes mellitus.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation ofthe disease.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
• the polynucleotides, polypeptides and agonists or antagonists ofthe invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury ofthe brain and/or nervous system.
• Nervous system disorders that can be treated with the compositions ofthe invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
• Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods ofthe invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries; (3) malignant lesions, in which a portion ofthe nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes
• the polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to protect neural cells from the damaging effects of hypoxia.
• the polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to protect neural cells from the damaging effects of cerebral hypoxia.
• the compositions ofthe invention are used to treat or prevent neural cell injury associated with cerebral hypoxia.
• the polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent neural cell injury associated with cerebral ischemia.
• the polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent neural cell injury associated with cerebral infarction.
• polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent neural cell injury associated with a stroke.
• polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
• polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent neural cell injury associated with a heart attack.
• polypeptides, polynucleotides, or agonists or antagonists ofthe invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.
• compositions ofthe invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.
• compositions ofthe invention which elicit any ofthe following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron- associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo.
• Such effects may be measured by any method known in the art.
• increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al, Proc Natl Acad Sci USA 91:3631-42 (2000) or in Arakawa et al, J. NeuroscL, 10:3501-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al, Exp. Neurol, 70:65-82 (1980), or Brown et al, Ann. Rev.
• NeuroscL, 4:11-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
• motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components ofthe nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
• disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components ofthe nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar
• compositions ofthe invention may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders.
• the compositions ofthe invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders.
• Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
• compositions ofthe invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.
• polypeptides, polynucleotides and/or agonists or antagonists of the invention may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasil
• polynucleotides or polypeptides as well as agonists or antagonists ofthe present invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, polynucleotides, polypeptides, agonists and/or antagonists ofthe invention may be used to treat and/or detect neurologic diseases. Moreover, polynucleotides or polypeptides, or agonists or antagonists ofthe invention, can be used as a marker or detector of a particular nervous system disease or disorder.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomal
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt- Jakob Syndrome, senile dementia such as
• Alzheimer's Disease and progressive supranuclear palsy vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St.
• Additional neurologic diseases which can be 1 treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AJDS Dementia Complex, Brain Abscess, subdural empyema, encephalomyehtis such as Equine Encephalomyehtis, Venezuelan Equine Encephalomyehtis, Necrotizing Hemorrhagic Encephalomyehtis, Visna, and cerebral malaria.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyehtis, necrotizing hemorrhagic ence
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists ofthe present invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdmg-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia,
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagomsts ofthe present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy ⁇ Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gu
• Polynucleotides or polypeptides, or agonists or antagonists ofthe present mvention may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases ofthe endocrine system.
• Hormones secreted by the glands ofthe endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones.
• the etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
• Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases ofthe menstrual cycle (e.g., dysmenorrhea and endometriosis).
• complications with pregnancy and labor e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor
• disorders and/or diseases ofthe menstrual cycle e.g., dysmenorrhea and endometriosis.
• Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases ofthe pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma— islet cell tumor syndrome; disorders and/or diseases ofthe adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases ofthe pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases ofthe thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (
• endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer.
• Other disorders and/or diseases ofthe testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias ofthe testis and neo-testis.
• endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
• a polypeptide ofthe invention may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide ofthe invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1, column 8 (Tissue Distribution Library Code).
• the polynucleotides or polypeptides, or agonists or antagonists ofthe invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders ofthe reproductive system.
• Reproductive system disorders that can be treated by the compositions ofthe invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
• Reproductive system disorders and/or diseases include diseases and/or disorders ofthe testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozo
• Reproductive system disorders also include disorders ofthe prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
• compositions ofthe invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases ofthe penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non- gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma;
• diseases and/or disorders ofthe vas deferens include vasculititis and CBAVD (congenital bilateral absence ofthe vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists, of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders ofthe seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
• disorders and/or diseases ofthe male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
• polynucleotides, polypeptides, and agonists or antagonists ofthe present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders ofthe vagina and vulva, including bacterial vaginosis, Candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibu
• disorders and/or diseases ofthe uterus include dysmenorrhea, retroverted . uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas.
• polypeptides, polynucleotides, or agonists or antagonists ofthe invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicomuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary hom, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary hom, arcuate uterus, uterine didelfus, and T-shaped uterus.
