WO2001023598A1 - 41 proteines humaines secretees - Google Patents

41 proteines humaines secretees Download PDF

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Publication number
WO2001023598A1
WO2001023598A1 PCT/US2000/026324 US0026324W WO0123598A1 WO 2001023598 A1 WO2001023598 A1 WO 2001023598A1 US 0026324 W US0026324 W US 0026324W WO 0123598 A1 WO0123598 A1 WO 0123598A1
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Prior art keywords
human
cgap
nci
seq
soares
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PCT/US2000/026324
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English (en)
Inventor
George Komatsoulis
Steven M. Ruben
Craig A. Rosen
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Human Genome Sciences, Inc.
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Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to CA002384662A priority Critical patent/CA2384662A1/fr
Priority to JP2001526980A priority patent/JP2003510090A/ja
Priority to EP00966857A priority patent/EP1220942A1/fr
Priority to AU77140/00A priority patent/AU7714000A/en
Publication of WO2001023598A1 publication Critical patent/WO2001023598A1/fr
Priority to US10/670,186 priority patent/US20070031842A1/en
Priority to US10/670,185 priority patent/US20070015162A1/en
Priority to US10/868,184 priority patent/US20070048818A1/en
Priority to US10/994,608 priority patent/US20070048297A1/en
Priority to US11/781,665 priority patent/US20070298491A1/en
Priority to US12/753,401 priority patent/US8410248B2/en
Priority to US13/848,789 priority patent/US20130203164A1/en

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    • 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
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.
  • One type of sorting signal directs a class of proteins to an organelle called the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • the ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, 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. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a "linker" holding the protein to the membrane.
  • the present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
  • a "secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
  • polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
  • the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • a "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ED NO:X or the cDNA contained within the clone deposited with the ATCC.
  • the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
  • a representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.
  • the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
  • the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC.
  • “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C. Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions.
  • Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
  • blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
  • polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • polypeptides may be branched , for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • SEQ ID NO:X refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in Table 1.
  • a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)
  • proteins and translated DNA sequences contain regions where the amino acid composition is highly biased toward a small subset of the 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.
  • Such regions can cause database similarity search programs such as BLAST to find high-scoring sequence matches that do not imply true homology.
  • the problem is exacerbated by the fact that most weight matrices (used to score the alignments generated by BLAST) give a match between any of a group of hydrophobic amino acids (L,V and I) that are commonly found in certain low complexity regions almost as high a score as for exact matches.
  • 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". This is then used as the input sequence (sometimes referred to herein as "Query” and/or "Q") to the BLASTX program. While this regime helps to ensure that high-scoring matches represent true homology, there is a negative consequence in that the BLASTX program uses the query sequence that has been masked by the filters to draw alignments.
  • a stretch of "X"s in an alignment shown in the following application does not necessarily indicate that either the underlying DNA 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 of the 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.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 93. Polynucleotides encoding these polypeptides are also provided. This gene is expressed primarily in Primary Dendritic Cells, lib 1.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2237 of SEQ ID NO:l 1, b is an integer of 15 to 2251, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO: 11 , and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 94 and/or SEQ ED NO: 95. Polynucleotides encoding these polypeptides are also provided.
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 53 as residues: Arg-31 to Asn-37. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2705 of SEQ ID NO: 12, b is an integer of 15 to 2719, 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.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Apoptotic T-cell, re-excision; Brain frontal cortex.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1589 of SEQ ED NO: 13, b is an integer of 15 to 1603, 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.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 55 as residues: Gly-25 to Asn-34. Polynucleotides encoding said polypeptides are also provided.
  • 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2040 of SEQ ID NO: 14, b is an integer of 15 to 2054, 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.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 15 Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2140 of SEQ ED NO: 15, b is an integer of 15 to 2154, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO: 15, and where b is greater than or equal to a + 14.
  • polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group:
  • fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%o, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides , or the complement there of are encompassed by the invention.
  • Antibodies that bind polypeptides of the invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the translation product of this gene is homologous to the complement receptors which play an important role in host defense against infection and in mediating unspecified immune responses.
  • This gene is expressed primarily in the following tissues/cDNA libraries:
  • polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include but are not limited to: vascular diseases and/or disorders.
  • polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s).
  • tissue or cell types e.g., vascular, reproductive, developmental, and cancerous and wounded tissues
  • bodily fluids e.g., serum, plasma, amniotic fluid, 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 homology to the complement receptors suggests a role for polypeptides and polynucleotides of the invention (eg delivered as a soluble receptor) in the treatment of immune disorders such as transplant rejection, sepsis, myocardial and intestinal ischaemia-reperfusion injury, ARDS, nephritis,rheumatoid arthritis and also collagen-induced arthritis, thrombolytic disease, allergy, asthma and multiple sclerosis.
  • tissue distribution and homology to complement receptors indicates the polynucleotides and polypeptides cooesponding to this gene would be useful for the diagnosis and treatment of a variety of immune system disorders.
  • Representative uses are described in the "Immune Activity” and “Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression indicates 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 AEDS, 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, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • AEDS immunodeficiency diseases
  • leukemia rheumatoid arthritis
  • granulomatous disease inflammatory bowel disease
  • sepsis sepsis
  • acne neutropenia
  • 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.
  • tissue distribution in placenta and homology to the fuoowed protein indicates that polynucleotides and polypeptides corresponding to this gene are useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis.
  • 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, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2915 of SEQ ID NO: 16, b is an integer of 15 to 2929, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO: 16, and where b is greater than or equal to a + 14.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 96 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Human Osteoclastoma and to a lesser extent in KMH2; Human Heart; Human 8 Week Whole Embryo; Soares_multiple_sclerosis_2NbHMSP; Human Rejected
  • the homology to B-cell antigen receptors indicates the protein product of this clone is useful for the diagnosis and treatment of a variety of immune system disorders.
  • this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AEDS, 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, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • AEDS immunodeficiency diseases
  • leukemia rheumatoid arthritis
  • granulomatous disease inflammatory bowel disease
  • sepsis sepsis
  • acne neutropenia
  • 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 1235 of SEQ DD NO: 17, b is an integer of 15 to 1249, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO: 17, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in PC3 Prostate cell line.
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO: 18 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2189 of SEQ ID NO: 18, b is an integer of 15 to 2203, 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.
  • Tumor PCA4 Tu; Human Thymus Stromal Cells; Infant brain, Bento Soares; Endothelial-induced; Human Colon Cancer,re-excision; H Female Bladder, Adult;
  • Stratagene colon (#937204); human ovarian cancer; Stratagene NT2 neuronal precursor 937230; Smooth muscle,control; Soares_fetal_lung_NbHL19W; Human
  • Meningima Ml; Human Whole Six Week Old Embryo; Stratagene neuroepithelium (#937231); Human pancreatic islet; Stratagene fetal retina 937202;
  • Soares_parathyroid_tumor_NbHPA 12 Week Old Early Stage Human, II;
  • NCI CGAP AA1 NCI_CGAP_Pr23; Early Stage Human Brain; Pancreatic Islet;
  • NCI CGAP Ewl Smooth muscle, serum induced,re-exc; Human Testes Tumor;
  • NCI_CGAP_Prl Human 8 Week Whole Embryo; pBMC stimulated w/ poly I/C;
  • Lymphoma Human Ovarian Cancer Reexcision; Soares placenta Nb2HP; Soares fetal liver spleen 1NFLS; NCI_CGAP_Lyml2; Human Pineal Gland; NCI_CGAP_Lym5; Stratagene colon (#937204); Jurkat T-cell Gl phase; Stratagene
  • Soares_fetal_lung_NbHL19W Human endometrial stromal cells
  • Cancer (4005522 A2); Human endometrial stromal cells-treated with progesterone; Human Colon, re-excision; TF-1 Cell Line GM-CSF Treated; Human Osteoblasts II;
  • NCI_CGAP_Pr2 NCI_CGAP_Alvl; Barstead spleen HPLRB2;
  • NCI_CGAP_Lyml2 Human Epididymus
  • NCI_CGAP_GC1 Human Epididymus
  • NCI_CGAP_Ut3 NCI_CGAP_Ut3;
  • Kidney Medulla re-excision; Human Bone Marrow, re-excision; Macrophage- oxLDL; Normal Human Trabecular Bone Cells; Stratagene NT2 neuronal precursor
  • Soares senescent fibroblasts NbHSF; Human fetal heart, Lambda ZAP Express; NCI_CGAP_Br3; NCI_CGAP_Pr22; NCI_CGAP_Thyl; Human pancreatic islet; Primary Dendritic Cells, lib 1 ; Human Fetal Brain; Soares ovary tumor NbHOT; Human White Adipose; NCI_CGAP_Ut2; NCI_CGAP_Lyml2; NCI_CGAP_Col4; CD40 activated monocyte dendridic cells; NCI_CGAP_Utl; NCI_CGAP_Brn35; Human endometrial stromal cells-treated with estradiol; NCI_CGAP_Ut2; LPS activated derived dendritic cells; H.
