EP1330466A1 - 24 proteines humaines secretees - Google Patents

24 proteines humaines secretees

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Publication number
EP1330466A1
EP1330466A1 EP01902080A EP01902080A EP1330466A1 EP 1330466 A1 EP1330466 A1 EP 1330466A1 EP 01902080 A EP01902080 A EP 01902080A EP 01902080 A EP01902080 A EP 01902080A EP 1330466 A1 EP1330466 A1 EP 1330466A1
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EP
European Patent Office
Prior art keywords
ofthe
seq
polypeptide
polypeptides
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01902080A
Other languages
German (de)
English (en)
Other versions
EP1330466A4 (fr
Inventor
Craig A. Rosen
George A. Komatsoulis
Kevin P. Baker
Charles E. Birse
Daniel R. Soppet
Henrik S. Olsen
Paul A. Moore
Ping Wei
Reinhard Ebner
Roxanne D. Duan
Yanggu Shi
Gil H. Choi
Michele Fiscella
Jian Ni
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
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Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP1330466A1 publication Critical patent/EP1330466A1/fr
Publication of EP1330466A4 publication Critical patent/EP1330466A4/fr
Withdrawn legal-status Critical Current

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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 ofthe 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 ofthe polynucleotide.
  • isolated does not refer to genomic or cD ⁇ A libraries, whole cell total or mR ⁇ A preparations, genomic D ⁇ A preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic D ⁇ A preparations or other compositions where the art demonstrates no distinguishing features ofthe polynucleotide/sequences ofthe 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 ofthe 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 ofthe invention comprise a portion ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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, Nirginia 20110-2209, USA. The ATCC deposit was made pursuant to the terms ofthe Budapest Treaty on the international recognition ofthe deposit of microorganisms for purposes of patent procedure.
  • a "polynucleotide” ofthe present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID ⁇ O: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 ofthe 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 ofthe 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).
  • polynucleotide ofthe 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 ofthe 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 JO 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 ofthe 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 ofthe polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide ofthe 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 ofthe present invention.) Many proteins (and translated DNA sequences) contain regions where the amino acid composition is highly biased toward a small subset ofthe available residues.
  • membrane spanning domains and signal peptides typically contain long stretches where Leucine (L), Valine (V), Alanine (A), and Isoleucine (I) predominate.
  • Poly-Adenosine tracts (polyA) at the end of cDNAs appear in forward translations as poly-Lysine (poly-K) and poly- Phenylalanine (poly-F) when the reverse complement is translated. These regions are often referred to as "low complexity" regions.
  • BLASTX.2 version 2.0a5MP-WashU
  • filters two filters which "mask” the low complexity regions in a particular sequence. These filters parse the sequence for such regions, and create a new sequence in which the amino acids in the low complexity region have been replaced with the character "X".
  • a stretch of "X"s in an alignment shown in the following application does not necessarily indicate that either the underlying D ⁇ A sequence or the translated protein sequence is unknown or uncertain. Nor is the presence of such stretches meant to indicate that the sequence is identical or not identical to the sequence disclosed in the alignment ofthe present invention. Such stretches may simply indicate that the BLASTX program masked amino acids in that region due to the detection of a low complexity region, as defined above.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence:
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: nervous system, reproductive system, and immune system disorders, particularly disorders involving neutrophils and other immune cells.
  • diseases and conditions which include but are not limited to: nervous system, reproductive system, and immune system disorders, particularly disorders involving neutrophils and other immune cells.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 69 as residues: Met-1 to Ser-6, Leu-88 to Gly-99, Ser-118 to Tyr-123, His-178 to Ala-185, Glu-193 to Gln-199, Thr-274 to Asp-284, Ser-292 to Ala-301, Leu-308 to Gln-313, Pro-323 to Gly-336, Asp-342 to Glu-347, His-352 to Asp-362, Leu-368 to Thr-374.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution and homology to fasciclin indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of immune system (particularly neutrophil related) disorders or diseases. For example, neutropenia, neutrophilia, tissue necrosis, neoplasia, granulomatous disease, and infectious diseases.
  • the tissue distribution in human endometrial tumor and homology to fasciclin also indicates the polynucleotides and polypeptides corresponding to this gene are useful for diagnosis, prevention, and/or treatment of tumorigenesis and metastasis (particularly ofthe female reproductive system).
  • Many polynucleotide sequences, such as EST sequences are publicly available and accessible through sequence databases.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2296 of SEQ ID NO: 11 , b is an integer of 15 to 2310, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a + 14.
  • polypeptides comprising the amino acid sequence of the open reading frame upstream ofthe predicted signal peptide are contemplated by the present invention.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence: SSGGQVGYLLPPSLASCELACDRISCPRCPLPSCHRAKAFDLMTVSSYLW (SEQ ID NO: 128).
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • This gene is expressed primarily in CD34 cord blood cells and activated T cells.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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 ofthe immune system including immune deficiencies and autoimmune conditions.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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 immune tissue distribution of this gene in CD34 positive cells and activated T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation, survival, differentiation, and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus eryfhematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, p
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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 tlirough sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 1695 of SEQ ID NO:12, b is an integer of 15 to 1709, 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.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence:
  • HCR HCR (SEQ ID NO: 96).
  • fragments and variants of this polypeptide 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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neoplasms of various origins (particularly those ofthe breast and ovaries) and immunological disorders (particularly those involving T-cells).
  • diseases and conditions which include but are not limited to: neoplasms of various origins (particularly those ofthe breast and ovaries) and immunological disorders (particularly those involving T-cells).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • tissue or cells particularly ofthe female reproductive system, including breast, ovary and uterus
  • expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ TD NO: 71 as residues: Thr-19 to Ala-33, Leu-54 to Asp-82, Pro-89 to Ala-97, Pro-100 to Lys-125, Ser-127 to Phe-135, Gly-164 to Leu-169, Cys-173 to Arg-178.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution and homology to murine ion channel RIC which is upregulated in various neoplasms indicates that polynucleotides and polypeptides corresponding to this gene are useful for treatment and diagnosis of tumors and neoplasms particularly ofthe breast, ovary and B-cell (lymphoma). Further, the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders. For example, the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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.
  • the sequence of this gene predicts that the encoded polypeptide encodes a transmembrane protein, which makes this a particularly attractive target for immune therapy, where antibodies are generated against this protein and are used to specifically kill cells which express this protein on their surface.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 927 of SEQ ID NO: 13, b is an integer of 15 to 941, 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.
  • This gene is expressed primarily in glioblastoma and to a lesser extent in tumors particularly ofthe uterus and ovary.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: cancer, neurological disorders (for example tumors ofthe brain, especially glioblastoma), as well as disorders involving the uterus and ovaries.
  • diseases and conditions which include but are not limited to: cancer, neurological disorders (for example tumors ofthe brain, especially glioblastoma), as well as disorders involving the uterus and ovaries.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 72 as residues: Ser-120 to Ala-125, Glu-143 to Tyr-152. Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution, protein sequence and homology to a transmembrane protein indicate that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of neoplasms particularly those originating from glial cells (glioblastomas) and female reproductive tract, e.g., uterus, endometrium, cervix and ovary.