• congenital uterine abnormalities such as bicomuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary hom, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a
• Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to,, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma ofthe ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
• Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
• diseases and/or disorders ofthe reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy.
• miscarriage and stillbirth such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion
• ectopic pregnancy anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation
• polynucleotides, polypeptides, and agonists or antagonists ofthe present invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis ofthe liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ova
• Complications associated with labor and parturition include premature rupture ofthe membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
• fetal distress e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position
• shoulder dystocia e.g., prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
• diseases and/or disorders ofthe postdelivery period including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
• disorders and/or diseases ofthe female reproductive system that maybe diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and agonists or antagonists ofthe present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittel inconvenience.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated.
• the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
• viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention.
• viruses include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adeno viridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxo viridae, Morbilli virus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parain
• Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
• arthritis bronchiollitis
• respiratory syncytial virus e.g., respiratory syncytial virus,
• polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to treat or detect any of these symptoms or diseases.
• polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B).
• hepatitis e.g., hepatitis B
• polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines.
• polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat AIDS.
• Enterobacter e.g. Enterobacter aerogenes
• Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legiqnella pheumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhe
• bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, l opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucell
• Polynucleotides or polypeptides, agonists or antagonists ofthe invention can be used to treat or detect any of these symptoms or diseases.
• polynucleotides, polypeptides, agonists or antagonists ofthe invention are used to tieat: tetanus, diptheria, botulism, and/or meningitis type B.
• parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and or agonist or antagonist ofthe present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale).
• polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to tieat, prevent, and/or diagnose any of these symptoms or diseases.
• polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose malaria.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention ofthe present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide ofthe present invention, and returning the engineered cells to the patient (ex vivo therapy).
• the polypeptide or polynucleotide ofthe present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
• the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
• Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
• organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
• muscle smooth, skeletal or cardiac
• vasculature including vascular and lymphatics
• nervous hematopoietic
• skeletal bone, cartilage, tendon, and ligament
• regeneration occurs without. or decreased scarring.
• Regeneration also may include angiogenesis.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after . damage.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could also be used prophylactically in an effort to avoid damage.
• Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.
• a further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
• nerve and brain tissue could also be regenerated by using polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, to proliferate and differentiate nerve cells.
• Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke).
• diseases associated with peripheral nerve injuries e.g., resulting from chemotherapy or other medical therapies
• peripheral neuropathy e.g., resulting from chemotherapy or other medical therapies
• localized neuropathies e.g., central nervous system diseases
• central nervous system diseases e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome
• Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome could all be treated using the polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention.
• Gastrointestinal Disorders Polynucleotides or polypeptides, or agonists or antagonists ofthe present invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor ofthe small intestine, non-Hodgkin's lymphoma ofthe small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
• cancers e.g., intestinal neoplasms (carcinoid tumor ofthe small intestine, non-Hodgkin's lymphoma ofthe small intestine, small bowl lymphoma)
• ulcers such as peptic ulcers.
• Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps ofthe stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atiophic, plasma cell, and Menetrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis,
• Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions ofthe ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation ofthe small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T.
• Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice
• hepatitis alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver failure
• cholangiocellular cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
• Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma ofthe pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
• pancreatic diseases e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency).
• Gallbladder diseases include gallstones (cholelithiasis and choledochohthiasis), postcholecystectomy syndrome, diverticulosis ofthe gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
• Diseases and/or disorders of the arge intestine include antibiotic-associated colitis, diverticuhtis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticuhtis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocohtis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy,
• biliary tract diseases such as, gastioschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma ofthe esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma ofthe pancreas, mucinous cystic neoplasm ofthe ' pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous biliary tract neoplasms,
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may have chemotaxis activity.
• a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutiophils, T-cells, mast cells, eosinophils, epithelial and/of endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation.
• the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
• Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to tieat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to tieat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules ofthe present invention can also attract fibroblasts, which can be used to treat wounds.
• polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may inhibit chemotactic activity. These molecules, could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used as an inhibitor of chemotaxis. Binding Activity
• a polypeptide ofthe present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
• the binding ofthe polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity ofthe polypeptide or the molecule bound.
• Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors),or small molecules.
• the molecule is closely related to the natural Iigand ofthe polypeptide, e.g., a fragment ofthe Iigand, or a natural substrate, a Iigand, a structural or functional mimetic.
