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EP1185700A1 - 49 proteines humaines secretees - Google Patents

49 proteines humaines secretees

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Publication number
EP1185700A1
EP1185700A1 EP00936426A EP00936426A EP1185700A1 EP 1185700 A1 EP1185700 A1 EP 1185700A1 EP 00936426 A EP00936426 A EP 00936426A EP 00936426 A EP00936426 A EP 00936426A EP 1185700 A1 EP1185700 A1 EP 1185700A1
Authority
EP
European Patent Office
Prior art keywords
human
seq
soares
sequence
cgap
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00936426A
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German (de)
English (en)
Inventor
Craig A. Rosen
Steven M. Ruben
George A. Komatsoulis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Human Genome Sciences Inc
Original Assignee
Human Genome Sciences Inc
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Filing date
Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Publication of EP1185700A1 publication Critical patent/EP1185700A1/fr
Withdrawn legal-status Critical Current

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    • 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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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides, 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 ID NO:X or the cDNA contained within the clone deposited with the ATCC.
  • the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
  • a representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.
  • the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
  • the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC.
  • “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 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.
  • 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 poIyA+ 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. (See, for instance,
  • 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.
  • 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.
  • AD7c-NTP neuronal thread protein
  • Length 375
  • the segment of gil3002527 that is shown as "S” above is set out in the sequence listing as SEQ ID NO. 109 . 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. 110 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 11 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 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 1873 of SEQ ID NO: 11, b is an integer of 15 to 1887, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 11, and where b is greater than or equal to a + 14.
  • 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. gil4102711 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "EH-binding protein [Homo sapiens]".
  • EH-binding protein [Homo sapiens] A partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil4102711 that is shown as "S” above is set out in the sequence listing as SEQ ID NO. 111 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 112 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 present invention comprise immunogenic epitopes shown in SEQ ID NO: 61 as residues: Met-1 to T ⁇ -9. 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 2271 of SEQ ID NO: 12, b is an integer of 15 to 2285, 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1791 of SEQ ID NO: 13, b is an integer of 15 to 1805, 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.
  • Soares_pregnant_uterus_NbHPU and to a lesser extent in Human Endometrial Tumor; Keratinocyte; Soares infant brain 1MB; Human Substantia Nigra; Human Amygdala; T Cell helper I; Human 8 Week Whole Embryo; Soares fetal liver spleen INFLS; Supt Cells, cyclohexamide treated; Human Ovary; Human Fetal Brain; Soares_NhHMPu_S 1 ; Human Fetal Heart; Human Osteoclastoma; Hodgkin's Lymphoma II; Human Fetal Brain, normalized c5-l l-26; Osteoclastoma-normalized A; Osteoclastoma-normalized B;
  • Soares_senescent_fibroblasts_NbHSF CD40 activated monocyte dendridic cells; Aorta endothelial cells + TNF-a; Soares retina N2b4HR;
  • 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 1519 of SEQ ID NO: 14, b is an integer of 15 to 1533, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 64 as residues: Gln-28 to Gly-36. Polynucleotides encoding said polypeptides are also provided.
  • 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 1744 of SEQ ID NO: 15, b is an integer of 15 to 1758, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 15, and where b is greater than or equal to a + 14.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 65 as residues: Trp- 19 to Ala-24, Gly-32 to Met-39. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 16 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1887 of SEQ ID NO: 16, b is an integer of 15 to 1901, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 16, and where b is greater than or equal to a + 14.
  • Many polynucleotide sequences, such as EST sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention.
  • such related polynucleotides are specifically excluded from the scope of the present invention.
  • a-b is any integer between 1 to 3131 of SEQ ID NO: 17, b is an integer of 15 to 3145, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, and where b is greater than or equal to a + 14.
  • 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. gil868189 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "No definition line found [Caenorhabditis elegans]". A partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil868189 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 113 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 114 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_NhHMPu_S 1 Smooth muscle, serum induced,re-exc; Macrophage-oxLDL, re-excision; NCl_CGAP_Prl2; Fetal Heart; PC3 Prostate cell line; Stratagene pancreas (#937208); Smooth muscle, serum treated; Human Fetal Heart; Activated T-
  • NCI_CGAP_Col l NCI_CGAP_GCB1; Osteoblasts; Soares fetal liver spleen
  • INFLS INFLS; Heart; Activated T-Cells, 12hrs, differentially expressed; Soares_NhHMPu_S 1 ; Soares_testis_NHT; Soares_fetal_liver_spleen_lNFLS_S 1 ; normalized infant brain cDNA; Human Amygdala Depression, re-excision; Activated T-Cells, 12 hrs, subtracted; Human rejected kidney; Tongue Normal; Human Microvascular Endothelial Cells, fract.
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 67 as residues: Pro-27 to Lys-34. 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 2954 of SEQ ID NO: 18, b is an integer of 15 to 2968, 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.
  • 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. gil204412 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "glutaminase [Rattus norvegicus]".
  • glutaminase [Rattus norvegicus] A partial alignment demonstrating the observed homology is shown immediately below.