  • glial cells glioblastomas
  • female reproductive tract e.g., uterus, endometrium, cervix and ovary.
  • a target for immune therapy i.e., a tumor antigen
  • the gene may also be useful for the diagnosis and treatment of other gynecologic disorders and diseases such as infertility, endometriosis, dysmenorrhea (primary or secondary), amenorrhea and ovarian cysts.
  • tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease,
  • elevated expression of this gene product in regions ofthe brain indicates it plays a role in normal neural function.
  • this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and are useful for the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions.
  • developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain degenerative disorders, such as spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • this gene product may be involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers.
  • this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides ofthe present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein would be useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein may also be used to determine biological activity, 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. Furthermore, the protein may also be used to determine biological activity, 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: 14 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 ofthe 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 1180 of SEQ TD NO: 14, b is an integer of 15 to 1194, 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.
  • polypeptides comprising the amino acid sequence ofthe open reading frame upstream ofthe predicted signal peptide are contemplated by the present invention.
  • polypeptides ofthe invention comprise, or alternatively consists of, an amino acid sequence selected from the group:
  • VLGQE SEQ ID NO: 130
  • fragments and variants of these polypeptides are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • This gene is expressed primarily in kidney, colon, and to a lesser extent in many other tissues/cells, for example, in reproductive, excretory and digestive systems.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: disorders ofthe kidney, gastrointestinal tract, and T-cell related immune disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • tissue or cells particularly ofthe immune, reproductive, excretory and digestive systems
  • expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or 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 and homology to LAK-4p indicates that polynucleotides and polypeptides corresponding to this gene are useful for immunity related diseases, for example, T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases; immunodeficiency diseases, such as acquired immunodeficiency syndrome; autoimmunity, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma; infections, and other inflammatory diseases and complications.
  • T-cell mediated cytotoxicity immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases
  • immunodeficiency diseases such as acquired immunodeficiency syndrome
  • autoimmunity such as autoimmune infertility, lens tissue injury, demye
  • kidney diseases including renal failure, nephritis, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome.
  • kidney diseases including renal failure, nephritis, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's
  • the protein may also be used to determine biological activity, 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 ofthe 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 1237 of SEQ ID NO:15, b is an integer of 15 to 1251, 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.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immunological disorders particularly those involving neutrophils (such as neutropenia).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 74 as residues: Met-1 to Leu-7. Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation, survival, differentiation, and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 593 of SEQ ID NO: 16, b is an integer of 15 to 607, 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 + 14.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: carcinogenesis and tumorigenesis (particularly hemangiopericytoma) and/or immune disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 75 as residues: His-35 to Ser-41.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of hemangiopericytoma or immune related disorders.
  • hypersensitivities such as T-cell mediated cytotoxicity
  • immune reactions to transplanted organs and tissues such as host-versus-graft and graft-versus-host diseases
  • immunodeficiency diseases such as acquired immunodeficiency syndrome
  • autoimmunity such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma; infections, and other inflammatory diseases and complications.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2017 of SEQ ID NO: 17, b is an integer of 15 to 2031, 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.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence:
  • fragments and variants of these polypeptides are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • This gene is expressed primarily in tongue and brain and to a lesser extent in many other tissues, such as, Human Testes, Reexcision, Nine Week Old Early Stage Human, Soares_testis_NHT, NCI_CGAP_Ewl, Human Testes, NCI_CGAP_Utl, Human Cerebellum, NCI_CGAP_Eso2, NCI_CGAP_Ut4, Synovial hypoxia-RSF subtracted, Human Pituitary, subt IX, Breast, Cancer: (4004943 A5), Human T-Cell Lymphoma, Human Adult Testes, Large Inserts, Reexcision, Human Placenta,
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: taste, neurological disorders, and muscular disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 76 as residues: Trp-15 to Gly-33, Ser-40 to Gln-46, Arg-74 to Leu-81.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neurological disorders or other diseases.
  • a secretory protein that can have functions in (i) modulation of cellular signal transduction, either in vitro or in vivo; (ii) regulation of communication between cells; (iii) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo (e.g. induction of cell differentiation); (iv) regulation of cellular proliferation, either in vitro or in vivo (e.g.
  • induction of cellular proliferation or inhibition of proliferation as in the case of suppression of tumorigenesis formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic (e.g. induction of head formation during vertebrate development or maintenance of hematopoietic progenitor cells);
  • modulation of cell death such as stimulation of cell survival;
  • regulating cell migration, and/or immune modulation, e.g. treating hyperproliferative diseases such as neoplastic and hyperplastic disease, e.g.
  • Parkinsoninson's disease Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis
  • demyelinating diseases multiple sclerosis, human immunodeficiency associated
  • tissue distribution also indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of various muscle disorders, such as muscular dystrophy, cardiomyopathy, fibroids, myomas, and rhabdomyosarcomas.
  • a-b is any integer between 1 to 1028 of SEQ ID NO: 18, b is an integer of 15 to 1042, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 18, and where b is greater than or equal to a + 14.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence: MRLRFWLLIWLLLGFISHQPTPVINSLAVYRHRETDFGVRVRDHPWX (SEQ ID NO: 133).
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • This gene is expressed primarily in Tongue Normal, Brain Amygdala Depression, eosinophils, colon, and prostate and to a lesser extent in many tissues/cells, for example, Human Testes (Reexcision), Synovial hypoxia-RSF
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: disorders related to the functions of brain (for example, involving the amygdala and disorders such as depression), immunological disorders (for example those involving eosinophils), disorders ofthe gastrointestinal tract (for example disorders ofthe colon such as colon cancer and diverticulitis), and disorders ofthe prostate (such as enlarged prostate or prostate cancer).
  • diseases and conditions which include but are not limited to: disorders related to the functions of brain (for example, involving the amygdala and disorders such as depression), immunological disorders (for example those involving eosinophils), disorders ofthe gastrointestinal tract (for example disorders ofthe colon such as colon cancer and diverticulitis), and disorders ofthe prostate (such as enlarged prostate or prostate cancer).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological
  • tissue or cells particularly ofthe nervous system (for example the amygdala), immune system (for example eosinophils), gastrointestinal system (for example the colon), and the prostate
  • expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 77 as residues: Arg-42 to Ser-53.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of disorders related to the functions of Brain Amygdala Depression, eosinophils, colon, and prostate.
  • the gene can be used in (i) modulation of cellular signal transduction, either in vitro or in vivo; (ii) regulation of communication between cells; (iii) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo (e.g. induction of cell differentiation); (iv) regulation of cellular proliferation, either in vitro or in vivo (e.g. induction of cellular proliferation or inhibition of proliferation as in the case of suppression of tumorigenesis; (v) formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic (e.g.