  • NCI_CGAP_Kid5 NCI_CGAP_Kid5; Stratagene hNT neuron (#937233); Macrophage-oxLDL, reexcision; Soares_multiple_sclerosis_2NbHMSP; Soares breast 3NbHBst; Human Fetal Kidney, Reexcision; Soares_fetal_heart_NbHH19W; Soares_parathyroid_tumor_NbHPA; Human Microvascular Endothelial Cells, fract.
  • Lymph node breast Cancer Spinal Cord, re-excision; HTCDL1; NCI_CGAP_Ut3; NCI_CGAP_Col2; NCI_CGAP_Kid5; NCI_CGAP_Thyl ; Gessler Wilms tumor; NCI_CGAP_Pr21 ; Epithelial-TNFa and INF induced; Stratagene NT2 neuronal precursor 937230; NCI_CGAP_Pr22; Stratagene colon (#937204); Fetal Liver, subtraction II;
  • NCI_CGAP_Pr25 Stratagene HeLa cell s3 937216; 22 week old human fetal liver cDNA library; NCI_CGAP_Pr3; NCI_CGAP_Col2;
  • Soares_multiple_sclerosis_2NbHMSP Human Parathyroid Tumor, subtracted; Human Macrophage; Human Astrocyte; Larynx carcinoma II; Human Bone Marrow;
  • NCI_CGAP_Col4 Jia bone marrow stroma; Stratagene schizo brain SI 1;
  • Ovarian Tumor IE, OV5232; Soares_fetal_heart_NbHH19W; Stratagene hNT neuron (#937233); Gessler Wilms tumor; Jia bone maoow stroma; NCI_CGAP_Ut2;
  • Adrenal Gland Tumor S, Human foetal Adrenals tissue; NCI_CGAP_Ut3;
  • NCI_CGAP_Gas4 Fetal Heart; Stratagene neuroepithelium (#937231); normalized infant brain cDNA; NCI_CGAP_Ut 1 ; Soares_fetal_lung_NbHL 19 W; Activated T-
  • Prostate-BPH subtracted II Human Leukocyte, control #2; H. Leukocytes, normalized cot 500 B; Human Normal Cartilage Fraction III; Human colon cancer, metaticized to liver, subtraction; Jurkat Cells; Thyroid Thyroiditis; Spleen/normal; Sinus piniformis Tumour; Larynx Normal; Pharynx Carcinoma; Colon Normal;
  • Stratagene hNT neuron (#937233); Human Umbilical Vein, Reexcision; Soares_fetal_lung_NbHL19W; NCI_CGAP_Ov23; NCI_CGAP_Pr28;
  • Prostate cell line Testis 5; Stratagene HeLa cell s3 937216; Human Substantia Nigra; Stratagene fetal retina 937202; Human Eosinophils; H Macrophage (GM-CSF treated), re-excision; NCI_CGAP_GC2; NCI_CGAP_Lu5; NCE_CGAP_Col2;
  • NCI_CGAP_Lym3 NCI_CGAP_Lym3; Stratagene pancreas (#937208); Stratagene fibroblast (#937212); Clontech human aorta polyA+ mRNA (#6572); Human heart cDNA (YNakamura);
  • Leukocytes normalized cot 5A; Prostate/LNCAP, subtraction I; Prostate, normal, subtraction I; Human Osteoarthritic Cartilage Fraction IV; Adrenal Gland,normal; Human Spleen; Kidney medulla; Human Striatum Depression, re-rescue; Namalwa Cells; Cheek Carcinoma; Prostate BPH,Lib 2, subtracted; Larynx carcinoma IV; Rectum normal; Stomach,normal; Rectum tumour; Messangial cell, frac 1; Colon, normal; Osteoclastoma-normalized B; Larynx Tumour; Human Hypothalamus, Alzheimer's; Human Pancreatic Langerhans; Pharynx carcinoma; Human Gastrocnemius; Stomach Normal; Human Tongue, frac 2; Human Placenta, subtracted; Stomach Tumour; Tongue Normal; Larynx Tumor; Normal trachea; Nasal polyps; K562 + PMA (36 hrs); Test
  • hypothalamus frac A; Ficolled Human Stromal Cells, 5Fu treated; Human Pre-Differentiated Adipocytes; Human B Cell 8866; Human Thymus Tumor, subtracted; Human Prostate BPH, re-excision; H. Striatum Depression, subtracted; Colon, Cancer: (9808C064R)-total RNA; Activated T-Cells, 8 hrs., ligation 2; Human Prostate, subtracted; H. Frontal Cortex, Epileptic; LNCAP + o.3nM R1881; H. Adipose Tissue; H.
  • NCI_CGAP_Brn25 Brain frontal cortex; NCI_CGAP_Brn25; Pancreatic Islet; Neutrophils control, reexcision; Primary Dendritic cells,frac 2; NCI_CGAP_Ut4;
  • NCE CGAP Col NCI_CGAP_Co9; NCE CGAP GC2; NCI_CGAP_Pr4; NCI_CGAP_Alvl; NCI_CGAP_HSC1; NCI_CGAP_Mel3; NCI_CGAP_PrlO;
  • NCI_CGAP_Brl.l Pancreatic Islet
  • NCI_CGAP_Pr2 NCI_CGAP_Pr3;
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 60 as residues: Leu-25 to Thr-31.
  • Polynucleotides encoding said polypeptides are also provided.
  • 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: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1975 of SEQ ED NO: 19, b is an integer of 15 to 1989, 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.
  • Prefeoed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 97. Polynucleotides encoding these polypeptides are also provided.
  • This gene is expressed primarily in the following tissues/cDNA libraries: HL- 60, PMA 4H, re-excision; Human Adult Testes, Large Inserts, Reexcision; Human Adult Pulmonary,re-excision and to a lesser extent in H.
  • Leukocytes normalized cot 5A; Human epithelioid sarcoma; Human endometrial stromal cells-treated with estradiol; Amniotic Cells - Primary Culture; Human Activated T-Cells, re-excision; Ulcerative Colitis; Synovial Fibroblasts (control); Soares breast 2NbHBst; Human Fetal Brain; Human Whole Six Week Old Embryo; Rejected Kidney, lib 4; NTERA2, control; Soares_total_fetus_Nb2HF8_9w; Colon Tumor;
  • 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:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1479 of SEQ ED NO:20, b is an integer of 15 to 1493, 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.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 98 and/or SEQ ID NO: 99. Polynucleotides encoding these polypeptides are also provided.
  • This gene is expressed primarily in the following tissues/cDNA libraries: NCI CGAP Co 14; Soares_multiple_sclerosis_2NbHMSP; Human Umbilical Vein Endothelial Cells, uninduced; Human Chondrosarcoma; Endothelial cells-control.