  • KIDNEY ISOFORM PRECURSOR (EC 3.5.1.2) (GLS) (L-GLUTAMINE AMIDOHYDROLASE) . >bbs
  • 174849 glutaminase, GA ⁇ C-terminal ⁇ [swine, LLC-PK1-F+ cells, Peptide Mitochondrial Partial, 175 aa] [Sus scrofa] ⁇ SUB 500-674 ⁇ Length 674 Plus Strand HSPs:
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 116 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • NCI_CGAP_GCB 1 NCI_CGAP_GCB 1 and to a lesser extent in T-Cell PHA 24 hrs; 12 Week Old Early Stage Human; Hodgkin's Lymphoma II; Human 8 Week Whole Embryo; Soares placenta Nb2HP; Primary Dendritic Cells, lib 1 ; Mo7e Cell Line GM-CSF treated (lng/ml); Human Eosinophils; Colon Carcinoma; 12 Week Early Stage Human II, Reexcision; Human T-cell lymphoma,re-excision; Stratagene endothelial cell 937223; Stratagene NT2 neuronal precursor 937230; Bone Marrow Stromal Cell, untreated; Stratagene lung (#937210);
  • Soares_pregnant_uterus_NbHPU Human Endometrial Tumor; Human fetal heart, Lambda ZAP Express; Osteoblasts; Human Cerebellum; C7MCF7 cell line, estrogen treated; Soares_pIacenta_8to9weeks_2NbHP8to9W; Bone Marrow Stroma,
  • TNF&LPS ind Larynx Normal; Human Microvascular Endothelial Cells, fract. B; Activated T-Cells, 8 hrs, subtracted; Thymus; Dermatofibrosarcoma Protuberance; Human Umbilical Vein Endothelial Cells, fract.
  • Soares_placenta_8to9weeks_2NbHP8to9W Human umbilical vein endothelial cells, IL-4 induced; Human Activated Monocytes; Human fetal heart, Lambda ZAP Express; Human retina cDNA randomly primed sublibrary; NCI_CG AP_GCB 1 ; Human Testes Tumor, re-excision; Human Adrenal Gland Tumor; Rejected Kidney, lib 4; Ovarian Tumor 10-3-95; Soares_fetal_liver_spleen_lNFLS_Sl; NTERA2, control; Gessler Wilms tumor; Liver HepG2 cell line.; PC3 Prostate cell line; Resting T-Cell Library ,11; Human T-Cell Lymphoma; NC1_CGAP_GCB1; Human Substantia Nigra; Colon Tumor II; Human fetal lung; Human Placenta; Human Fetal Heart; Human Neutrophil, Activated; Endo
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO: 19 Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • a-b is any integer between 1 to 3939 of SEQ ID NO: 19
  • b is an integer of 15 to 3953, 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.
  • 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. gil2731377 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "similar to alcohol dehydrogenase/ribitol dehydrogenase [Caenorhabditis elegans]".
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil2731377 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 117 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 118 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Umbilical Vein Endothelial Cells IL4 induced; Human Adult Spleen, subtracted; Human Fetal Kidney; Soares_parathyroid_tumor_NbHPA; Human Astrocyte; Stratagene fibroblast (#937212); Skin, burned; Human Adult Spleen, fractionll; H.
  • HCC Human Colon Carcinoma
  • Stratagene muscle 937209 and Stratagene endothelial cell 937223 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.
  • a-b is any integer between 1 to 1949 of SEQ ID NO:20
  • b is an integer of 15 to 1963
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20
  • b is greater than or equal to a + 14.
  • T-cell(12h)/Thiouridine-re- excision Activated T-cell(12h)/Thiouridine-re- excision; Stratagene lung (#937210); Smooth muscle, serum induced,re-exc; 12 Week Early Stage Human II, Reexcision; Activated T-Cell (12hs)/Thiouridine labelledEco; Soares_pregnant_uterus_NbHPU; Human Adult Heart,re-excision; Stratagene fetal spleen (#937205); Human Placenta (re-excision); Human Testes Tumor, re-excision; Fetal Liver, subtraction II; Soares_fetal_liver_spleen_lNFLS_Sl; Clontech human aorta polyA+ mRNA (#6572); Dendritic cells, pooled; Human Fetal Lung III; Endothelial-induced; Soares_fetal_liver_spleen_lNFLS_Sl;
  • Lymph node breast Cancer Brain Frontal Cortex, re-excision; Human Adult Small Intestine; Human Neutrophil; Human Chronic Synovitis; Human Umbilical Vein Endothelial Cells, uninduced; Human Hippocampus; Human Adipose; Human Activated Monocytes; Human Rhabdomyosarcoma; NCI_CGAP_GCB1; Soares_NFL_T_GBC_Sl; Soares_fetal_heart_NbHH19W; Soares_parathyroid_tumor_NbHPA; Hepatocellular Tumor, re-excision; Soares_fetal_liver_spleen_lNFLS_Sl; Fetal Heart; PC3 Prostate cell line; Human Eosinophils; Human Synovial Sarcoma; Neutrophils control, re- excision; Soares_senescent_fibroblasts_NbHSF; human tonsils; Human Fetal Heart; Endothelial cells-control; Anergic T-
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:21 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope 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 3031 of SEQ ID NO:21, b is an integer of 15 to 3045, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:21, and where b is greater than or equal to a + 14.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 432 of SEQ ID NO:22, b is an integer of 15 to 446, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a + 14.