  • hyperproliferative diseases such as neoplastic and hyperplastic disease, (for example, various cancers and leukemias), and fibroproliferative disorders, malignancy, premalignant and benign conditions, to stimulate tissue repair or wound healing or trea spinal cord injuries, brain injuries, lesions associated with surgery, ischemic lesions, malignant lesions, infectious lesions, degenerative lesions (Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis), demyelinating diseases (multiple sclerosis, human immunodeficiency associated myelopathy, pontine myelinolysis), motor neuron injuries, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular at
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 19 Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 1494 of SEQ ID NO: 19, b is an integer of 15 to 1508, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 19, and where b is greater than or equal to a + 14.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immunological disorders and diseases (for example those involving neutrophil functions).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 78 as residues: Asn-54 to Leu-62, Tyr-66 to Trp-74.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of disorders related to neutrophils. For example, neutropenia, neutrophilia, tissue necrosis, neoplasia, granulomatous disease and infectious diseases.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation, survival, differentiation, and/or activation of hematopoietic cell lineages (including blood stem cells).
  • hematopoietic cell lineages including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:20 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 1644 of SEQ ID NO:20, b is an integer of 15 to 1658, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in prostate and to a lesser extent in pancreas, brain, and placenta.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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 involving the prostate, pancreas, placenta, and brain.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 79 as residues: Asp-33 to Leu-39.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of prostate related diseases or conditions, for example, prostate inflammatory disorders, such as chronic prostatitis, granulomatous prostatitis and malacoplakia.
  • Prostatic hyperplasia and Prostate neoplastic disorders including adenocarcinoma, transitional cell carcinoma, ductal carcinoma, squamous cell carcinoma.
  • tissue distribution also indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • this gene product in regions ofthe brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the protein may also be used to determine biological activity, 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:21 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 2527 of SEQ ID NO:21, b is an integer of 15 to 2541, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a + 14.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immunological disorders (for example those involving neutrophil function).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 80 as residues: Pro-20 to Phe-26, Ala-39 to Phe-46.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution in neutrophils indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neutrophil related disorders. For example, neutropenia, neutrophilia, tissue necrosis, neoplasia, granulomatous disease, and infectious diseases.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host- versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, p
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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 750 of SEQ ID NO:22, b is an integer of 15 to 764, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in prostate, eosinophils, and T cells and to a lesser extent in Human umbilical vein endothelial cells, Gessler Wilm's tumor, Human Chondrosarcoma, and infant brain cDNA.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immune disorders (for example disorders and diseases involving neutrophil and T cell functions).
  • diseases and conditions which include but are not limited to: immune disorders (for example disorders and diseases involving neutrophil and T cell functions).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 81 as residues: Pro-39 to Cys-47. Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation, survival, differentiation, and/or activation of hematopoietic cell lineages (including blood stem cells).
  • hematopoietic cell lineages including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:23 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 732 of SEQ ID NO:23, b is an integer of 15 to 746, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:23, and where b is greater than or equal to a
  • polypeptides comprising the amino acid sequence ofthe open reading frame upstream ofthe predicted signal peptide are contemplated by the present invention.
  • polypeptides ofthe invention comprise, or alternatively consists of, an amino acid sequence selected from the group: FSPNVLQSLLPLSHPGSCVGMSVEDGGMPGLGRPRQARWTLMLLLSTAMYG AHAPLLALCHVDGRVPFRPSSAVLLTELTKLLLCAFSLLVGWQAWPQGPPPW RQAAPFALSALLYGANNNLVIYLQRYMDPSTYQVLSNLKIGSTAVLYCLCLR HRLSVRQGLALLLLMAAGACYAAGGLQVPGNTLPSPPPAAAASPMPLHITPL GLLLLILYCLISGLSSVYTELL (SEQ ID NO: 134) and
  • LLIGLAMRLYYGSR SEQ ID NO: 135.
  • fragments and variants of these polypeptides are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neurological conditions (for example neoplasms of neural system), immunological disorders, and developmental disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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 and homology to UDP N-acetylglucosamine transporters indicates that polynucleotides and polypeptides corresponding to this gene are useful for recombinant protein production, especially for correct protein glycosylation.
  • the gene may also be used for diagnostic purposes to detect specific changes in protein glycosylation during disease states such as cancer, inflammation and infection.
  • the tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • elevated expression of this gene product in regions ofthe brain indicates it plays a role in normal neural function.
  • this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host- versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, p
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:24 Some of these sequences are related to SEQ ID NO:24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 2815 of SEQ ID NO:24, b is an integer of 15 to 2829, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:24, and where b is greater than or equal to a + 14.
  • polypeptides ofthe invention comprise, or alternatively consist of, the following amino acid sequence:
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neurological conditions (such as Alzheimer's and Parkinson's disease) and cancer and other cell proliferative disorders (such as ovarian cancer).
  • diseases and conditions which include but are not limited to: neurological conditions (such as Alzheimer's and Parkinson's disease) and cancer and other cell proliferative disorders (such as ovarian cancer).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 83 as residues: Arg-33 to Gly-38, His-56 to Gly-61, Pro-116 to Ala-121.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • this gene product in regions ofthe brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. For example, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
  • Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain degenerative disorders, such as spinal muscular atrophy (SMA).
  • this gene product may be involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification.
  • the polynucleotides and polypeptides ofthe present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein would be useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein may also be used to determine biological activity, 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:25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 2109 of SEQ ID NO:25, b is an integer of 15 to 2123, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a + 14.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: reproductive disorders (for example male infertility and testicular cancer) and immunological disorders, particularly those involving neutrophils (such as neutropenia).
  • reproductive disorders for example male infertility and testicular cancer
  • immunological disorders particularly those involving neutrophils (such as neutropenia).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 84 as residues: Pro-40 to Leu-47, Gln-60 to Ala-65.
  • polypeptides encoding said polypeptides are also encompassed by the invention. expression of this gene product in the testis and testicular cancer tissue may implicate this gene product in normal testicular function, hi addition, this gene product would be useful in the treatment of male infertility, and/or could be used as a male contraceptive. Therefore, polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g. endocrine function, sperm maturation), as well as cancer. Therefore, this gene product would be useful in the treatment of male infertility and/or impotence.
  • testicular function e.g. endocrine function, sperm maturation
  • This gene product is also useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents.
  • the protein is believed to be useful in the treatment and/or diagnosis of testicular cancer, male infertility syndromes, as well as inflammatory conditions including orchitis, nonspecific epididymitis and tumors ofthe testis.
  • the testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues ofthe body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
  • the tissue distribution in activated neutrophils indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:26 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:26 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 1212 of SEQ ID NO:26, b is an integer of 15 to 1226, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a + 14.
  • polypeptides ofthe invention comprise, or alternatively consists of, the following amino acid sequence: MDLWTTSFFFFAVMHNAAMNINVQVSESGFSFWGRYLGVELLGCVVNLYLF l ⁇ WPNCFLNGCIILFlTHQQYIRVSCFSTSYLLMAFKNYRHSXKCEVXPHCSLX CXFLITMM (SEQ ID NO: 117).
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention.
  • This gene is expressed primarily in neural/sensory, musculoskeletal, and digestive systems.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neural/sensory, musculoskeletal, and digestive system diseases.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 85 as residues: Asn-88 to Cys-93.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for neural/sensory, musculoskeletal, digestive systems.
  • tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • elevated expression of this gene product in regions ofthe brain indicates it plays a role in normal neural function.
  • this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the protein may also be used to determine biological activity, 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:27 Some of these sequences are related to SEQ ID NO:27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 2171 of SEQ ID NO:27, b is an integer of 15 to 2185, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:27, and where b is greater than or equal to a + 14.