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1677 of SEQ ED NO:21, b is an integer of 15 to 1691, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:21, and where b is greater than or equal to a + 14.
  • S The segment of dbj
  • Prefeoed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 101 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Preferred polypeptides of the invention comprise the following amino acid sequence: MVPLVAVVSGPRAQLFACLLRLGTQQVGPLQLHTGASHAARNHYEVLVLGG GSGGITMAAJAMKRKVGAENVAIVEPSERHFYQPIWTLVGAGAKQLSSSGRPT ASVEPSGVEWEKARVTELNPDKNCEHTDDDEKISYRYLIEALGIQLDYEKEKGLP EGFAHPKIGSNYSVKTVEKTWKALQDFKEGNAEFTFPNTPVKCAGAPQKIMY LSEAYFRKTGKRSK.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Epithelial-TNFa and INF induced and to a lesser extent in Activated T-cells, 24 hrs,re-excision; Monocyte activated; Primary Dendritic Cells, lib 1 ; Colon Normal III; Soares fetal liver spleen 1NFLS; Stratagene lung (#937210); Keratinocyte; Macrophage-oxLDL; T cell helper II; NCI_CGAP_Kid5; H Macrophage (GM-CSF treated), re-excision; Human OB MG63 control fraction I; Soares NhHMPu S 1 ; LPS activated derived dendritic cells; Human T-cell lymphoma,re-excision; T-Cell PHA 16 hrs; Human Pancreas Tumor; Macrophage (GM-CSF treated); Normal colon; Human Endometrial Tumor; Soares infant brain lNEB; Human Colon, subtraction
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 63 as residues: Met-1 to Pro-7. Polynucleotides encoding said polypeptides are also provided.
  • the tissue distribution in activated T-cells, monocytes, dendritic cells, and spleen indicates the protein product of this clone is useful for the diagnosis and treatment of a variety of immune system disorders. Representative uses are described in the "Immune Activity” and "Infectious Disease” sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells.
  • This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions.
  • immunological disorders including arthritis, asthma, immunodeficiency diseases such as AEDS, 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, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.
  • AEDS immunodeficiency diseases
  • leukemia rheumatoid arthritis
  • granulomatous disease inflammatory bowel disease
  • sepsis sepsis
  • acne neutropenia
  • 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 1721 of SEQ ID NO:22, b is an integer of 15 to 1735, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:22, and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 104. Polynucleotides encoding these polypeptides are also provided. This gene is expressed primarily in the following tissues/cDNA libraries:
  • Chondrosarcoma NTERA2, control; Pancreas Islet Cell Tumor; Primary Dendritic cells,frac 2; Human Fetal Heart; Endothelial-induced; Human Primary Breast Cancer Reexcision; Human Bone Maoow, treated; Soares infant brain lNIB; Kidney medulla; Human colon carcinoma (HCC) cell line, remake; Soares_placenta_8to9weeks_2NbHP8to9W; Human Soleus; B Cell lymphoma;
  • STROMAL -OSTEOCLASTOMA H Female Bladder, Adult; NTERA2 + retinoic acid, 14 days; Breast Cancer Cell line, angiogenic; Apoptotic T-cell; Human Activated T-Cells; Human Pancreas Tumor; T-Cell PHA 24 hrs; Rejected Kidney, lib 4; Resting T-Cell Library,II; Colon Carcinoma; Human Placenta; Dendritic cells, pooled; Soares melanocyte 2NbHM; Normal Human Trabecular Bone Cells; Soares_NhHMPu_S 1 ; Soares_total_fetus_Nb2HF8_9w;
  • Lymph node breast Cancer Human Adult Small Intestine; Hippocampus, Alzheimer Subtracted; Ovary, Cancer: Poorly differentiated adenocarcinoma (9809C332); B-Cells; Human Brain, Striatum; Human Osteoblasts II; Human Uterine Cancer; Soares_placenta_8to9weeks_2NbHP8to9W; Stromal cell TF274; Macrophage-oxLDL; Human Hypothalmus,Schizophrenia; Human umbilical vein endothelial cells, IL-4 induced; Human Pancreas Tumor, Reexcision; Liver, Hepatoma; Human Activated T-Cells, re-excision; Ulcerative Colitis; Macrophage (GM-CSF treated); Ovarian Tumor 10-3-95; Stratagene lung (#937210);
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 64 as residues: Ser-32 to Ser-39. Polynucleotides encoding said polypeptides are also provided.
  • 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2040 of SEQ ID NO:23, b is an integer of 15 to 2054, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:23, and where b is greater than or equal to a + 14.
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 65 as residues: Pro-75 to Asn-80. Polynucleotides encoding said polypeptides are also provided.
  • 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1684 of SEQ ID NO:24, b is an integer of 15 to 1698, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:24, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Human Neutrophil, Activated; human ovarian cancer.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:25 amino acid sequences
  • amino acid 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2385 of SEQ ID NO:25, b is an integer of 15 to 2399, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:25, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • S The segment of gb
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 106 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). This gene is expressed primarily in the following tissues/cDNA libraries:
  • 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 Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, epilepsy, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemoohages, 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 of the 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.
  • Many polynucleotide sequences, such as EST sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO:26 and may have been publicly available prior to conception of the present invention.
  • polynucleotides are specifically excluded from the scope of the present invention.
  • a-b is any integer between 1 to 925 of SEQ ID NO:26
  • b is an integer of 15 to 939, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:26, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Neutrophils control, re-excision and to a lesser extent in Soares infant brain 1NIB; Human Adult Testes, Large Inserts, Reexcision; Human Cerebellum; Early Stage Human Brain; 12 Week Early Stage Human II, Reexcision; Human Testes Tumor, re- excision; Soares_NFL_T_GBC_Sl ; Human Prostate Cancer, Stage C, re-excission; Human Prostate Cancer, Stage B2, re-excision; H.
  • NCI CGAP Ewl Human Activated Monocytes
  • Human Chondrosarcoma Human Chondrosarcoma
  • Soares_testis_NHT Human adult testis, large inserts; Pancreas Islet Cell Tumor; NCI_CGAP_Lu5 ; NCI_CGAP_Kid5 ; Fetal Liver, subtraction II; Human T-Cell
  • Lymphoma Human Substantia Nigra; Colon Carcinoma; breast lymph node CDNA library; Colon Normal II; Adipocytes; Human Fetal Lung III;
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 809 of SEQ ED NO:27, b is an integer of 15 to 823, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:27, and where b is greater than or equal to a
  • the translation product of this clone was deterined to have homology to the okadaic acid-induciple phosphoprotein. Based upon the sequence similarity, it is expected that the polynucleotides and polypeptides of this clone will share at least some of the biological activities to phosphoproteins, and specifically okadaic acid- induciple phosphoprotein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 107. Polynucleotides encoding these polypeptides are also provided. When tested against PC 12 cell lines, supernatants removed from cells containing this gene activated the EGRl (early growth response gene 1) promoter element. Thus, it is likely that this gene activates sensory neuron cells through the EGRl signal transduction pathway. EGRl is a separate signal transduction pathway from Jak-STAT, genes containing the EGRl promoter are induced in various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation.
  • This gene is expressed primarily in the following tissues/cDNA libraries: 12 Week Early Stage Human II, Reexcision and to a lesser extent in 12 Week Old Early Stage Human; Human 8 Week Whole Embryo ; Soares_NhHMPu_S 1 ;
  • NCI CGAP GCBl Human Endometrial Tumor; Morton Fetal Cochlea; Human fetal heart, Lambda ZAP Express; 12 Week Old Early Stage Human, II; CD34 positive cells (Cord Blood); Soares testis NHT; Soares_parathyroid_tumor_NbHPA; NCI_CGAP_Lu5; Human Eosinophils; Soares breast 3NbHBst; Soares infant brain lNEB; Soares adult brain N2b4HB55Y; Gessler Wilms tumor;
  • Soares_pregnant_uterus_NbHPU H. Frontal cortex,epileptic,re-excision; Soares_fetal_lungJSrbHL19W; Soares_fetal_heart_NbHH19W; Human adult (K.Okubo); Colon Tumor II; T cell helper II and Soares placenta Nb2HP.