  • 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. gil206886 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "homologue to sec61 [Rattus rattus]".
  • sequence homology is shown immediately below.
  • the segments of gil206886 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 119 and SEQ ID NO. 121 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 120 and/or SEQ ID NO. 122 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • CD34 positive cells CD34 positive cells (Cord Blood); PRMIX; Brain, Kozak;
  • Soares Soares; Soares_testis_NHT; Soares_pineal_gland_N3HPG;
  • NCI_CGAP_Pr9 Human Brain, Striatum; NCI_CGAP_Pr9; NCI_CGAP_PrlO; NCI_CGAP_Pr22; Human Fetal Kidney; NCI_CGAP_AA1; NCI_CGAP_Br2; NCI_CGAP_Br3 ;
  • Soares_NhHMPu_Sl Human Activated T-Cells, re- excision; Soares_testis_NHT; Synovial Fibroblasts (control); Hemangiopericytoma;
  • CHME Cell Line treated 5 hrs; NTERA2, control; normalized infant brain cDNA;
  • Soares_multiple_sclerosis_2NbHMSP Human Fetal Lung III; 12 Week Early Stage
  • NCI_CGAP_GC3 Spleen, Chronic lymphocytic leukemia
  • NCI_CGAP_Co8
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 72 as residues: Leu-36 to Ser-49. 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 3383 of SEQ ID NO:23, b is an integer of 15 to 3397, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:23, and where b is greater than or equal to a + 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. gnllPIDIe225817 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "unknown [Schizosaccharomyces pombe]".
  • gnllPIDIe225817 all information available through the recited accession number is incorporated herein by reference
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • the segment of gnllPIDIe225817 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 123 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 124 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 present invention comprise immunogenic epitopes shown in SEQ ID NO: 73 as residues: Phe-11 to Gly-16, Pro-33 to Ser-42.
  • 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: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 2822 of SEQ ID NO:24, b is an integer of 15 to 2836, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:24, and where b is greater than or equal to a + 14.
  • 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. gil575681 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "unknown orf, len: 442, CAI: 0.13 [Saccharomyces cerevisiae]".
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil575681 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 125 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 126 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • E2 fraction I; Human Adult Pulmonary; H. Atrophic Endometrium; Breast Lymph node cDNA library; Human Lung; H. Epididiymus, cauda;
  • Soares_NFL_T_GBC_Sl Human Pancreas Tumor, Reexcision; Human Activated Monocytes; Epithelial-TNFa and INF induced; Synovial Fibroblasts (control); Human
  • Thymus Stromal Cells Human Whole Six Week Old Embryo; Human Gall Bladder; Resting T-Cell Library ,11; Smooth muscle, serum treated; Early Stage Human Brain; Human Testes Tumor; Human Synovial Sarcoma; Primary Dendritic cells rac 2; Human Placenta; human tonsils; Human Fetal Heart; Human Primary Breast Cancer Reexcision; Soares_fetal_heart_NbHH19W; CD34 depleted Buffy Coat (Cord Blood), re-excision; Colon Normal III; Human Microvascular Endothelial Cells, fract.
  • A Monocyte activated; HUMAN B CELL LYMPHOMA; T Cell helper I; T cell helper II; Soares placenta Nb2HP; Human retina cDNA Tsp509I-cleaved sublibrary; NCI_CGAP_Co9; NCI_CGAP_GC3; NCI_CGAP_GC4; NCI_CGAP_ColO; NCI_CGAP_Col2; NCI_CGAP_Brl .l and Soares_fetal_heart_NbHH19W.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 74 as residues: Pro-67 to Ser-78, Thr-89 to T ⁇ -96, Arg-114 to Ser-125, Ser-147 to Ala-155. Polynucleotides encoding said polypeptides are also provided.
  • 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.
  • a-b is any integer between 1 to 2087 of SEQ ID NO:25
  • b is an integer of 15 to 2101, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a + 14.
  • 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. gil 1825601 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "weak similarity to rat TEGT protein (G 456207) [Caenorhabditis elegans]".
  • G 456207 rat TEGT protein
  • segment of gill825601 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 127 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 128 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Progenitor Cell Line Progenitor Cell Line; Stratagene muscle 937209; Stratagene endothelial cell 937223; Brain Frontal Cortex, re-excision; Human Adult Small Intestine; T-Cell PHA 16 hrs; Soares_NhHMPu_Sl; Stratagene HeLa cell s3 937216; Soares_fetal_liver_spleen_lNFLS_Sl; NCl_CGAP_Brn25; Human Thymus; L428; NCI_CGAP_GCB 1 ; Human Umbilical Vein Endothelial Cells, uninduced;
  • Macrophage-oxLDL Human Hypothalmus,Schizophrenia; Human Placenta (reexcision); Human Thymus Stromal Cells; Human Whole Six Week Old Embryo; NCI_CGAP_Kid5; Ovarian Tumor 10-3-95; PC3 Prostate cell line; Colon Tumor; Human T-Cell Lymphoma; Smooth muscle, serum treated; Human Placenta; Stratagene hNT neuron (#937233); NCI_CGAP_Kid3; Stratagene neuroepithelium (#937231); Human Fetal Lung III; human tonsils; Activated T-Cell (12hs)/Thiouridine labelledEco; Soares_fetal_liver_spleen_lNFLS_Sl; Human Microvascular Endothelial Cells, fract.