• the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment ofthe receptor capable of being bound by the polypeptide (e.g., active site).
• the molecule can be rationally designed using known techniques.
• the screening for these molecules involves producing appropriate cells which express the polypeptide.
• Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
• Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
• the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide. Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
• an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
• the antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
• the receptor to which the polypeptide ofthe present invention binds can be identified by numerous methods known to those of skill in the art, for example, Iigand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).
• polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to tiansfect COS cells or other cells that are not responsive to the polypeptides.
• Transfected cells which are grown on glass slides are exposed to the polypeptide ofthe present invention, after they have been labeled.
• the polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
• the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors ofthe polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
• DNA shuffling may be employed to modulate the activities ofthe polypeptide ofthe present invention thereby effectively generating agonists and antagonists ofthe polypeptide ofthe present invention. See generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al, Curr. Opinion Biotechnol. 8:724- 33 (1997); Harayama, S. Trends Biotechnol 16(2):76-82 (1998); Hansson, L.
• alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination.
• polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc., ofthe polypeptide ofthe present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• the heterologous molecules are family members.
• the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF- beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF- beta5, and glial-derived neurotrophic factor (GDNF).
• PDGF platelet-derived growth factor
• IGF-I insulin-like growth factor
• TGF transforming growth factor
• EGF epidermal growth factor
• FGF fibroblast growth factor
• TGF- beta TGF- beta
• BMP
• Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity ofthe polypeptide ofthe present invention.
• the biological activity ofthe fragments may include an improved desired activity, or a decreased undesirable activity.
• this invention provides a method of screening compounds to identify those which modulate the action ofthe polypeptide ofthe present invention.
• An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide ofthe present invention, the compound to be screened and 3[H]
• a control assay may be performed in the absence ofthe compound to be screened and compared to the amount of fibroblast proliferation in the presence ofthe compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case.
• the amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of 3rjj] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
• a mammalian cell or membrane preparation expressing a receptor for a polypeptide ofthe present invention is incubated with a labeled polypeptide ofthe present invention in the presence ofthe compound.
• the ability of the compound to enhance or block this interaction could then be measured.
• the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist.
• second messenger systems include but are not limited to, cAMP guanylate cyclase,. ion channels or phosphoinositide hydrolysis.
• All of these above assays can be used as diagnostic or prognostic markers.
• the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule.
• the assays can discover agents which may inhibit or enhance the production ofthe polypeptides ofthe invention from suitably manipulated cells or tissues.
• the invention includes a method of identifying compounds which bind to a polypeptide ofthe invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide ofthe present invention; and (b) determining if binding has occurred.
• the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide ofthe present invention, (b) assaying a biological activity, and (b) determining if a biological activity ofthe polypeptide has been altered.
• the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide ofthe invention, or cells expressing a cell bound form of a polypeptide ofthe invention.
• polypeptides or antibodies ofthe invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
• the invention provides a method for the specific delivery of compositions ofthe invention to cells by administering polypeptides ofthe invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids.
• the invention provides a method for delivering a therapeutic, protein into the targeted cell.
• the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
• a single stranded nucleic acid e.g., antisense or ribozymes
• double stranded nucleic acid e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed
• the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides ofthe invention (e.g., polypeptides ofthe invention or antibodies ofthe invention) in association with toxins or cytotoxic prodrugs.
• toxins is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
• Toxins that may be used according to the methods ofthe invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
• radioisotopes known in the art
• compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
• cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
• Cytotoxic prodrugs that may be used according to the methods ofthe invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
• polypeptides ofthe present invention or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities ofthe polypeptides ofthe present invention.
• a method would include contacting the polypeptide ofthe present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
• This invention is particularly useful for screening therapeutic compounds by using the polypeptides ofthe present invention, or binding fragments thereof, in any of a variety of drug screening techniques.
• the polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly.
• One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays.
• One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide ofthe present invention.
• the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agent with a polypeptide ofthe present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art.
• the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure ofthe ability of a particular agent to bind to the polypeptides ofthe present invention.
• Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in European Patent Application 84/03564, published on September 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides ofthe present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
• This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides ofthe present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a pofypeptide ofthe invention.