  • 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 neurodegenerative disorders, such as spinal muscular atrophy (SMA).
  • this gene product may be involved in the pattern of cellular proliferation that accompanies early embryogenesis.
  • abeoant expression of this gene product in tissues - particularly adult tissues - may cooelate 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 of the 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 is useful in modulating the immune response to abeoant polypeptides, as may exist in proliferating and cancerous cells and tissues.
  • the protein can also be used to gain new insight into the regulation of cellular growth and proliferation.
  • 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, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3020 of SEQ ED NO:28, b is an integer of 15 to 3034, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in neutrophils control.
  • 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 407 of SEQ ED NO:29, b is an integer of 15 to 421, 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 the following tissues/cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Soares placenta Nb2HP; Soares senescent fibroblasts NbHSF; Primary Dendritic Cells, lib 1; 12 Week Old Early Stage Human, II; Stromal cell TF274; normalized infant brain cDNA; Synovial Fibroblasts (Ill/TNF), subt; Synovial Fibroblasts (control); Soares breast 3NbHBst; Soares fetal lung NbHLl 9W; Soaresjparathyroid tumor NbHPA; Soares_multiple_sclerosis_2NbHMSP; Soares_placenta_8to9weeks_2NbHP8to9W; NCI_CGAP_Co8; Jurkat Cells, cyclohexamide treated, subtraction;
  • Soares_parathyroid_tumor_NbHPA Smooth muscle, serum treated; Human Testes Tumor; Human Fetal Lung III; 12 Week Early Stage Human II, Reexcision; Human Osteoclastoma; Human Amygdala; Human Microvascular Endothelial Cells, fract. A; HUMAN B CELL LYMPHOMA; T Cell helper I; Colon Normal III; Stratagene hNT neuron (#937233); Hodgkin's Lymphoma II; Osteoblasts; Colon Tumor II; Nine Week Old Early Stage Human; NCI_CGAP_Brn35 and Human Cerebellum.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:30 Some of these sequences are related to SEQ ID NO:30 and may have been publicly available prior to conception of the present invention.
  • related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • a-b is any integer between 1 to 2003 of SEQ ID NO:30
  • b is an integer of 15 to 2017, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:30, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. dbj
  • D4 D4 dopamine receptor (D4DR) [Nycticebus coucang] A partial alignment demonstrating the observed homology is shown immediately below.
  • PR R APGPL S 1 PRSRPAPGPL 10
  • S The segments of dbj
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 109 and/or SEQ ID NO: 111 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Soares infant brain 1NEB and to a lesser extent in normalized infant brain cDNA; Primary Dendritic Cells, lib 1; Activated T-cell(12h)/Thiouridine-re-excision; Human Cerebellum; H.
  • Ovarian Tumor II, OV5232; Soares_pregnant_uterus_NbHPU; Human Thymus Stromal Cells; CHME Cell Line,treated 5 hrs; NTERA2, control; Macrophage-oxLDL, re-excision; Fetal Heart; Human Eosinophils; Human Fetal Kidney, Reexcision; Human Placenta; Activated T- Cell (12hs)/Thiouridine labelledEco; Endothelial cells-control; Human Microvascular Endothelial Cells, fract. A; T Cell helper I; NCI_CGAP_Lu5; Soares placenta Nb2HP; H.
  • Leukocytes normalized cot 500 A; Human Eosinophils; Soares NbHFB; Soares testis NHT; Soares NFL T GBC Sl ; Soares ovary tumor NbHOT; Nasal polyps; CD34+ cell, I, frac II; H. Adipose Tissue; HL-60, RA 4h, Subtracted; Human Adult Spleen; Human OB HOS control fraction I; Soares retina N2b5HR; Human
  • Meningima Ml; Human Infant Brain; Apoptotic T-cell; Human Osteoblasts II; Human Activated T-Cells; Human Heart; Human Hypothalmus,Schizophrenia; Liver, Hepatoma; Stratagene colon (#937204); Human Adipose; Human Thymus; Human Whole Six Week Old Embryo; Rejected Kidney, lib 4; Ovarian Tumor 10-3-95; Human Liver, normal; Human T-Cell Lymphoma; breast lymph node CDNA library; Colon Normal II; Human Placenta; Adipocytes; Dendritic cells, pooled; Human Synovial Sarcoma; 12 Week Early Stage Human II, Reexcision; human tonsils; Human Primary Breast Cancer Reexcision; CD34 depleted Buffy Coat (Cord Blood), re-excision; Spleen, Chronic lymphocytic leukemia; Human Testes; NCI_CGAP_Utl; NCI_CGAP_CLL1; Soares_
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1964 of SEQ ED NO:31, b is an integer of 15 to 1978, 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.
  • This gene is expressed primarily in the following tissues/cDNA libraries: NCI CGAP GCB1; Hodgkin's Lymphoma II and to a lesser extent in Hodgkin's Lymphoma I; Monocyte activated, re-excision; human ovarian cancer;
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 582 of SEQ ID NO:32, b is an integer of 15 to 596, 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
  • Prefeoed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 113 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group: DGKRGKPRGTGGLWVKAEANMGFGATLAVGLTEFVLSVVTIIICFTCSCCCLY KTCRRPRPSKCAAQLPWTKLPGLPHHAASARDASSTLPNAVPTTLPSPAHGPT GLPRDPGWRSSRALPRQPASLQPGLHGCPEAAL (SEQ ID NO: ) and MGFGATLAVGLTEFVLSVVTIIICFTCSCCCLYKTCRRPRPSKCAAQLPWTKLP GLPHHAASARDASSTLPNAVPTTLPSPAHGPTGLPRDPGWRSSRALPRQPASL QPGLHGCPEAAL (SEQ ID NO: ).
  • fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides , or the complement there of are encompassed by the invention.
  • Antibodies that bind polypeptides of the invention are also encompassed by the invention.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Soares ovary tumor NbHOT and to a lesser extent in Keratinocyte; Osteoblasts; Human Primary Breast Cancer Reexcision; Soares adult brain N2b4HB55Y; NTERA2, control; Soares_fetal_heart_NbHH19W; NCI_CGAP_Pr22; NCI_CGAP_GC3; Human Thymus; Stratagene lung (#937210); Soares_placenta_8to9weeks_2NbHP8to9W; Activated T-cell(l 2h)/Thiouridine-re- excision; Colon Tumor II; T cell helper II; Human Cerebellum; Soares fetal liver spleen 1NFLS; Human Primary Breast Cancer,re-excision; Smooth muscle, ILlb induced; NTERA2 + retinoic acid, 14 days; Human Thymus; Clontech human aorta polyA+ mRNA
  • TF-1 Cell Line GM-CSF Treated Human Fetal Kidney; Human Osteoblasts II; Soares_fetal_lung_NbHL19W; Soares_fetal_heart_NbHH19W; Human Umbilical Vein Endothelial Cells, uninduced; Human pancreatic islet; Human retina cDNA Tsp5091-cleaved sublibrary; NCI_CGAP_Br2; NCI_CGAP_GC4; NCI_CGAP_Col2; NCI_CGAP_GCB1; NCI_CGAP_Kid5; NCI_CGAP_Kid6;
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 74 as residues: Ser-62 to Gly-75. Polynucleotides encoding said polypeptides are also provided.
  • Expression of this gene in keratinocytes suggests a role for the encoded protein in the treatment and/or detection of diseases involving the skin including psoriasis, eczema, warts and wound healing, while expression in ovarian tumor suggests a role in the treatment of ovarian cancer, and expression in osteoblasts suggests a role in the treatment of bone disorders such as osteoporosis.