  • polypeptides ofthe invention comprise, or alternatively consist of, the following amino acid sequence: MASWPFLSPMGPIALALLTQALSSPVGLCLALTCSRRPSPDSVCASCRFPLVPL CCQPSLPALLRPVSHCRYPGTSLGIMLXVYCLFLPMRMSDCEGRDLCXIRCRD PSPWNDA (SEQ ID NO: 137).
  • fragments and variants of this polypeptide (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 hybridize, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding this polypeptide are also encompassed by the invention. This gene is expressed primarily in neutrophils.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immunological disorders particularly those involving neutrophils (such as neutropenia).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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 the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:28 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 487 of SEQ ID NO:28, b is an integer of 15 to 501, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a + 14.
  • the translation product of this gene shares sequence homology with a yeast membrane protein, SUR4, which encodes for APAl that acts on a glucose-signaling pathway that controls the expression of several genes that are transcriptionally regulated by glucose.
  • polypeptides ofthe invention are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and for diagnosis, prevention, and/or treatment of diseases and disorders ofthe liver (particularly fetal liver), disorders involving glucose-regulated ATPase activities in tissues, disorders involving the placenta, and breast tissue (for example breast cancer).
  • polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • tissue or cells particularly of the fetal immune/hematopoietic system, reproductive system, and mammary tissues
  • expression of this gene at significantly higher or lower levels may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
  • tissues e.g., cancerous and wounded tissues
  • bodily fluids e.g., serum, plasma, urine, synovial fluid or spinal fluid
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 87 as residues: Met-1 to Gln-9, His-196 to Leu-204.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • yeast SUR4 membrane protein suggests that the protein product of this clone would be useful for diagnosis and treatment of defects of fetal liver or defects of glucose-regulated ATPase activities.
  • the expression within fetal tissue and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions.
  • developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.
  • Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain degenerative disorders, such as spinal muscular atrophy (SMA).
  • this gene product may be involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • aberrant expression of this gene product in tissues - particularly adult tissues - may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification.
  • the polynucleotides and polypeptides ofthe present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions.
  • this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein would be useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing models and/or tissue trauma.
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drag induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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:29 Some of these sequences are related to SEQ ID NO:29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 1457 of SEQ ID NO:29, b is an integer of 15 to 1471, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:29, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in neural/sensory, connective/epithelial, reproductive, and immune/hematopoetic tissues.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neural/sensory, connective/epithelial, reproductive, and immune/hematopoetic diseases.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe tissue(s) or cell type(s).
  • tissue or cells particularly ofthe neural/sensory, connective/epithelial, reproductive, and immune/hematopoetic systems
  • expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 88 as residues: Ile-55 to Leu-60.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for neural/sensory, connective/epithelial, reproductive, and immune/hematopoetic system diseases.
  • tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception.
  • elevated expression of this gene product in regions ofthe brain indicates it plays a role in normal neural function.
  • this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival.
  • the tissue distribution also indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, p
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1783 of SEQ ID NO:30, b is an integer of 15 to 1797, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:30, and where b is greater than or equal to a
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: immunological disorders (particularly, but not limited to, disorders involving neutrophil and fibroblast functions and synovial fibroblast functions; such as, for example, rheumatoid arthritis).
  • diseases and conditions which include but are not limited to: immunological disorders (particularly, but not limited to, disorders involving neutrophil and fibroblast functions and synovial fibroblast functions; such as, for example, rheumatoid arthritis).
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 89 as residues: Met-1 to Arg-9, Pro-31 to Pro-36, Pro-61 to Pro-66, Pro-81 to Trp-86, Arg-97 to His- 106.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of neutrophil and synovial fibroblast related disorders. For example, neutropenia, neutrophilia, tissue necrosis, neoplasia, granulomatous disease and infectious diseases.
  • the gene can be used in (i) modulation of cellular signal transduction, either in vitro or in vivo; (ii) regulation of communication between cells; (iii) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo (e.g. induction of cell differentiation); (iv) regulation of cellular proliferation, either in vitro or in vivo (e.g.
  • induction of cellular proliferation or inhibition of proliferation as in the case of suppression of tumorigenesis (v) formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, both adult and embryonic (e.g. induction of head formation during vertebrate development or maintenance of hematopoietic progenitor cells); (vi) modulation of cell death, such as stimulation of cell survival; (vii) regulating cell migration; and/or, (viii) immune modulation.
  • hyperproliferative diseases such as neoplastic and hyperplastic disease, e.g.
  • Parkinsoninson's disease Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis
  • demyelinating diseases multiple sclerosis, human immunodeficiency associated
  • the gene expression in synovial and immune related functions also indicate its uses in diseases like arthritis.
  • tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drag induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the protein may also be used to determine biological activity, 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 964 of SEQ ID NO:31, b is an integer of 15 to 978, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:31, and where b is greater than or equal to a + 14.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: hepatic disorders, particularly liver cancer and other liver neoplasms.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 90 as residues: Pro-25 to Trp-36, Lys-38 to Tyr-46, His-49 to His-56, Ala-68 to Ser-73, Asp-87 to Arg-93.
  • Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection and treatment of liver disorders and cancers.
  • the protein can be used for the detection, treatment, and/or prevention of Wilson's disease, cirrhosis, fibrosis, bilirabin metabolism, hepatomegaly, cholestasis, liver cancer (for example, hepatoblastoma), jaundice, hepatitis (acuta and chronic) and liver metabolic diseases and conditions attributable to the differentiation of hepatocyte progenitor cells.
  • polynucleotide sequences such as EST sequences, are publicly available and accessible tlirough sequence databases. Some of these sequences are related to SEQ ID NO:32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 990 of SEQ ID NO:32, b is an integer of 15 to 1004, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:32, and where b is greater than or equal to a
  • polypeptides comprising the amino acid sequence of the open reading frame upstream ofthe predicted signal peptide are contemplated by the present invention, hi specific embodiments, polypeptides ofthe invention comprise, or alternatively consists of, an amino acid sequence selected from the group: NGVPLSTAPRSNASVNNSHPAPGDWFVAAJTLPPSSQKIELKGLAPTCAYVFQ PELLVTRVVEIS ⁇ MEPDVPLPQTLLSHPSYLKVFVPDYTRELLLELRDCVS ⁇ GS LGCPVRLTVGPVTLPS ⁇ FQKVLTCTGAPWPCRLLLPSPPWDRWLQVTAESLV GPLGTVAFSAVAALTACRPRSVTXQPLLQSSQ ⁇ QSF ⁇ ASSGLLSPSPDHQDLG RSGRVDRSPFCLT ⁇ YPVTREDMDVVSVHFQPLDRVSVRVCSDTPSVMRLRL ⁇ TGMDSGGSLTISLRA ⁇ KTEMR ⁇ ETV
  • LGSGAAIWVTILC (SEQ ID NO: 139).
  • 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 ) are encompassed by the invention.
  • Antibodies that bind polypeptides ofthe invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • This gene is expressed primarily in tumors, especially in cancers of breast, ovarian and pancreatic origin.
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: carcinogenesis and rumorigenesis, particularly neoplasms ofthe breast, ovary and pancreas.