  • Adrenal Gland Subtracted; Human Fetal Heart, Differential (Fetal-Specific); Thyroid Tumour; Normal Prostate; Human Adult Heart; K562 + PMA (36 hrs); Human Normal Cartilage.Fraction I; Human Normal Cartilage Fraction II; NCI_CGAP_GCB1; Human osteoarthritis,fraction I; Human Prostate, subtracted; Colorectal Tumor; NCI_CGAP_Co3 ; Saos2, Dexamethosome Treated; Human OB HOS treated (1 nM E2) fraction I; LNCAP untreated; Frontal Lobe, Dementia; Human Colon; Human (HCC) cell line liver (mouse) metastasis, remake; Human Colon Carcinoma (HCC) cell line; Hodgkin's Lymphoma I; Human Pituitary, subtracted; Human Neutrophils, Activated, re-excision; Aorta endothelial cells + TNF-a; Activated T-cells; Human
  • Soares_testis_NHT Soares_parathyroid_tumor_NbHPA; Hepatocellular Tumor,re- excision; Hepatocellular Tumor; pBMC stimulated w/ poly I/C; Alzheimers, spongy change; NTERA2 + retinoic acid, 14 days; H. Kidney Cortex, subtracted; Human Stomach,re-excision; LNCAP prostate cell line; Human Osteosarcoma; healing groin wound, 6.5 hours post incision; H.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2032 of SEQ ID NO:26, b is an integer of 15 to 2046, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a + 14.
  • 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. gnllPIDIe214699 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ARF-like protein 5 [Rattus norvegicus]."
  • This rat protein is thought to be a novel member of the ARF- family of Ras-related GTPases. A partial alignment demonstrating the observed homology is shown immediately below.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 130 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • this protein in vivo where the ras proto-oncogene has undergone a mutation to the oncogene may reduce or eliminate the activity of the ras protein.
  • the ras proto-oncogene undergoes a mutation to the oncogene, the activity of the associated GTPases is greatly reduced.
  • expression of this protein in vivo may reduce or terminate the activity of the ras oncogene.
  • Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:27 Some of these sequences are related to SEQ ID NO:27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope 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 3555 of SEQ ID NO:27, b is an integer of 15 to 3569, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:27, and where b is greater than or equal to a + 14.
  • 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. gnllPIDId 1021323 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "peptide/histidine transporter [Rattus norvegicus]."
  • This rat peptide/histidine transporter has been shown to be expressed in the brain and retina, and is thought to be involved in histidine transport. A partial alignment demonstrating the observed homology is shown immediately below.
  • the segment of gnllPIDIdl021323 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 131 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 132 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_pineal_gland_N3HPG Stratagene lung carcinoma 937218; Activated T- cell(12h)/Thiouridine-re-excision; Soares infant brain INIB; Spleen/normal; Human
  • Soares_NhHMPu_Sl Stratagene HeLa cell s3 937216; Merkel Cells; Pancreatic
  • the gene or gene product may also play a role in the treatment and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:28 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • a-b is any integer between 1 to 2542 of SEQ ID NO:28
  • b is an integer of 15 to 2556, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a + 14.
  • the 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. gil 1872200 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "alternatively spliced product using exon 13A [Homo sapiens]".
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gill872200 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 133 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 134 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_NhHMPu_S 1 Soares ovary tumor NbHOT; Normal Prostate; Tongue Tumour; Bone Cancer; H. Frontal Cortex, Epileptic; Supt Cells, cyclohexamide treated; Smooth muscle, control, reexcision; Smooth muscle-ILb induced; Aorta endothelial cells + TNF-a; H.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 78 as residues: Thr-28 to Leu-34, Pro-36 to Trp-47. Polynucleotides encoding said polypeptides are also provided.
  • 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.
  • a-b is any integer between 1 to 1135 of SEQ ID NO:29
  • b is an integer of 15 to 1149, 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.
  • NCI_CG AP_GCB 1 Soares_testis_NHT and to a lesser extent in Soares_fetal_liver_spleen_lNFLS_Sl ; Soares placenta Nb2HP; Soares fetal liver spleen INFLS; Human Adult Pulmonary; NCI_CGAP_Alvl; Human Colon, reexcision; Spleen metastic melanoma; Spinal Cord, re-excision; Bone Marrow Stromal Cell, untreated; Human Ovarian Cancer Reexcision; Human Osteoclastoma and Soares_parathyroid_tumor_NbHPA.