  • 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 of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1721 of SEQ ID NO:33, b is an integer of 15 to 1735, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:33, and where b is greater than or equal to a + 14.
  • Meningima Ml; Human Neutrophil; Human Prostate; Soares_NhHMPu_S 1 ; KMH2; B-Cells; Soares_total_fetus_Nb2HF8_9w; Human Fetal Dura Mater; T-Cell PHA 24 hrs; Human fetal heart, Lambda ZAP
  • Kidney Medulla re-excision; Gessler Wilms tumor; Soares_fetal_heart_NbHH19W; L428; Human Pancreas Tumor; Human Hypothalmus,Schizophrenia; Human Rhabdomyosarcoma; Bone Maoow Stromal Cell, untreated; Human Adrenal Gland Tumor; Pancreatic Islet; Fetal Liver, subtraction II; Colon Tumor; Resting T-Cell Library,II; 12 Week Old Early Stage Human; Soares_multiple_sclerosis_2NbHMSP;
  • Adipose subtracted; Human Testes; Human Eosinophils; PCR, pBMC E/C treated; Aryepiglottis Normal; Testis, normal; brain stem; Human Umbilical Vein Endothelial cells, frac B, re-excision; Salivary Gland, Lib 3; Human Pre-Differentiated Adipocytes; CD34+cells, II, FRACTION 2; Human Leukocytes; LNCAP + 30nM R1881; H.
  • Meniingima M6; Dermatofibrosarcoma Protuberance; Human Adult Liver, subtracted; Human Fetal Brain, random primed; Morton Fetal Cochlea; Human Gall Bladder, fraction II; Human OB HOS control fraction I; Human (HCC) cell line liver (mouse) metastasis, remake; Human Cerebellum, subtracted; Human Umbilical Vein Endothelial Cells, fract. A; H. Atrophic Endometrium; Human Placenta; NCI_CGAP_Brn35; Fetal Heart, re-excision; Lung, Cancer: Poorly-Differentiated Squamous Cell carcinoma (4005313 A3); Human Primary Breast Cancer;
  • Kidney Cortex subtracted; Human Stomach,re-excision; Human Adipose Tissue, re-excision; Human Osteosarcoma; Myoloid Progenitor Cell Line; Jurkat T-Cell, S phase; wilm's tumor; H.
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO: 34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1981 of SEQ ID NO:34, b is an integer of 15 to 1995, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:34, and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 115 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Soares NFL T GBC Sl and to a lesser extent in Soares_testis_NHT; Human Testes; Soares_NhHMPu_S 1 ; Soares_total_fetus_Nb2HF8_9w;
  • 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:35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2488 of SEQ ID NO:35, b is an integer of 15 to 2502, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:35, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • S: 428 VPTT 431 The segment of gb
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 117 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polynucleotides are specifically excluded from the scope of the present invention.
  • a-b is any integer between 1 to 3181 of SEQ ED NO:36
  • b is an integer of 15 to 3195, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. emb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 119 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries:
  • NCI_CGAP_Kid5 Human Fetal Heart
  • Human colon cancer metaticized to liver, subtraction
  • Human fetal heart Lambda ZAP Express
  • NCI_CGAP_Co3 NCI_CGAP_Co3;
  • Soares_NFL_T_GBC_Sl Soares_fetal_heart_NbHH19W;
  • Osteoclastoma CD34 positive cells (Cord Blood); Human Testes; Activated T- cell(12h)/Thiouridine-re-excision; neutrophils control; Nine Week Old Early Stage
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 78 as residues: Ala-41 to Pro-57.
  • Polynucleotides encoding said polypeptides are also provided.
  • 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:37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1575 of SEQ ED NO:37, b is an integer of 15 to 1589, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:37, and where b is greater than or equal to a + 14.
  • Soares_pregnant_uterus_NbHPU H. Kidney Cortex, subtracted; LNCAP prostate cell line; Ovary, Cancer: (4004576 A8); Jurkat T-cell Gl phase; Normalized infant brain,
  • Prefeoed polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 79 as residues: Glu-35 to Phe-44. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3133 of SEQ ID NO:38, b is an integer of 15 to 3147, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:38, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. dbjlBAAl 9005.11 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "lectin-like oxidized LDL receptor [Bos taurus]".
  • sequence homology is shown immediately below.
  • BAAl 9005.11 that is shown as "S” above is set out in the sequence listing as SEQ ID NO: 120. Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 121 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Primary Dendritic Cells, lib 1 and to a lesser extent in Human Neutrophil, Activated;
  • Soares melanocyte 2NbHM Smooth muscle, serum treated; Soares placenta Nb2HP;
  • T-cell(12h)/Thiouridine-re-excision activated monocyte dendridic cells; Keratinocyte; HSC172 cells; Human Activated Monocytes;
  • LPS Human Activated Macrophage
  • Amniotic Cells - TNF induced Smooth muscle, ILlb induced
  • Human Umbilical Vein, Endo. remake Human endometrial stromal cells-treated with progesterone; Epithelial-TNFa and INF induced; Human
  • hypothalamus, frac A re- excision; Skin, burned; Soares_parathyroid_tumor_NbHPA; Larynx Tumour; Human Tongue, frac 1 ; Stomach Tumour; Human Adult Heart; Human White Fat;
  • NCI_CGAP_Kid5 Soares_senescent_fibroblasts_NbHSF; Soares_placenta_8to9weeks_2NbHP8to9W; NTERA2 teratocarcinoma cell line+retinoic acid (14 days); Messangial cell, frac 2; Stomach cancer (human),re- excision; NCI_CGAP_GC6; NCI_CGAP_Panl; Human
  • NCI_CGAP_Kidl Human adult (K.Okubo); NCI_CGAP_Kidl; Fetal Heart; NCE_CGAP_Utl;
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO:39 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2436 of SEQ ED NO:39, b is an integer of 15 to 2450, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:39, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 123 and/or SEQ ID NO: 125 which cooespond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Human Adult Testes, Large Inserts, Reexcision; Soares_senescent_fibroblasts_NbHSF and to a lesser extent in Early Stage Human Brain; Soares retina N2b4HR; Soares fetal heart NbHH 19W; Human adult testis, large inserts; Stratagene lung (#937210); Human Fetal Kidney, Reexcision; Human Primary Breast Cancer Reexcision; HL-60, unstimulated; H.
  • Macrophage-oxLDL re-excision; Colon Carcinoma; Barstead spleen HPLRB2; Gessler Wilms tumor; Soares_total_fetus_Nb2HF8_9w; Soares senescent fibroblasts NbHSF; normalized infant brain cDNA; Human Testes, Reexcision; human tonsils; Activated T-Cell (12hs)/Thiouridine labelledEco; NC1_CGAP_GC6; Soares_fetal_lung_NbHLl 9W; Human Testes; Keratinocyte;
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 81 as residues: Pro-46 to Arg-63. Polynucleotides encoding said polypeptides are also provided.
  • testes The relatively high expression of this gene in testes suggests a role in the detection and treatment of male reproductive disorders or dysfunctions such as testicular cancer, and male infertility.
  • 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 Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, epilepsy, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemoohages, 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 of the 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1500 of SEQ ID NO:40, b is an integer of 15 to 1514, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:40, and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 82 as residues: His-1 to Gln-6, Glu-28 to Pro-35.
  • Polynucleotides encoding said polypeptides are also provided.
  • 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:41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2191 of SEQ ID NO:41, b is an integer of 15 to 2205, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:41, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • AF031588 WASP interacting protein [Homo sapiens] A partial alignment demonstrating the observed homology is shown immediately below.
  • S The segment of gb
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 127 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). This gene is expressed primarily in the following tissues/cDNA libraries:
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 83 as residues: Cys-31 to Leu-37.
  • Polynucleotides encoding said polypeptides are also provided.