  • diseases and conditions which include but are not limited to: carcinogenesis and rumorigenesis, particularly neoplasms ofthe breast, ovary and pancreas.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 91 as residues: Arg-52 to Cys-59, Thr-90 to Thr-97, Pro-115 to Glu-121, Ile-135 to Glu-141. Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions.
  • developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain degenerative disorders, such as spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • this gene product may be involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • this gene product may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides ofthe present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus this protein may modulate apoptosis or tissue differentiation and would be useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases.
  • the protein would be useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. Furthermore, the protein may also be used to determine biological activity, 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:33 Some of these sequences are related to SEQ ID NO:33 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 3159 of SEQ ID NO:33, b is an integer of 15 to 3173 , where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a + 14.
  • the translation product of this clone is expected to share at least some biological activities with RNA helicase proteins. Such activities are known in the art, some of which are described elsewhere herein.
  • This gene is expressed primarily in brain and placenta and to a lesser extent in germinal center B-cells and kidney
  • Polynucleotides and polypeptides ofthe invention are useful as reagents for differential identification ofthe 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: neurological disorders (for example, neurodegenerative diseases such as Alzheimer's and Parkinson's disease), immunological disorders, and reproductive disorders, particularly those involving the placenta.
  • neurological disorders for example, neurodegenerative diseases such as Alzheimer's and Parkinson's disease
  • immunological disorders immunological disorders
  • reproductive disorders particularly those involving the placenta.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification ofthe 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.
  • Preferred polypeptides ofthe present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO: 92 as residues: Arg-44 to Pro-57, Arg-72 to Ala-80, Gln-95 to Asp-105. Polynucleotides encoding said polypeptides are also encompassed by the invention.
  • tissue distribution indicates polynucleotides and polypeptides corresponding to this gene would be useful for the detection, treatment, and/or prevention of neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
  • the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease,
  • this gene product may also be useful for the treatment and diagnosis of learning and cognitive diseases, depression, dementia, psychosis, mania, bipolar syndromes, schizophrenia and other psychiatric conditions.
  • elevated expression of this gene product in regions ofthe brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Expression of this gene the placenta suggests a role for the protein product of this clone in the treatment and/or detection of disorders associated with the female reproductive system and disorders associated with pregnancy (for example, but not limited to, placenta abraptio, placenta previa, placental failure, placental insufficiency).
  • the tissue distribution indicates the polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • the expression pattern indicates this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests a usefulness for treatment of cancer (e.g. by boosting immune responses).
  • Expression in cells of lymphoid origin indicates the natural gene product would be involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, ps
  • the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recraits hematopoietic cells to sites of injury.
  • this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1970 of SEQ LD NO:34, b is an integer of 15 to 1984, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:34, 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 ofthe 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 ofthe putative start codon (methionine) is identified as "5' NT of Start Codon.”
  • the nucleotide position of SEQ ID NO:X ofthe 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 ofthe 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 ofthe secreted portion is identified as "Predicted First AA of Secreted Portion.”
  • amino acid position of SEQ ID NO:Y ofthe last amino acid in the open reading frame is identified as "Last AA of ORF.”
  • SEQ ID NO:X (where X may be any ofthe polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any ofthe 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 ofthe 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 ofthe 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. ⁇ , but also a sample of plasmid DNA containing a human cDNA ofthe 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 ofthe 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.
  • 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 ofthe invention and polynucleotide sequences that may be disclaimed according to certain embodiments ofthe 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 "ntB” 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.
  • the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded from the invention.
  • Polypeptides ofthe 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 ofthe 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 ofthe 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 4. 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe polypeptide having the polypeptide sequence of SEQ ID 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 ofthe 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 ofthe 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 ofthe polypeptide.
  • the deduced amino acid sequence ofthe secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are inco ⁇ orated. The analysis ofthe amino acid sequences ofthe secreted proteins described herein by this program provided the results shown in Table 1.
  • the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) ofthe predicted cleavage point.
  • SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) ofthe predicted cleavage point.
  • cleavage ofthe 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 ofthe 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 ofthe 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 ofthe polypeptide sequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.
  • “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide ofthe present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide ofthe 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%, 97%, 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%, 97%, 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%, 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 ofthe nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides ofthe reference nucleotide sequence encoding the polypeptide.
  • nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% ofthe nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% ofthe 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 ofthe 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 ofthe 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 ofthe subject sequence when calculating percent identity.
  • the percent identity is corrected by calculating the number of bases ofthe query sequence that are 5' and 3' ofthe subject sequence, which are not matched/aligned, as a percent ofthe total bases ofthe query sequence. Whether a nucleotide is matched/aligned is determined by results ofthe 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 ofthe present invention.
  • 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' ofthe 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' ofthe 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 ofthe present invention.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence ofthe present invention it is intended that the amino acid sequence ofthe 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 ofthe query amino acid sequence.
  • up to 5% ofthe 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 ofthe 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 ofthe query sequence that are N- and C-tenninal ofthe subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent ofthe total bases ofthe query sequence. Whether a residue is matched/aligned is determined by results ofthe 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 ofthe present invention.
  • the 10 unpaired residues represent 10% ofthe 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%.
  • 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 ofthe subject sequence which are not matched/aligned with the query, hi 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 ofthe encoded polypeptide are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino 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 ofthe polypeptides ofthe present invention.
  • one or more amino acids can be deleted from the N-terminus or C-terminus ofthe 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 ofthe aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement ofthe hydroxyl residues Ser and Thr; replacement ofthe acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement ofthe aromatic residues Phe, Tyr, and Trp, and replacement ofthe small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants ofthe present invention include (i) substitutions with one or more ofthe 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 ofthe mature polypeptide with another compound, such as a compound to increase the stability and/or solubility ofthe polypeptide (for example, polyethylene glycol), or (iv) fusion ofthe 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 ofthe polypeptide with another compound, such as albumin (including, but not limited to, recombinant albumin (see, e.g., U.S.
  • a further embodiment ofthe invention relates to a polypeptide which comprises the amino acid sequence ofthe 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 to have an amino acid sequence which comprises the amino acid sequence ofthe 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, hi specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence ofthe present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), 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 ofthe polynucleotides ofthe 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, or 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 ofthe 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.
  • “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • nucleotide fragments have uses that 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.
  • representative examples of polynucleotide fragments ofthe 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
  • 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 may be "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 ofthe 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.
  • Preferred 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 ofthe secreted protein or mature form. Furthermore, any combination ofthe above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural or 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 ofthe polypeptide ofthe present invention.
  • the biological activity ofthe 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 ofthe 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 ofthe invention for binding) to an antibody to the polypeptide ofthe invention], immunogenicity (ability to generate antibody which binds to a polypeptide ofthe invention), ability to form multimers with polypeptides ofthe invention, and ability to bind to a receptor or ligand for a polypeptide ofthe invention.