• the invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides ofthe invention, and the polypeptide of the invention binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists ofthe polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
• This method comprises the steps of ontacting a polypeptide ofthe invention with a plurality of molecules; and identifying a molecule that binds the polypeptide of the invention.
• the step of contacting the polypeptide ofthe invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptide ofthe invention on a solid support and bringing a solution ofthe plurality of molecules in contact with the immobilized polypeptide of the invention. Such a procedure would be akin to an affinity chromato graphic process, with the affinity matrix being comprised ofthe immobilized polypeptide ofthe invention.
• the molecules having a selective affinity for the polypeptide ofthe invention can then be purified by affinity selection.
• the nature ofthe solid support, process for attachment ofthe polypeptide ofthe invention to the solid support, solvent, and conditions ofthe affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
• the individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be "probed" by the polypeptide ofthe invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptide of the invention and the individual clone.
• the polypeptides Prior to contacting the polypeptide ofthe invention with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience.
• a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon.
• positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for a polypeptide ofthe invention.
• the amino acid sequence ofthe polypeptide having a selective affinity for the polypeptide ofthe invention can be determined directly by conventional means or the coding sequence ofthe DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
• any unbound polypeptide ofthe invention may be desirable to wash away any unbound polypeptide ofthe invention, or altemtatively, unbound polypeptides, from a mixture ofthe polypeptide ofthe invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction.
• Such a wash step may be particularly desirable when the polypeptide ofthe invention or the plurality of polypeptides is bound to a solid support.
• the plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to a polypeptide ofthe invention.
• diversity libraries such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to a polypeptide ofthe invention.
• Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
• phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
• In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91 :9022-9026.
• a benzodiazepine library see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91 :4708-4712
• Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used.
• Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers.
• Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
• Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order ofthe monomers.
• monomer units that have been used are carbamates, pyrrolinones, and morpholinos.
• Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon form the basis of another version of non-peptide oligomer libraries.
• the first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility. Screening the libraries can be accomplished by any of a variety of commonly known methods.
• screening to identify a molecule that binds a polypeptide ofthe invention can be carried out by contacting the library members with a polypeptide ofthe invention immobilized on a solid phase and harvesting those library members that bind to the polypeptide ofthe invention.
• panning techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
• the two-hybrid system for selecting interacting proteins in yeast can be used to identify molecules that specifically bind to a polypeptide ofthe invention.
• the polypeptide ofthe invention binding molecule is a polypeptide
• the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries.
• bias is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity ofthe resulting collection of molecules, in this case peptides.
• a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position ofthe peptide is the same for all 20 amino acids.
• a bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine.
• biases can be contemplated, and the present invention is not restricted to any particular bias.
• the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
• the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids.
• a polypeptide of the invention binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
• the selected polypeptide ofthe invention binding polypeptide can be obtained by chemical synthesis or recombinant expression.
• antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone.
• antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation.
• Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• c-myc and c-myb antisense RNA constructs to inhibit the growth ofthe non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the ohgoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580.
• a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases ofthe open reading frame is flanked by an EcoRl site on the 5 end and a Hindlll site on the 3 end. Next, the pair of oligonucleotides is heated at 90°C for one minute and then annealed in 2X ligation buffer (20mM TRIS HC1 pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl/Hind III site ofthe retroviral vector PMV7 (WO 91/15580).
• 2X ligation buffer 20mM TRIS HC1 pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP
• the 5' coding portion of a polynucleotide that encodes the mature polypeptide ofthe present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region ofthe gene involved in transcription thereby preventing transcription and the production ofthe receptor.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.
• the antisense nucleic acid ofthe invention is produced intracellularly by transcription from an exogenous sequence.
• a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) ofthe invention.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the antisense nucleic acid ofthe invention.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells.
• Expression ofthe sequence encoding a polypeptide ofthe invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells.
• promoters can be inducible or constitutive.
• Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci.
• the antisense nucleic acids ofthe invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required.
• a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids ofthe invention, a single strand ofthe duplex DNA may thus be tested, or triplex formation may be assayed.
• the ability to hybridize will depend on both the degree of complementarity and the length ofthe antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence ofthe invention it may contain and still form a stable duplex (or triplex as the case may be).
• Oligonucleotides that are complementary to the 5 ' end ofthe message e.g., the 5 ' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation.