  • 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:42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1233 of SEQ ID NO:42, b is an integer of 15 to 1247, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:42, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 129 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries:
  • SoaresJNfhHMPu Sl Soares_fetal_liver_spleen_lNFLS_Sl; Stratagene ovarian cancer (#937219); Stratagene neuroepithelium NT2RAMI 937234; Soares infant brain 1NEB; stomach cancer (human); Human Lung Cancer,re-excision; LNCAP prostate cell line; Human Prostate; Normal Human Trabecular Bone Cells;
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 84 as residues: Asn-54 to Thr-61. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:43 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:43 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3019 of SEQ ID NO:43, b is an integer of 15 to 3033, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:43, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent in Soares infant brain lNIB; Soares_total_fetus_Nb2HF8_9w; Soares_pregnant_uterus_NbHPU; Soares_fetal_liver_spleen_lNFLS_S 1 ; Soares NhHMPu S 1 ; Soares_fetal_liver_spleen_lNFLS_Sl ; NCI_CGAP_Pr28; Soares_total_fetus_Nb2HF8_9w; Soares melanocyte 2NbHM; normalized infant brain cDNA; NCI_CGAP_Col2; Larynx normal #10 261-273; stromal cell clone 2.5; Morton Fetal Cochlea; Smooth muscle-ILb induced; NCI_CGAP_Brn23 ;
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:44 amino acid sequence sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1438 of SEQ ID NO:44, b is an integer of 15 to 1452, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:44, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. sp
  • G957282 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "TOXIN 7, TOXEN II-13.3 BETA-TYPE NA+ CHANNEL-BLOCKENG TOXEN".
  • S The segments of sp
  • Prefened polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 131 and/or SEQ ID NO: 133 which cooespond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). This gene is expressed primarily in Salivary Gland, Lib 2.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:45 amino acid sequence sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2106 of SEQ ED NO:45, b is an integer of 15 to 2120, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:45, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. emb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • Prefeoed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 135 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). This gene is expressed primarily in the following tissues/cDNA libraries: H.
  • Kidney Cortex subtracted and to a lesser extent in Soares NhHMPu Sl;
  • NCI CGAP GCBl Colon Carcinoma; human tonsils; Human Osteoclastoma;
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO:46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2303 of SEQ ED NO:46, b is an integer of 15 to 2317, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. emb
  • testis nuclear RNA binding protein [Mus musculus] >pir 11488401148840 testis nuclear RNA binding protein ⁇ sp
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 137 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent in Human adult testis, large inserts; Human Liver, normal; Liver, Hepatoma; Stratagene liver (#937224); breast lymph node CDNA library; Human osteoarthritis,fraction I; Human Cerebellum; Human Amygdala; Human Testes; Human Endometrial Tumor; Human Pineal Gland; Human Epididymus; Synovial Fibroblasts (control); H. Frontal cortex,epileptic,re- excision; H.
  • Kidney Cortex subtracted; Human Chronic Synovitis; Human Substantia Nigra; Soares ovary tumor NbHOT; Human Manic Depression Tissue; Soares breast 3NbHBst; Human Hypothalmus,Schizophrenia; human tonsils; Spleen, Chronic lymphocytic leukemia; Human Amygdala,re-excision; Human Brain, Striatum; Spinal cord; Human osteoarthritic,fraction II; Human Pituitary, subt IX; Soares placenta Nb2HP; Human Normal Cartilage,Fraction I; Breast Lymph node cDNA library; H. Epididiymus, cauda; Human Hypothalamus,schizophrenia, re-excision; Amniotic
  • Hepatocellular Tumor Hepatocellular Tumor, re-excision; Smooth muscle,control; Nine Week Old Early Stage Human; Soares infant brain INEB; Human Fetal Liver, subtracted; STRIATUM DEPRESSION; Weizmann Olfactory Epithelium; Soares_pineal_gland_N3HPG; Soares adult brain N2b4HB55Y; Hepatocellular Tumor; Synovial IL-l/TNF stimulated; Glioblastoma; Human Whole Brain #2 - Oligo dT > 1.5Kb; H.
  • Meningima Ml; Prostate BPH; Stratagene HeLa cell s3 937216; H. Kidney Medulla, re-excision; Stratagene lung (#937210); Human T-Cell Lymphoma; Hodgkin's Lymphoma II; Human 8 Week Whole Embryo; Human Hippocampus, prescreened; Brain Amygdala Depression; H. Frontal Cortex, Epileptic; Human Cerebellum, subtracted; H.
  • Atrophic Endometrium Human Thyroid; Stratagene ovarian cancer (#937219); Stratagene ovary (#937217); H Female Bladder, Adult; Soares_fetal_lung_NbHL19W; Human Adipose Tissue, re-excision; H.
  • Lymph node breast Cancer human ovarian cancer; Human Pancreas Tumor; Soares_fetal_lung_NbHL19W; Epithelial-TNFa and INF induced; Rejected Kidney, lib 4; Pancreas Islet Cell Tumor; Brain frontal cortex; Human Testes Tumor; Human Fetal Kidney, Reexcision; Human Synovial Sarcoma; Human Placenta; Human Hippocampus; Human kidney Cortex, subtracted; H.
  • hypothalamus frac A,re- excision; Human Osteoarthritic Cartilage Fraction III; Human Amygdala Depression, re-excision; Human Infant Adrenal Gland; Human Normal Cartilage Fraction II; Testes; Hypothalamus; Human Pituitary, re-excision; Human Colon; Human
  • Pancreatic Carcinoma Human Pituitary, subtracted; Human Fetal Bone; Smooth Muscle- HASTE normalized; H. Whole Brain #2, re-excision; B Cell lymphoma; Human T-cell lymphoma,re-excision; Human Prostate; Soares_fetal_liver_spleen_lNFLS_Sl ; Stratagene fetal spleen (#937205); Soares_total_fetus_Nb2HF8_9w; Human Rhabdomyosarcoma; Human Chondrosarcoma; Stratagene HeLa cell s3 937216; Ulcerative Colitis; Soares_fetal_heart_NbHH19W; Human Fetal Brain; Stratagene NT2 neuronal precursor 937230; Stratagene ovarian cancer (#937219); H Macrophage (GM-CSF treated), re-excision; Endothelial-induced; Soares_pregnant_uterus_Nb
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 88 as residues: Gly-23 to Gln-30, Asn-57 to Ala-62, Pro-161 to Leu- 168. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:47 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:47 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 943 of SEQ ID NO:47, b is an integer of 15 to 957, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:47, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. emb
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 139 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). This gene is expressed primarily in the following tissues/cDNA libraries:
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 89 as residues: Phe-30 to Lys-37, Pro-43 to Lys-75. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1067 of SEQ ID NO:48, b is an integer of 15 to 1081, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:48, and where b is greater than or equal to a + 14.
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. emb
  • BolA [Vibrio alginolyticus] A partial alignment demonstrating the observed homology is shown immediately below.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO: 141 which cooesponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries:
  • Soares infant brain INEB; Smooth muscle-ILb induced; Messangial cell, frac 2;
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 90 as residues: Ala-21 to Ser-27, Pro-33 to Pro-41.
  • Polynucleotides encoding said polypeptides are also provided.
  • 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:49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 911 of SEQ ED NO:49, b is an integer of 15 to 925, where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:49, and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ED NO: 143 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • This gene is expressed primarily in the following tissues/cDNA libraries:
  • Soares_NFL_T_GBC_Sl Human Primary Breast Cancer Reexcision and to a lesser extent in Soares ovary tumor NbHOT; Soares_NFL_T_GBC_Sl; Human Brain; Breast, Cancer: (9806C012R); Hodgkin's Lymphoma I; NTERA2 teratocarcinoma cell line+retinoic acid (14 days); Soares adult brain N2b4HB55Y; Synovial hypoxia-RSF subtracted; wilm's tumor; Olfactory epithelium,nasalcavity; breast lymph node CDNA library;
  • NCI_CGAP_GC6 Endothelial cells-control; NCI_CGAP_GC6; NCI_CGAP_Gas4; NCI_CGAP_Brn25; Soares_testis_NHT; Soares_NFL_T_GBC_Sl; NCI_CGAP_Co3; Hodgkin's Lymphoma II; NCI_CGAP_GCB1 and T cell helper II.