  • 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1853 of SEQ ID NO: 30, b is an integer of 15 to 1867, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:30, and where b is greater than or equal to a + 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. pirlJC5238IJC5238 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "galactosylceramide- like protein, GCP - human".
  • GCP - human galactosylceramide- like protein
  • S The segment of pirlJC5238IJC5238 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 135 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 136 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 present invention comprise immunogenic epitopes shown in SEQ ID NO: 80 as residues: Arg-84 to Cys-90, Gln-138 to Leu- 155. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:31 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:31 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 1258 of SEQ ID NO:31, b is an integer of 15 to 1272, 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.
  • 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. gil500832 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "Yhr074wp [Saccharomyces cerevisiae]".
  • Yhr074wp [Saccharomyces cerevisiae]
  • a partial alignment demonstrating the observed homology is shown immediately below. >gi
  • S46811 hypothetical protein YHR074w - yeast (Saccharomyces cerevisiae) Length 714
  • segment of gil500832 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 137 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 138 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 present invention comprise immunogenic epitopes shown in SEQ ID NO: 81 as residues: Arg-36 to Ala-43. 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 2221 of SEQ ID NO:32, b is an integer of 15 to 2235, 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 + 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. gil531829 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "beta-galactosidase alpha peptide [Cloning vector pSportl]".
  • 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. 140 and/or SEQ ID NO. 142 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Lambda ZAP Express Normal colon; Soares melanocyte 2NbHM; Primary Dendritic cells,frac 2; Human Fetal Lung III; Human Amygdala; Normal Human Trabecular Bone Cells; Soares_NhHMPu_Sl; Soares_NFL_T_GBC_S 1 ; Soares_total_fetus_Nb2HF8_9w; Soares_fetal_liver_spleen_lNFLS_Sl and Human Cerebellum.
  • 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 1949 of SEQ ID NO:33, b is an integer of 15 to 1963, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a + 14.
  • 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. gil3643809 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "zinc finger protein
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 144 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_testis_NHT Human Bone Marrow, treated; Osteoblasts; Keratinocyte; Soares_fetal_lung_NbHL19W; A-14 cell line; Human Colon Carcinoma (HCC) cell line; Human Thyroid; Human T-cell lymphoma,re-excision; Stratagene colon HT29 (#937221); Stratagene ovary (#937217); Human Lung Cancer,re-excision; Morton Fetal; H. Kidney Cortex, subtracted; Human Osteoclastoma, re-excision;
  • Placenta Atrium cDNA library Human heart; Human Activated T-Cells, re-excision; Synovial Fibroblasts (control); Human Thymus Stromal Cells; Human Adrenal Gland Tumor; CHME Cell Line,treated 5 hrs; Hepatocellular Tumor, reexcision; Human Gall Bladder; Smooth muscle, serum treated; Human Testes Tumor; Human Fetal Lung III; 12 Week Early Stage Human II, Reexcision; Soares_pineal_gland_N3HPG; Bone marrow; Smooth muscle,control; HUMAN B CELL LYMPHOMA; Spleen, Chronic lymphocytic leukemia; Hodgkin's Lymphoma II; Activated T-cell(12h)/Thiouridine-re-excision; Human fetal heart, Lambda ZAP Express; Human 8 Week Whole Embryo; Human Cerebellum and 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 1753 of SEQ ID NO:34, b is an integer of 15 to 1767, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 34, and where b is greater than or equal to a + 14.
  • 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. gil 1519392 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "leptin receptor [Homo sapiens]".
  • sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gil 1519392 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "leptin receptor [Homo sapiens]".
  • Leptin receptor [Homo sapiens] A partial alignment demonstrating the observed homology is shown immediately below.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 146 and/or SEQ ID NO. 148 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Kidney Cortex subtracted; Human Primary Breast Cancer Reexcision; CD34 depleted Buffy Coat (Cord Blood), re-excision; Spleen, Chronic lymphocytic leukemia; human colon cancer; Human Pineal Gland; CD34 positive cells (cord blood),re-ex; Human Stomach,re-excision; Jurkat T-cell Gl phase; Human Fetal Dura Mater; Human Ovary; Human Adipose; breast lymph node CDNA library; Early Stage Human Brain; Human Fetal Lung III; Human Microvascular Endothelial Cells, fract. A; CD34 positive cells (Cord Blood); H.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:35 amino acid sequences
  • amino acid 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 1803 of SEQ ID NO:35, b is an integer of 15 to 1817, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID 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. gil4153872 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "structure confirmed by Genscan, human EST AA447021 (MD:g2159686) and mouse EST AA119040 (NID:gl676735) [Homo sapiensl". A partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil4153872 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 149 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 150 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • SEQ ID NO. 150 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • supernatants removed from cells containing this gene activated the NF-kB transcription factor.
  • nuclear factor kB is a transcription factor activated by a wide variety of agents, leading to cell activation, differentiation, or apoptosis. Reporter constructs utilizing the NF-kB promoter elementare used to screen supernatants for such activity.