• sequences complementary to the 3 ' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature,
• oligonucleotides complementary to either the 5 ' - or 3 ' - non- translated, non-coding regions of a polynucleotide sequence ofthe invention could be used in an antisense approach to inhibit translation of endogenous mRNA.
• Oligonucleotides complementary to the 5' untranslated region ofthe mRNA should include the complement ofthe AUG start codon.
• Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5' -, 3' - or coding region of mRNA,.
• antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
• the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• the polynucleotides ofthe invention can be DNA or RNA or chimeric mixtures br derivatives or modified versions thereof, single-stranded or double- stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability ofthe molecule, hybridization, etc.
• the oligonucleot de may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell ' membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
• the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
• the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• the antisense oligonucleotide is an a-anomeric oligonucleotide.
• An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contiary to the usual b-units, the stiands run parallel to each other (Gautier et al, Nucl. Acids Res., 15:6625-6641 (1987)).
• the oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al, FEBS Lett. 215:327-330 (1987)).
• Polynucleotides ofthe invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
• an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
• phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988))
• methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.
• antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region are most preferred.
• Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published
• ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides ofthe invention, the use of hammerhead ribozymes is preferred.
• Hammerhead ribozymes cleave mRNAs at locations dictated " by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5 ' -UG-3 ' .
• hammerhead ribozymes The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature, 334:585-591 (1988).
• the ribozyme is engineered so that the cleavage recognition site is located near the 5 ' end ofthe mRNA corresponding to the polynucleotides ofthe invention; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• the ribozymes ofthe invention can be composed of modified oligonucleotides (e.g.
• DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
• a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
• Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects ofthe polypeptides ofthe present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
• the antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention ofthe mitogenic activity ofthe polypeptides ofthe present invention may also be desirous in cases such as restenosis after balloon angioplasty.
• the antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
• the antagonist/agonist may also be employed to treat, prevent, and/or diagnose the diseases described herein.
• the invention provides a method of treating or preventing diseases, disorders, and/or conditions, including but not limited to the diseases, disorders, and/or conditions listed throughout this application, associated with overexpression of a polynucleotide ofthe present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide ofthe present invention, and/or (b) a ribozyme directed to the polynucleotide ofthe present invention. invention, and/or (b) a ribozyme directed to the polynucleotide ofthe present invention
• polypeptide ofthe present invention may be employed in treatment for stimulating re- vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
• the polypeptide may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogemc to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
• the polypeptide ofthe present invention may also be employed stimulate neuronal growth and to treat, prevent, and/or diagnose neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AJDS-related complex.
• the polypeptide ofthe invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
• the polypeptide ofthe present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
• the polypeptide ofthe invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth.
• the polypeptides ofthe present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
• the polypeptide ofthe invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues.
• the polypeptide ofthe present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
• the polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
• polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).
• polypeptides or polynucleotides and/or agonist or antagonists ofthe present mvention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
• Polypeptide or polynucleotides and/or agonist or antagonists ofthe present mvention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive diseases, disorders, and/or conditions), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
• Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
• Preferred Embodiments include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table.1.
• nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position ofthe 5' Nucleotide of the Clone Sequence and ending with the nucleotide at about the position ofthe 3' Nucleotide ofthe Clone Sequence as defined for SEQ ID NO:X in Table 1.
• nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position ofthe 5 ' Nucleotide of the Start Codon and ending with the nucleotide at about the position ofthe 3' Nucleotide ofthe Clone Sequence as defined for SEQ ID NO:X in Table 1.
• nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position ofthe 5' Nucleotide ofthe First Amino Acid ofthe Signal Peptide and ending with the nucleotide at about the position of the 3' Nucleotide ofthe Clone Sequence as defined for SEQ ID NO:X in Table 1.
• nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ LD NO:X.
• nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
• a further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position ofthe 5' Nucleotide of the First Amino Acid ofthe Signal Peptide and ending with the nucleotide at about the position ofthe 3 ' Nucleotide ofthe Clone Sequence as defined for SEQ ID NO.X in Table 1.
• a further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X.
• nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
• composition of matter comprising a DNA molecule which comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1, which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1 for said cDNA Clone Identifier.
• an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by a cDNA Clone Identifier in Table 1 , which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1.
• nucleic acid molecule wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence ofthe complete open reading frame sequence encoded by said human cDNA clone.
• nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
• a further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95 %> identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
• a further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone.