  • Prefeoed polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ED NO: 91 as residues: His-49 to His-57. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 788 of SEQ ID NO:50, b is an integer of 15 to 802, where both a and b cooespond to the positions of nucleotide residues shown in SEQ ED NO:50, and where b is greater than or equal to a + 14.
  • This gene is expressed primarily in the following tissues/cDNA libraries: normalized infant brain cDNA and to a lesser extent in Soares_NFL_T_GBC_Sl ; Soares infant brain INEB; Human Whole Brain #2 - Oligo dT > 1.5Kb; Barstead spleen HPLRB2; Human fetal heart, Lambda ZAP Express; NCI_CGAP_Ewl; Human Adult Spleen; Soares_pregnant_uterus_NbHPU; Stratagene neuroepithelium NT2RAMI 937234; Human Fetal Kidney; Stratagene lung (#937210); Human Testes, Reexcision; Bone maoow; Neutrophils IL-1 and LPS induced; Human Testes;
  • Soares_testis_NHT H. Frontal cortex,epileptic,re-excision; Hodgkin's Lymphoma II; Human 8 Week Whole Embryo; Colon Tumor II and Human Cerebellum.
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ED NO:51 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2297 of SEQ ED NO:51, b is an integer of 15 to 2311 , where both a and b correspond to the positions of nucleotide residues shown in SEQ ED NO:51, 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 ED NO:X” was assembled from partially homologous ("overlapping") sequences obtained from the "cDNA clone ED” 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 ED was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones cooesponding to the same gene. "Vector” refers to the type of vector contained in the cDNA Clone ID.
  • Total NT Seq refers to the total number of nucleotides in the contig identified by "Gene No.”
  • the deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5' NT of Clone Seq.” and the "3 ' NT of Clone Seq.” of SEQ ID NO:X.
  • the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon.”
  • the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep.”
  • the translated amino acid sequence beginning with the methionine, is identified as "AA SEQ ED NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques.
  • the polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
  • SEQ ID NO:Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep.”
  • the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion.”
  • amino acid position of SEQ ED NO:Y of the last amino acid in the open reading frame is identified as "Last AA of ORF.”
  • SEQ ED NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ED NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below.
  • SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ED NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention.
  • polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1. Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing eoors.
  • the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
  • the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence.
  • the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
  • the present invention provides not only the generated nucleotide sequence identified as SEQ ED NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1.
  • the nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits.
  • amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
  • the present invention also relates to the genes corresponding to SEQ ED
  • 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 ED NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
  • Table 2 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 Codes” shows the expression profile of tissue and/or cell line libraries which express the polynucleotides of the invention.
  • Each Library Code in column 2 represents a tissue/cell source identifier code corresponding to the Library Code and Library description provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested.
  • One of skill in the art could routinely use this information to identify tissues which show a predominant expression pattern of the cooesponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue expression.
  • Table 3 column 1 provides a nucleotide sequence identifier, "SEQ ID NO:X,” that matches a nucleotide SEQ ID NO:X disclosed in Table 1, column 5.
  • Table 3, column 2 provides the chromosomal location, "Cyto logic Band or Chromosome,” of polynucleotides cooesponding to SEQ ED NO:X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIMTM.
  • Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 3, column 3.
  • OMEM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMEM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
  • Column 2 provides diseases associated with the cytologic band disclosed in Table 3, column 2, as determined using the Morbid Map database.
  • polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.
  • the present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ED NO:X, and/or a cDNA contained in ATCC deposit Z.
  • the present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z.
  • Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ED NO:Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.
  • the present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ED NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone.
  • Polynucleotides encoding the mature forms are also encompassed by the invention.
  • proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • the deduced amino acid sequence of the 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 of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1. As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty.
  • the present invention provides secreted polypeptides having a sequence shown in SEQ ED NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • SEQ ED NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below).
  • a mammalian cell e.g., COS cells, as desribed below.
  • the present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ED NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.
  • the present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ED NO:Y and/or encoded by a deposited clone.
  • Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
  • the present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 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).
  • 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
  • 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%o, 96%), 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ED 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).
  • nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
  • nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • the query sequence may be an entire sequence shown inTable 1, the ORF (open reading frame), or any fragment specified as described herein.
  • nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)).
  • a sequence alignment the query and subject sequences are both DNA sequences.
  • An RNA sequence can be compared by converting U's to T's.
  • the result of said global sequence alignment is in percent identity.
  • the percent identity is cooected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This cooected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
  • the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
  • the 10 unpaired bases represent 10%) of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • a 90 base subject sequence is compared with a 100 base query sequence.
  • deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the pu ⁇ oses of the present invention.
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • the amino acid sequence of the subject polypeptide may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%,, 96%, 97%, 98% or 99% identical to, for instance, an amino acid sequences shown in Table 1 (SEQ ID NO:Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs.
  • a prefeoed method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)).
  • the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is in percent identity.
  • Penalty l
  • Window Size sequence length
  • Window Size 500 or the length of the subject amino acid sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to N- or C- te ⁇ ninal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a cooesponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the pu ⁇ oses of the present invention.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence.
  • deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually cooected.
  • residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually cooected for. No other manual corrections are to made for the pu ⁇ oses of the present invention.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also prefeoed.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host
  • 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.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
  • Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
  • 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. For example, 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. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and T ⁇ , and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification or (v) fusion of the polypeptide with another compound, such as albumin (including, but not limited to, recombinant albumin (see, e.g., U.S.
  • a further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions.
  • a peptide or polypeptide it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
  • the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
  • the present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, 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 of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:X.
  • “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 of the invention include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650
  • 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 of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region.
  • polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further prefeoed polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1 - 60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural 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 of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity.
  • a polypeptide demonstrating a "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein.
  • Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
  • polypeptides of the invention and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123.
  • physiological cooelates of binding of a polypeptide of the invention to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo).
  • Other methods will be known to the skilled artisan and are within the scope of the invention.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ED NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ED NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ED NO:X), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as 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.
  • Prefeoed polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
  • Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art.
  • immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intiadermal 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 adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof), or albumin (including but not limited to recombinant albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
  • antigens e.g., insulin
  • FcRn binding partner such as IgG or Fc fragments
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
  • Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • HA hemagglutinin
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Cuo. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention may be altered by being subjected to random mutagenesis by eoor-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody- antigen binding).
  • TCR T-cell antigen receptors
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, EgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulin molecules of the invention are IgGl .
  • the immunoglobulin molecules of the invention are IgG4.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. 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 of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-teoninal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
  • Antibodies that bind polypeptides with at least 95%>, at least 90%, at least 85%, at least 80%), at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%) identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the cooesponding epitopes thereof.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%>, and less than 50%> identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
  • Prefeoed binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 “2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 "4 M, 10 “4 M, 5 X 10 "5 M, 10 “5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "n M, 10 " ⁇ M, 5 X 10 " ' 2 M, 10"12 M, 5 X 10 "13 M, 10 “13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 "15 M, or 10 "15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%), at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
  • the invention features both receptor-specific antibodies and ligand-specific antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.
  • receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • phosphorylation e.g., tyrosine or serine/threonine
  • 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% of the activity in absence of the 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 of the 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 of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Haoop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (inco ⁇ orated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
  • the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular 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.
  • the derivative may contain one or more non-classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art. For example, 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).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology
  • “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which caoy the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are 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 cooesponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska.
  • 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 of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinj ected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice reaoange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a 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 of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ED NO:Y.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non- human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by 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).
  • a number of viral -based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the 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.