  • polypeptides ofthe 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 ofthe 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 ofthe invention to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of polypeptides ofthe invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that ofthe polypeptide ofthe 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 ofthe polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement ofthe 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 ofthe invention (such as, for example, the sequence disclosed in SEQ ID NO:X), polynucleotide sequences ofthe complementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • polypeptide sequence ofthe invention such as, for example, the sequence disclosed in SEQ ID NO:X
  • polynucleotide sequences ofthe complementary strand of a polynucleotide sequence encoding an epitope ofthe 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 immimogenic 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 determined 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 ofthe 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 iimnunization, 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 maybe 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 ofthe selected antibodies according to methods well known in the art.
  • polypeptides ofthe present invention can be fused to heterologous polypeptide sequences.
  • polypeptides ofthe 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 ofthe 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 ofthe 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 inco ⁇ orated by reference in its entirety.
  • polypeptides and or antibodies ofthe 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.
  • Polypeptides and/or antibodies ofthe present invention may be fused to either the N- or C-terminal end ofthe heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).
  • the heterologous protein e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide.
  • Polynucleotides encoding fusion proteins ofthe 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 ofthe first two domains ofthe human CD4-polypeptide and various domains ofthe constant regions ofthe 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., Fountoulalds 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 ofthe 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 ofthe invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists ofthe 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 ED 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 ofthe 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 ofthe invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • polypeptides ofthe invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, ofthe present invention (as determined by immunoassays well known in the art for assaying specific antibody- antigen binding).
  • TCR T-cell antigen receptors
  • Antibodies ofthe 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 ofthe 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 ofthe 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 ofthe invention are IgGl .
  • the immunoglobulin molecules ofthe invention are IgG4.
  • the antibodies are human antigen-binding antibody fragments ofthe 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 ofthe 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 ofthe 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 ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide ofthe present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
  • Antibodies ofthe present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide ofthe 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-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide ofthe present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides ofthe present invention, and allows for the exclusion ofthe same.
  • Antibodies ofthe 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 ofthe 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 ofthe present invention are also included in the present invention.
  • antibodies ofthe 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 ofthe 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 ofthe specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide ofthe 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 IO “5 M, IO “5 M, 5 X IO “6 M, 10 “6 M, 5 X IO “7 M, IO 7 M, 5 X 10 "8 M, IO “8 M, 5 X 10 "9 M, IO "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "11 M, 10 "11 M, 5 X IO "12 M, 10"12 M, 5 X 10 "13 M, IO “13 M, 5 X 10 "14 M, IO “14 M, 5 X IO “15 M, or IO “15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope ofthe 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 ofthe present invention may act as agonists or antagonists ofthe polypeptides ofthe present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides ofthe invention either partially or fully.
  • antibodies ofthe 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 ligand 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) ofthe receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • antibodies are provided that inhibit ligand 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% ofthe activity in absence ofthe antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand 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 ligand.
  • receptor-specific antibodies which both prevent ligand 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 ligand.
  • neutralizing antibodies which bind the ligand and prevent binding ofthe ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand 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 ofthe biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization ofthe receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities ofthe 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 ofthe present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides ofthe 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 ofthe polypeptides ofthe present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (inco ⁇ orated by reference herein in its entirety).
  • the antibodies ofthe 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 ofthe 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 ofthe 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 ligand 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 tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies ofthe 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 inco ⁇ orated by reference in their entireties).
  • mice can be immunized with a polypeptide ofthe invention or a cell expressing such peptide.
  • 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 ofthe 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 ofthe invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen ofthe 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 ofthe invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments ofthe 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 ofthe heavy chain.
  • the antibodies ofthe 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 ofthe 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 ofthe 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 inco ⁇ orated 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 ofthe interactions ofthe 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. 4,444,887 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 inco ⁇ orated 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 ofthe 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 ofthe 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 ofthe invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides ofthe 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 ofthe invention to a ligand 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 ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide ofthe 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 ofthe 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 ofthe invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y.
  • the polynucleotides maybe obtained, and the nucleotide sequence ofthe polynucleotides detennined, by any method known in the art.
  • a polynucleotide encoding the antibody maybe 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 ofthe 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 ofthe 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 ofthe invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends ofthe 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 ofthe 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 ofthe heavy and/or light chain variable domains may be inspected to identify the sequences ofthe 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 ofthe invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody ofthe invention or a single chain antibody ofthe 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), ofthe invention has been obtained, the vector for the production ofthe 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 ofthe 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 ofthe 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 ofthe invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chain antibody ofthe 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 ofthe entire immunoglobulin molecule, as detailed below.
  • a variety of host-expression vector systems may be utilized to express the antibody molecules ofthe 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 ofthe 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; pTN 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 adso ⁇ tion 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 Spodoptera frugiperda 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, hi cases where an adenovirus is used as an expression vector, 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 he ⁇ es 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 (Crouse 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.
  • 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, h drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
  • human proteins such as hIL-5
  • Fc portions for the pu ⁇ ose 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 a pQE vector (QIAGEN, hie, 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • a pQE vector QIAGEN, hie, 9259 Eton Avenue, Chatsworth, CA, 91311
  • hexa- histidine provides for convenient purification ofthe fusion protein.
  • 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 acetylcholmesterase
  • 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, 1 llln or 99Tc.
  • 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 pachtaxol, 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-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, 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.g.,
  • 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, ATM 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 inco ⁇ orated 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 may be 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
  • these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
  • 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 PJPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in
  • a lysis buffer such as PJPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% S
  • 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 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 1251) 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 1251
  • 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 limited 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 ofthe 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 ofthe invention maybe administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that ofthe patient is preferred. Thus, in a preferred embodiment, 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 IO "2 M, IO “2 M, 5 X 10 "3 M, 10 "3 M, 5 X 10 " M, IO “4 M, 5 X 10 "5 M, IO “5 M, 5 X IO “6 M, IO “6 M, 5 X IO “7 M, IO “7 M, 5 X 10 “8 M, IO “8 M, 5 X IO “9 M, IO "9 M, 5 X 10 "10 M, 10 “10 M, 5 X IO "11 M, IO “11 M, 5 X IO '12 M, IO "12 M, 5 X 10 "13 M, 10 " 13 M, 5 X IO "14 M, IO “14 M, 5 X IO "15 M, and IO “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 of the 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 ligand 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 ligand 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 inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad.
  • 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 tecliniques 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 pu ⁇ oses 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 pu ⁇ oses 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 abso ⁇ tion tlirough 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.
  • 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 inco ⁇ orated 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.
  • the term “earner” 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 of the invention can be determined by standard clinical techniques, hi addition, 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 mg/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 maybe 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 container(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 pu ⁇ oses 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 immunohisto logical 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 99mTc.
  • 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., "hnmunopharmacokinetics 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
  • 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 adso ⁇ tion 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. Alternatively, streptavidin 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 o the 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, may be 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 ofthe 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
  • Fusion proteins having disulfide-linked dimeric structures can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
  • Fusion proteins having disulfide-linked dimeric structures due to the IgG
  • Polynucleotides comprising or alternatively consisting of nucleic acids which encode these fusion proteins are also encompassed by the invention.
  • EP-A-O 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof, hi many cases, 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, hi drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
  • human proteins such as hIL-5
  • Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
  • the polypeptides ofthe 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.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311)
  • hexa-histidine provides for convenient purification ofthe fusion protein.
  • Another peptide tag useful for purification, the "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).)