  • NTERA2 + retinoic acid 14 days; Healing groin wound, 7.5 hours post incision;
  • KMH2 Human Umbilical Vein Endothelial Cells, uninduced; Human Hippocampus; Human Chondrosarcoma; H Macrophage (GM-CSF treated), re-excision;
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:36 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:36 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 2474 of SEQ ID NO:36, b is an integer of 15 to 2488, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a
  • 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. gil307307 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "nueroendocrine- specific protein A [Homo sapiens]".
  • nueroendocrine- specific protein A [Homo sapiens] A partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil307307 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 151 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 152 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Keratinocyte Keratinocyte; Stratagene fetal spleen (#937205); Bone Marrow Stromal Cell, untreated; Smooth muscle, serum induced, re-exc; Soares melanocyte 2NbHM; Nine
  • Amygdala Soares fetal liver spleen INFLS; Soares infant brain INIB; Stratagene pancreas (#937208); Human Umbilical Vein Endothelial Cells, uninduced;
  • Macrophage (GM-CSF treated), re-excision; Colon Tumor II; Normal colon; Soares_fetal_heart_NbHH19W; Human Microvascular Endothelial Cells, fract. A;
  • CHME Cell Line treated 5 hrs; Macrophage-oxLDL, re-excision; CHME Cell
  • Kidney Cortex subtracted; Glioblastoma; Human Whole Brain #2 - Oligo dT > 1.5Kb; Stratagene endothelial cell 937223; Prostate BPH; Spinal Cord, re-excision; Human Prostate; NCI_CGAP_GCB 1 ; L428; 12 Week Old Early Stage Human, II; HUMAN JURKAT MEMBRANE BOUND POLYSOMES; NCI_CGAP_Br2; Human Hypothalmus,Schizophrenia; Soares_testis_NHT; Human Pancreas Tumor, Reexcision; Human Hippocampus; Human Adipose; NCI_CGAP_GCB 1 ; Rejected Kidney, lib 4; Soares_parathyroid_tumor_NbHPA; Stratagene liver (#937224); Colon Tumor; Soares_fetal_lung_NbHL19W; Colon Normal II; Soares breast 3NbHBst; Early Stage Human Brain; normalized infant
  • Osteosarcoma Human endometrial stromal cells; Stratagene fetal retina 937202; Stratagene ovarian cancer (#937219); Stratagene neuroepithelium (#937231); Stratagene colon (#937204); Stratagene neuroepithelium NT2RAMI 937234; Myoloid Progenitor Cell Line; Spleen metastic melanoma; Stratagene muscle 937209; Brain Frontal Cortex, re-excision; Human Infant Brain; Human Chronic Synovitis; H.
  • Kidney Medulla re-excision; Human promyelocyte; normalized infant brain cDNA; Human Bone Marrow, re-excision; KMH2; Human Brain, Striatum; NCI_CGAP_Alvl; NCI_CGAP_Kid5; NCI_CGAP_Pr22; Monocyte activated, reexcision; Human Fetal Kidney; NCI_CGAP_GC2; NCI_CGAP_Col l; Human Activated T-Cells; T-Cell PHA 24 hrs; Barstead spleen HPLRB2; Gessler Wilms tumor; Human pancreatic islet; Soares_pregnant_uterus_NbHPU; Soares_total_fetus_Nb2HF8_9w; Olfactory epithelium,nasalcavity; Soares_multiple_sclerosis_2NbHMSP; Spinal cord; Human Activated T-Cells, reexcision; Human
  • Soares_testis_NHT Soares_fetal_heart_NbHH19W
  • Soares_total_fetus_Nb2HF8_9w Stratagene fetal retina 937202; Stratagene hNT neuron (#937233); Human Liver, normal; Human Gall Bladder; NCI_CGAP_GC4; PC3 Prostate cell line; Resting T-Cell Library ,11; Human T-Cell Lymphoma; Human pancreatic islet; Jia bone marrow stroma;
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:37 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 1744 of SEQ ID NO:37, b is an integer of 15 to 1758, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:37, 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. gil3002527 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "neuronal thread protein AD7c-NTP [Homo sapiens]". A partial alignment demonstrating the observed homology is shown immediately below.
  • AD7c-NTP neuronal thread protein
  • AF010144 neuronal thread protein
  • AD7c-NTP Homo sapiens
  • segment of gil3002527 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 153 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 154 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1144 of SEQ ID NO:38, b is an integer of 15 to 1158, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:38, 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. 156 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 88 as residues: Cys-64 to Lys-70. Polynucleotides encoding said polypeptides are also provided.
  • EGR1 Early growth response 1
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:39 Some of these sequences are related to SEQ ID 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 2176 of SEQ ID NO:39, b is an integer of 15 to 2190, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID 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. gil3094014 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "Homo sapiens protein".
  • Homo sapiens protein 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. 158 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 present invention comprise immunogenic epitopes shown in SEQ ID NO: 89 as residues: Asn-2 to Trp-13. 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 2562 of SEQ ID NO:40, b is an integer of 15 to 2576, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:40, 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. gnllPIDIe 1288408 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AJ005678) SA3 [Mus musculus]".