  • 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 of the desired coding sequence to ensure translation of the entire insert. These 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 of the 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 of the protein.
  • Different host cells have characteristic and specific mechanisms for the posttranslational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the cooect modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the 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.
  • 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. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) 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. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 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 of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
  • the host cell may be co-transfected with two expression vectors of the 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 ah 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 of the 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, or by any other standard technique for the purification of proteins.
  • the antibodies of the 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 of the polypeptide) of the 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 of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the 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 of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the 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 of the present invention can form dimers through disulfide bonding between the Fc portions.
  • polypeptides cooesponding to a polypeptide, polypeptide fragment, or a variant of SEQ ED NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides cooesponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification.
  • chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins.
  • polypeptides of the 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.
  • Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the 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, 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 of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which cooesponds 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. Examples of 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. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin; and
  • suitable radioactive material include 1251, 1311, 11 lln 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, 213Bi.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabohtes (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
  • the conjugates of the 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, AEM 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 of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • 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 of the invention may be utilized for immunophenotyping of cell lines and biological samples.
  • the translation product of the gene of the 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. Patent 5,985,660; and Morrison et al, Cell, 96:131-49 (1999)).
  • hematological malignancies i.e. minimal residual disease (MRD) in acute leukemic patients
  • 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 of the 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 REP A 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 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 SDS/sample buffer.
  • a period of time e.g., 1-4 hours
  • 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 SDS/sample buffer e.g., western blot analysis.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%>- 20% SDS- PAGE depending on the molecular weight of the 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 non- fat 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 of the 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 of the 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 of the antigen of interest to the coated well.
  • ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • labeled antigen e.g., 3H or 1251
  • the affinity of the 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 of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions.
  • Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
  • the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with abeoant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.
  • the treatment and/or prevention of diseases, disorders, or conditions associated with abeoant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
  • a summary of the ways in which the antibodies of the 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 of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
  • 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.
  • the antibodies of the invention may be 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 of the patient is preferred.
  • 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, of the present invention.
  • Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
  • Prefeoed binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 ' 4 M, 10 ⁇ 4 M, 5 X 10 "5 M, 10 "5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 '7 M, 10 “7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 " “ M, 10 “ “ M, 5 X 10 "12 M, 10 “12 M, 5 X 10 ",3 M, 10 “ 13 M, 5 X 10 "I4 M, 10 "14 M, 5 X 10 "15 M, and l0 "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. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • 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 of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.
  • 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, of the antibody.
  • Delivery of the 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. Sci. USA 86:8932-8935 (1989); Zijlstra et al " ., ' Nature 342:435-438 (1989)).
  • viral vectors that contains nucleic acid sequences encoding an antibody of the 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 of the 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 of the 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, Cuo. 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 transfeoed gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinj ection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
  • the technique should provide for the stable transfer of the 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 maoow, 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.
  • 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 of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • the compounds or pharmaceutical compositions of the 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 of the 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 of the invention, preferably an antibody of the 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 of the 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 through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neural. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the 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.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example
  • pharmaceutically acceptable means approved by a regulatory agency of the 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 "caoier” 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.
  • the 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 foonulated 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 of the 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 of the 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, feoic hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with abeoant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the 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 of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the 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 of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the 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 of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the 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 of the invention.
  • the invention provides for the detection of abeoant expression of a polypeptide of interest, comprising (a) assaying the expression of the 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 of the 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 of the 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 of the disease, or may provide a means for detecting the disease prior
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101 :976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the 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 of the 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
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or
  • time interval following administration is 5 to 20 days or 5 to 10 days.
  • monitoring of the 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 of the 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 of the invention, preferably a purified antibody, in one or more containers.
  • the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
  • the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
  • kits of the 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 of the kit may also be attached to a solid support.
  • the detecting means of the 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 of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
  • the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the 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 of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

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Abstract

La présente invention concerne de nouvelles protéines humaines sécrétées ainsi que des acides nucléiques isolés contenant les régions codantes des gènes codant ces protéines. L'invention concerne aussi des vecteurs, des cellules hôtes, des anticorps ainsi que des méthodes de recombinaison permettant la production des protéines humaines sécrétées. L'invention concerne également des méthodes diagnostiques et thérapeutiques utiles pour le diagnostic et le traitement de maladies, de troubles et/ou d'états liés à ces nouvelles protéines humaines sécrétées.
PCT/US2000/026324 1999-03-12 2000-09-26 41 proteines humaines secretees WO2001023598A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
CA002384662A CA2384662A1 (fr) 1999-09-27 2000-09-26 41 proteines humaines secretees
JP2001526980A JP2003510090A (ja) 1999-09-27 2000-09-26 ４１個のヒト分泌タンパク質
EP00966857A EP1220942A1 (fr) 1999-09-27 2000-09-26 41 proteines humaines secretees
AU77140/00A AU7714000A (en) 1999-09-27 2000-09-26 41 human secreted proteins
US10/670,185 US20070015162A1 (en) 1999-03-12 2003-09-25 99 human secreted proteins
US10/670,186 US20070031842A1 (en) 1999-03-12 2003-09-25 379 human secreted proteins
US10/868,184 US20070048818A1 (en) 1999-03-12 2004-06-16 Human secreted proteins
US10/994,608 US20070048297A1 (en) 1999-03-12 2004-11-23 Human secreted proteins
US11/781,665 US20070298491A1 (en) 1999-03-12 2007-07-23 Human Secreted Proteins
US12/753,401 US8410248B2 (en) 1999-03-12 2010-04-02 HWBAO62 polypeptides
US13/848,789 US20130203164A1 (en) 1999-03-12 2013-03-22 Human Secreted Proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15580799P 1999-09-27 1999-09-27
US60/155,807 1999-09-27

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2000/026371 Continuation-In-Part WO2001023409A2 (fr) 1999-03-12 2000-09-26 38 proteines secretees humaines
PCT/US2000/026323 Continuation-In-Part WO2001023546A1 (fr) 1999-03-12 2000-09-26 37 proteines secretees humaines

Related Child Applications (4)

Application Number Title Priority Date Filing Date
PCT/US2000/026371 Continuation-In-Part WO2001023409A2 (fr) 1999-03-12 2000-09-26 38 proteines secretees humaines
PCT/US2000/026323 Continuation-In-Part WO2001023546A1 (fr) 1999-03-12 2000-09-26 37 proteines secretees humaines
US95008201A Continuation-In-Part 1999-03-12 2001-09-12
US95008301A Continuation-In-Part 1999-03-12 2001-09-12

Publications (1)

Publication Number Publication Date
WO2001023598A1 true WO2001023598A1 (fr) 2001-04-05

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EP (1) EP1220942A1 (fr)
JP (1) JP2003510090A (fr)
AU (1) AU7714000A (fr)
CA (1) CA2384662A1 (fr)
WO (1) WO2001023598A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995022604A1 (fr) * 1994-02-17 1995-08-24 The Wistar Institute Of Anatomy And Biology Enzyme d'edition d'arn et procedes d'utilisation
US5547856A (en) * 1992-05-18 1996-08-20 Genentech, Inc. Hepatocyte growth factor variants
WO1998022577A1 (fr) * 1996-11-15 1998-05-28 Maria Grazia Masucci Proteines de fusion a demi-vie allongee

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5547856A (en) * 1992-05-18 1996-08-20 Genentech, Inc. Hepatocyte growth factor variants
WO1995022604A1 (fr) * 1994-02-17 1995-08-24 The Wistar Institute Of Anatomy And Biology Enzyme d'edition d'arn et procedes d'utilisation
WO1998022577A1 (fr) * 1996-11-15 1998-05-28 Maria Grazia Masucci Proteines de fusion a demi-vie allongee

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CA2384662A1 (fr) 2001-04-05
AU7714000A (en) 2001-04-30
JP2003510090A (ja) 2003-03-18
EP1220942A1 (fr) 2002-07-10

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