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides ofthe present invention.
  • 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.
  • 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, tap, 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, pRIT5 available from Phannacia 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, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from hivitrogen, Carlbad, CA).
  • Suitable vectors will be readily apparent to the skilled artisan.
  • Introduction ofthe construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking a recombinant vector.
  • 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-tenninal 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 2 .
  • 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 . 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, pPIC3.5K, 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 ligand, 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 derivitization 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, 16,000, 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; Mo ⁇ urgo 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 inco ⁇ orated 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 inco ⁇ orated 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 pu ⁇ oses 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.
  • One or more 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.
  • 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-terminally 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 maybe 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. Tlxera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol 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 inco ⁇ orated 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 3 ).
  • MPEG monmethoxy polyethylene glycol
  • ClSO CH 2 CF 3 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-
  • 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 detennining 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
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers
  • 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 inco ⁇ orated by reference in its entirety).
  • two or more polypeptides ofthe invention are joined through peptide linkers.
  • peptide linkers include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby inco ⁇ orated by reference). Proteins comprising multiple polypeptides ofthe 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 inco ⁇ orated 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.
  • a leucine zipper derived from lung surfactant protein D SPD
  • SPD lung surfactant protein D
  • Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides ofthe invention.
  • 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 inco ⁇ orated 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 inco ⁇ orated by reference in its entirety).
  • polypeptides ofthe invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus ofthe polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer ofthe invention (see, . e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • multimers ofthe invention may be generated using genetic engineering teclmiques 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 inco ⁇ orated by reference in its entirety).
  • polynucleotides coding for a homodimer ofthe invention are generated by ligating a polynucleotide sequence encoding a polypeptide ofthe 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 inco ⁇ orated 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 inco ⁇ orated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated 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 polymo ⁇ hisms), 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 ID 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 ID 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 tecliniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby inco ⁇ orated 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
  • teclmiques and others known in the art see, e.g., Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483- 492 (1998); Herrick et al, Chromosome Res. 7:409-423 (1999); Hamilton et al, Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered.
  • 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 ofthe 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.
  • biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide ofthe present invention, and other tissue sources found to express the polypeptide ofthe present invention.
  • body fluids such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid
  • tissue sources found to express the polypeptide ofthe present invention.
  • Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a 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 polymo ⁇ hisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymo ⁇ hisms (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 prefened form if the polynucleotides are inco ⁇ orated 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).
  • PNAs phosphorus, phosphorus oxides, or deoxyribose derivatives
  • 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.
  • 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.
  • 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.
  • 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, hi particular the invention is useful during diagnosis of 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.
  • 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, prefened 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 conect 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 polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • 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 cunent limitations of "Dog Tags" which can be lost, switched, or stolen, 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 detennining 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 ID 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 polymo ⁇ hic loci such as DQa class II HLA gene, are used in forensic biology to identify individuals.
  • polynucleotides ofthe present invention can be used as polymo ⁇ hic markers for forensic pu ⁇ oses.
  • 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 inco ⁇ orated 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, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneaily) into the mammal.
  • a radioisotope for example, 1311, 112In, 99mTc
  • a radio-opaque substance for example, parenterally, subcutaneously, or intraperitoneaily
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
  • 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., "hnmunopharmacokinetics 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 a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (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).
  • a 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 ofthe present invention can bind and reduce ove ⁇ roduction ofthe polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • polypeptides ofthe 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 ofthe 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) sequences into an animal to achieve expression of a polypeptide ofthe present invention.
  • 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.
  • Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein inco ⁇ orated 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 art. For example, see Belldegrun et al., J. Natl. Cancer Inst, 85:207-216 (1993); Fenantini et al., Cancer Research, 53: 107-1112 (1993); Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., hit. J.
  • the cells which are engineered are arterial cells.
  • the arterial cells may be reintroduced into the patient tlirough direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
  • the polynucleotide constructs can be 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 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 inco ⁇ orated 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 pEFlN5, pcDNA3.1, and pRc/CMV2 available from Invitrogen.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Suitable promoters include adeno viral promoters, such as the adeno viral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) 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 He ⁇ es 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 manow, 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 prefened 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. Preferably 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. Of course, as the artisan of ordinary skill will appreciate, 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 inco ⁇ orated by reference); mRNA (Malone et al, Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein inco ⁇ orated 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 GIB CO BRL, Grand Island, N.Y. (See, also, Feigner et al, Proc. Natl Acad. Sci. USA , 84:7413-7416 (1987), which is herein inco ⁇ orated 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)piOpane 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 inco ⁇ orated 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 choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.
  • DOPC dioleoylphosphatidyl choline
  • 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 prefened.
  • 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 inco ⁇ orated by reference.
  • MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid 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. Natl.
  • 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 inco ⁇ orated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
  • WO 94/9469 (which are herein inco ⁇ orated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.
  • U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein inco ⁇ orated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.
  • cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides ofthe invention.
  • Retroviruses 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, Myeloproliferative 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, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy , 1:5-14 (1990), which is inco ⁇ orated 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 4 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 unifonnly 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 inco ⁇ orated 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 he ⁇ es 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 of the 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 ofthe 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 constract 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 ofthe present invention may be administered along with other polynucleotides encoding other angiongenic proteins.
  • Angio genie 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. Additionally, the signal sequence may be chemically synthesized using methods known in the art. Any mode of administration of any of the 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 prefened 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 inco ⁇ orated herein by reference).
  • Oral delivery can be performed by complexing 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. Prefened 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 conesponding 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 ofthe present invention can be used as a marker or detector of a particular immune system disease or disorder.
  • a polypeptide ofthe 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),
  • 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, 22ql 1.2 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 immunodeficiencies
  • 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, 1 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 an 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 hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia pu ⁇ ura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia pu ⁇ ura, pu ⁇ ura (e.g., Henloch-Scoenlein pu ⁇ ura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
  • Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the 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, polyendocrmopathies, Reiter' s Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
  • 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
  • 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)
  • 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 cinhosis (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 pu ⁇ ura 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 chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, overproduction 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); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis
  • 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 maybe 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 maybe 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 agonists 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.
  • 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: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B).
  • 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: HIN/ ADDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, he ⁇ es simplex, and yellow fever.
  • a virus, disease, or symptom selected from the group consisting of: HIN/ ADDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, he ⁇ es 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, Enterohemonhagic 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 manow 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 manow transplant, and B cell chronic lymphocytic leukemia (CLL).
  • CLL B cell chronic lymphocytic leukemia
  • 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-rumor 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 manow 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 pu ⁇ ura, 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
  • polypeptides, antibodies, polynucleotides and/or agonists or antagonists ofthe present invention may be employed for instance to inliibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD 8 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 agonists or antagonists ofthe 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 of the 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, recunent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, he ⁇ es zoster (e.g., severe he ⁇ es zoster), and/or pneumocystis carnii.
  • blood-borne infections e.g., sepsis, meningitis, septic arthritis
  • 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, HIN 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 ("CVID"; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.
  • CVID 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 ' ⁇ ype ⁇ roliferative 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 canier, 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 limited to, the prevention of occlusions in extrco ⁇ oreal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
  • extrco ⁇ oreal 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 maybe 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, neutrophils, mast cells, macrophages) and platelets.