  • sequence 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. 160 and/or SEQ ID NO. 162 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • EGR1 Early growth response 1
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:41 amino acid sequences
  • amino acid 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.
  • a-b is any integer between 1 to 1138 of SEQ ID NO:41
  • b is an integer of 15 to 1152, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID 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. gil2088839 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AF003386) No definition line found [Caenorhabditis elegans]". A partial alignment demonstrating the observed homology is shown immediately below.
  • the segments of gil2088839 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 163 and SEQ ID NO. 165 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 164 and/or SEQ ID NO. 166 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • T- cell(12h)/Thiouridine-re-excision Activated T- cell(12h)/Thiouridine-re-excision; Soares placenta Nb2HP; Soares fetal liver spleen INFLS; Soares_fetal_heart_NbHH19W; Infant brain, Bento Soares; Normal Human Trabecular Bone Cells; Human Eosinophils; Early Stage Human Brain; Colon Tumor II; Activated T-Cell (12hs)/Thiouridine labelledEco;
  • Kidney Medulla re-excision; Human Umbilical Vein, Reexcision; Human Dermal Endothelial Cells,untreated; HM1; Human adult (K.Okubo); NCI_CGAP_AA1; NCI_CGAP_Co2; NCI_CGAP_Ew 1 ; NCI_CGAP_GC4; NCI_CGAP_Lu5; NCI_CGAP_Pr2; NCI_CGAP_Pr3; NCI_CGAP_CLL1 ; NCI_CGAP_Col0; NCI_CGAP_Kidl ; NCI_CGAP_Kid6; NCI_CGAP_Pr25; Soares_fetal_heart_NbHH19W; Human Umbilical Vein Endothelial Cells, uninduced; Human Adult Testes, Large Inserts, Reexcision; Stromal cell TF274; Human Hypothalmus,Schizophrenia; STRATAGENE Human skeletal muscle cDNA library, cat.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3839 of SEQ ID NO:42, b is an integer of 15 to 3853, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:42, and where b is greater than or equal to a + 14.
  • 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 542 of SEQ ID NO:43, b is an integer of 15 to 556, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:43, 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. gil3411185 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AF076240) putative ferredoxin reductase MocF [Rhizobium leguminosarum bv. viciae]".
  • AF076240 putative ferredoxin reductase MocF [Rhizobium leguminosarum bv. viciae]
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • the segment of gil3411185 that is shown as "S” above is set out in the sequence listing as SEQ ID NO. 167 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 168 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_parathyroid_tumor_NbHPA Human adult small intestine,re-excision; Human Adult Small Intestine; Human heart cDNA (YNakamura); T cell helper II; Soares fetal liver spleen INFLS; Human Cardiomyopathy, subtracted; Stratagene colon
  • Soares_NhHMPu_Sl Human Microvascular Endothelial Cells, fract. A; Smooth muscle,control; Monocyte activated; HUMAN B CELL LYMPHOMA; Human Endometrial Tumor and Human 8 Week Whole Embryo.
  • 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.
  • a-b is any integer between 1 to 2136 of SEQ ID NO:44
  • b is an integer of 15 to 2150
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:44
  • 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. gil4103490 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AF024519) glucocorticoid-induced leucine zipper GILZ protein [Mus musculus].”
  • This novel murine protein is thought to protect T lymphocytes from TCR/CD3 activated cell death. A partial alignment demonstrating the observed homology is shown immediately below.
  • VKNHLMYAVREEVEILKEQIRELVEKNSQLERENTLLKTLASPEQLEKFQSCLSPEEPAP 630 VKNHLMYAVREEVE+LKEQIREL+EKNSQLERENTLLKTLASPEQLEKFQS LSPEEPAP S: 61 VKNHLMYAVREEVEVLKEQIRELLEKNSQLERENTLLKTLASPEQLEKFQSRLSPEEPAP 120
  • segment of gil4103490 that is shown as "S” above is set out in the sequence listing as SEQ ID NO. 169 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 170 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • EGR1 Early growth response 1
  • Thymus Thymus; Stratagene lung (#937210); Human Bone Marrow, treated;
  • Soares_multiple_sclerosis_2NbHMSP Human adult small intestine,re-excision;
  • Soares_senescent_fibroblasts_NbHSF H. Meningima, Ml; Human Uterine Cancer; NCI_CGAP_CLL1 ; Soares_testis_NHT; Fetal Heart; NCI_CGAP_Lu5;
  • Leukocytes normalized cot > 500A; Human Fetal Kidney; Human Normal Cartilage Fraction III; Human Lung Cancer, subtracted; Human Osteoarthritic Cartilage Fraction III; Human Gastrocnemius; Human Placenta, subtracted; Bone marrow stroma,treated; Human Normal Cartilage Fraction II; Human Leukocytes; H.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 94 as residues: VaI-80 to Leu-92, Ser-98 to Lys- 104, Pro-111 to Pro-122. Polynucleotides encoding said polypeptides are also provided. Given the homology of this protein to the murine leucine zipper protein, this gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the gene or 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.