• a further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95%o identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1 ; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1 ; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
• said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group.
• said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group.
• the nucleic acid molecules can comprise DNA molecules or RNA molecules.
• a further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95%) identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1 ; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• the method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
• a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1 comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• the method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95%) identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
• composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1 ; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• the nucleic acid molecules can comprise DNA molecules or RNA molecules.
• an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ JD NO:Y wherein Y is any integer as defined in Table 1.
• polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO: Y in the range of positions beginning with the residue at about the position ofthe First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid ofthe Open Reading Frame as set forth for SEQ ID NO:Y in Table 1.
• an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.
• an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ JD NO:Y. Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95%> identical to the complete amino acid sequence of SEQ ID NO:Y.
• an isolated polypeptide comprising an amino acid sequence at least 90%> identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a secreted portion ofthe secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence ofthe secreted portion ofthe protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence ofthe secreted portion ofthe protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence ofthe secreted portion ofthe protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ JD NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA.
• said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ JD NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
• a method for identifying the species, tissue or cell type of a biological sample comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• the above method for identifying the species, tissue or cell type of a biological sample comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
• a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1 comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90%> identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• the step of detecting said polypeptide molecules includes using an antibody.
• an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90%o identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• nucleic acid molecule wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
• polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
• a method of making a recombinant vector comprising inserting any ofthe above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of • making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
• a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ JD NO:Y beginning with the residue at the position ofthe First Amino Acid of the Secreted Portion of SEQ JD NO:Y wherein Y is an integer set forth in Table 1 and said position ofthe First Amino Acid ofthe Secreted Portion of SEQ JD NO:Y is defined in Table 1; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA
• the isolated polypeptide produced by this method is also preferred. Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical. composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody ofthe claimed invention effective to increase the level of said protein activity in said individual.
• hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non- human primate, and human.
• the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat.
• the host is a mammal.
• the host is a human.
• each "Contig ID" listed in the fourth column of Table 5 preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, a nucleotide sequence referenced in the fifth column of Table 5 and described by the general formula of a-b, whereas a and b are uniquely determined for the corresponding SEQ JD NO:X referred to in column 3 of Table 5.
• Further specific embodiments are directed to polynucleotide sequences excluding one, two, three, four, or more ofthe specific polynucleotide sequences referred to in the fifth column of Table 5. In no way is this listing meant to encompass all ofthe sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
• HKZAH22 14 1125739 AI761413, AI815855, AI269862, AI362637, AI887194, AI924971, AI886124, AI886753, AI648663, AL036403, AW071417, AI906328, AL041772, AI922901, AW059837, A1434468, AW403717, AA508692,AI816947, AI289937, AI284131, AL036146, AI499285, AI561299, AI446373, AI571909, AI439717, AI174394, AI539771, AL039086, ALl 19863, AW162071, AI869367, AI345608, AL120853, AW051258, AI680498, AI921248, AI445165, AL040243, AI564247, AI796743, AI097248, AL036214, AI269696, AW190042, AI922676, AI800453, AI784252, AI521012,
• HKZAH22 25 1120159 AI761413, AI269862, AI815855, AI824576, AI097248, AI886124, AL036403, AW059837, AW170635, AL039086, AI886753, AI648663, AI345608, AW071417, AI866770, AW149925, AI906328, AL041772, AI929108, AI571909, AI439717, AI922901, AI345471, AI366992, AI932794, AI287704, AL038445, F27788, AI816947, AW403717, AI344928, AL134999, AI680498, AI445165, AA493923, AW235745, AI269696, H89138, AI784252, AI800453, AI362637, AI431909, AI689420, AI284131, AI499285, AWl 69653, AI249962, AI561299, AA833760, AI25
• HAOST94 17 1132398 AI929674 AW157771, AW157572, AA675913, AWl 60730, AA675927, AW160709, AW163812, AW163448, AI341602, AI768042, AI499043, AI198154, AW291471, AI738504, F37450, AW303089, AI282355, AI915243, AI073952, AI920782, AI335411, AI955906, AW023590, AI554343, AW089275, AI927755, AA983883, AW074869, AI345224, AW152469, AW129929, AI311892, AI932638, AI494201, AI829990, AW172723, AL041772, AW 193134, AI890806, AI687166, AI950664, AA427700, AI923422, AL120819, AL041150, AW169653, AI573167

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