  • blood cells such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets.
  • myeloid cells e.g., basophils, eosinophils, neutrophils, 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, neutrophils, mast cells, macrophages) and platelets.
  • blood cells such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, 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 ofthe 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 antagonists ofthe 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 pmpura, and thrombotic thrombocytopenic pmpura), 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 pmpura, and thrombotic thrombocytopenic pm
  • Hemorhhagic Telangiectsia also known as Rendu-Osler- Weber syndrome, allergic pu ⁇ ura (Henoch Schonlein pu ⁇ ura) 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, cinhosis 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 ofthe 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 ofthe 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,
  • 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 hypereosinophilic 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.
  • ALL acute lymphocytic leukemia
  • acute myeloid my
  • 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, macroglobulinemia, 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 myeloproliferative 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.
  • myeloproliferative 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 neutrophils, 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 hype ⁇ roliferative 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 inliibit the hype ⁇ roliferative disorder.
  • hype ⁇ roliferative 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 hype ⁇ roliferative disorders, such as a chemotherapeutic agent.
  • Examples of hype ⁇ roliferative 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, s
  • hype ⁇ roliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists ofthe present invention.
  • hype ⁇ roliferative 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, AHOS-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 hype ⁇ lasia, 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.
  • Hype ⁇ lasia 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.
  • Hype ⁇ lastic 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, angiofollicular mediastinal lymph node hype ⁇ lasia, angiolymphoid hype ⁇ lasia with eosinophilia, atypical melanocytic hype ⁇ lasia, basal cell hype ⁇ lasia, benign giant lymph node hype ⁇ lasia, cementum hype ⁇ lasia, congenital adrenal hype ⁇ lasia, congenital sebaceous hype ⁇ lasia, cystic hype ⁇ lasia, cystic hype ⁇ lasia ofthe breast, denture hype ⁇ lasia, ductal hype ⁇ lasia, endometrial hype ⁇ lasia, fibromuscular hype ⁇ lasia, focal epithelial hype ⁇
  • 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 pleomo ⁇ hism. 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 ectodennal dysplasia, craniodiaphysial dysplasia, cranioca ⁇ otarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysp
  • 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 ofthe 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 antagonists 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 thyroidit
  • 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 including,
  • 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/reperfusion injury, cholestosis (bile duct injury) and liver cancer
  • toxin- induced liver disease such as that caused by alcohol
  • septic shock cachexia and anorexia.
  • Hype ⁇ roliferative 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
  • hype ⁇ roliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists ofthe invention.
  • hype ⁇ roliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, pu ⁇ ura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hype ⁇ roliferative disease, besides neoplasia, located in an organ system listed above.
  • Another prefened embodiment utilizes polynucleotides of the present invention to inhibit abenant 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.
  • 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 invention may be 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 of the 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.
  • a 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 ofthe 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, rumor 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 of the 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.
  • Prefened binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 "6 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, tlirough the inhibition of hematopoietic, tumor- specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer h st, 90(21):1648-53 (1998), which is hereby inco ⁇ orated 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 inco ⁇ orated 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 ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, etal., Eur J Biochem 254(3) :439-59 (1998), which is hereby inco ⁇ orated by reference).
  • TNF tumor necrosis factor
  • TRAMP TNF-receptor-related apoptosis-mediated protein
  • TRAIL TNF-related apoptosis
  • 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, thioredoxms, 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., Cun Top Microbiol Immunol 1998;231:125-41, which is hereby inco ⁇ orated by reference).
  • Such thereapeutic affects ofthe present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
  • the invention 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 of the 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, atheroembohc 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, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis,
  • 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
  • Goodpasture syndrome IgA nephropathy
  • 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 of the 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., neplirocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hype ⁇ hosphatemia).
  • 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 anhythmias, 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 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.
  • heart disease such as anhythmias,
  • Arrhythmias include, but are not limited to, sinus anhythmia, 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 reentry 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, hemonhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein
  • 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 hemonhage, epidural hematoma, subdural hematoma, subaraxhnoid hemonhage, 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 pu ⁇ ura, 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
  • 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, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Asp ergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydi
  • 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. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
  • 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 agonists 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.
  • 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, antagonists 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 bom 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) ofthe eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; 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, retrolental fibroplasia and macular degeneration.
  • neovascular diseases ofthe eye including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia 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 periconieal 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, he ⁇ es 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, he ⁇ es 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-angio genie 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 maybe injected directly into the corneal stroma by an ophthalmologist under microscopic guidance.
  • the prefened site of injection may vary with the mo ⁇ hology 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 corneal 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 fonnation 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 bom 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,
  • an amount ofthe compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occuned, 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 inco ⁇ orated 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.
  • tlie 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 inco ⁇ orated 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 fonn 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-dipyr ⁇ dyl, 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, 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,
  • 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 macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas
  • 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 cinhosis, 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/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.

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  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des protéines humaine sécrétées et des acides nucléiques isolés contenant les régions codantes des gènes qui codent pour ces protéines. Elle concerne aussi des vecteurs, des cellules hôtes, des anticorps, et des procédés de recombinaison destinés à produire des protéines humaines sécrétées. Elle concerne enfin des méthodes thérapeutiques et des procédés de diagnostic utiles pour diagnostiquer et soigner des maladies, des troubles et/ou des conditions associés à ces protéines humaines sécrétées.
EP01902080A 2000-09-29 2001-01-17 24 proteines humaines secretees Withdrawn EP1330466A4 (fr)

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US20040010134A1 (en) * 2000-04-12 2004-01-15 Rosen Craig A. Albumin fusion proteins

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] EBI; 14 September 1999 (1999-09-14) ROBERTSON ET AL.: "df53b05.y1 Morton Fetal Cochlea Homo sapiens cDNA clone IMAGE:2487104 5', mRNA sequence." Database accession no. AW023308 XP002267268 *
DATABASE EMBL [Online] EBI; 26 April 1999 (1999-04-26) MAHAIRAS ET AL.: "HS_5165_A1_F05_T7A RPCI-11 Human Male BAC Library Homo sapiens genomic clone Plate=741 Col=9 Row=K, genomic survey sequence." Database accession no. AQ455625 XP002267267 *
DATABASE GENESEQ [Online] DERWENT; 27 August 2002 (2002-08-27) ROSEN ET AL.: "Human albumin fusion protein #1446" Database accession no. ABG64771 XP002267269 & WO 01 77137 A (HUMAN GENOME SCIENCE INC.) 18 October 2001 (2001-10-18) *
JACOBS K A ET AL: "A GENETIC SELECTION FOR ISOLATING CDNAS ENCODING SECRETED PROTEINS" GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER SCIENCE PUBLISHERS, BARKING, GB, vol. 198, 1 October 1997 (1997-10-01), pages 289-296, XP002045919 ISSN: 0378-1119 *
KLEIN R D ET AL: "SELECTION FOR GENES ENCODING SECRETED PROTEINS AND RECEPTORS" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 93, 1996, pages 7108-7113, XP002911164 ISSN: 0027-8424 *
See also references of WO0228877A1 *

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AU2001227920A1 (en) 2002-04-15

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