  • this gene product may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis.
  • this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
  • Many polynucleotide sequences, such as EST sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:45 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 1994 of SEQ ID NO:45
  • b is an integer of 15 to 2008
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:45
  • b is greater than or equal to a + 14.
  • hypothalamus frac A,re-excision; Human adult (K.Okubo); Human fetal heart, Lambda ZAP Express; NCI_CGAP_Lu5; NCI_CGAP_Kid3; Soares_testis_NHT; HUMAN STOMACH; Human Tonsils, Lib 2; Human Prostate; Stratagene lung (#937210) and Soares infant brain INIB.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 95 as residues: Arg-15 to Ser-27, Ser-29 to Tyr-41, Thr-55 to Phe-62.
  • 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: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 1460 of SEQ ID NO:46, b is an integer of 15 to 1474, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a
  • NTERA2 control; breast lymph node CDNA library; Soares melanocyte 2NbHM;
  • INIB normalized infant brain cDNA
  • Infant brain Bento Soares
  • Soares_testis_NHT Thyroid Tumour; Stomach Tumour; CD34+ cell, I, frac II;
  • 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 1879 of SEQ ID NO:47, b is an integer of 15 to 1893, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:47, and where b is greater than or equal to a + 14.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2404 of SEQ ID NO:48, b is an integer of 15 to 2418, 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.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:49 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 1145 of SEQ ID NO:49, b is an integer of 15 to 1159, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:49, 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. gil 182221 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ORF 3 [Homo sapiens]".
  • ORF 3 Homo sapiens
  • segment of gil 182221 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 171 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 172 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Soares_NFL_T_GBC_Sl Stromal cell TF274; Soares adult brain N2b5HB55Y; Human Adrenal Gland Tumor; Pancreas Islet Cell Tumor; Human Osteoclastoma; H. Frontal cortex,epileptic,re -excision; Soares_pregnant_uterus_NbHPU; Human Lung Cancer, subtracted; Thyroid Normal (SDCA2 No); H. Striatum Depression, subt; Activated T-cells; Papillary serous cystic neoplasm of low malignant potential
  • Soares_pregnant_uterus_NbHPU Human Normal Breast; B Cell lymphoma; Human Hypothalamus,schizophrenia, re-excision; Hepatocellular Tumor; pBMC stimulated w/ poly I/C; H Female Bladder, Adult; H. Kidney Cortex, subtracted; Healing groin wound, 7.5 hours post incision; Pancreas normal PCA4 No; Synovial hypoxia; Soares_pregnant_uterus_NbHPU; Spinal Cord, re-excision; Human
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2778 of SEQ ID NO:50, b is an integer of 15 to 2792, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:50, 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. gil3170458 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AF053003) diphthamide biosynthesis protein-2; DPH2 [Homo sapiens]".
  • AF053003 diphthamide biosynthesis protein-2
  • DPH2 [Homo sapiens] A partial alignment demonstrating the observed homology is shown immediately below.
  • segment of gil3170458 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 173 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 174 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • 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:51 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 1907 of SEQ ID NO:51
  • b is an integer of 15 to 1921
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:51
  • 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. gil296560 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ORF YBR1729 [Saccharomyces cerevisiae]". A partial alignment demonstrating the observed homology is shown immediately below.
  • polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 176 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 101 as residues: Gly-80 to Gly-88. 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 1480 of SEQ ID NO:52, b is an integer of 15 to 1494, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:52, and where b is greater than or equal to a + 14.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:53 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:53 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 1686 of SEQ ID NO:53, b is an integer of 15 to 1700, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:53, and where b is greater than or equal to a + 14.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:54 Some of these sequences are related to SEQ ID NO:54 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 1163 of SEQ ID NO:54, b is an integer of 15 to 1 177, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:54, 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. gnllPIDIel254905 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AJ002308) synaptogyrin 2 [Homo sapiens]".
  • sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gnllPIDIel254905 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "(AJ002308) synaptogyrin 2 [Homo sapiens]".
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • VGFCFLTNQWAVTNPK VLVGADSVRAAIT GVLASLAYQRYKAGVDDFIQN S: 121 VGFCFLTNQ AVTNPKDVLVGADSVRAAITFSFFSIFSWGVLASLAYQRYKAGVDDFIQN 180
  • S sequence listing as SEQ ID NO. 177 .
  • these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 178 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • NCI_CGAP_Co9 Human Fetal Kidney; Human umbilical vein endothelial cells, IL-4 induced; Dendritic cells, pooled; Activated T-Cell (12hs)/Thiouridine labelledEco; NCl_CGAP_Kid5; Human Bone Marrow, treated; NCI_CGAP_Co8; Soares_fetal_heart_NbHH19W; NCI_CGAP_Co8; Human Primary Breast Cancer; Human Quadriceps; Human Tonsils, Lib 2; pBMC stimulated w/ poly I/C; Stratagene placenta (#937225); Breast Cancer cell line, MDA 36; Human Pituitary, subt IX; NCI_CGAP_Co3; NCI_CGAP_Pr22; Human Thymus; Human Testes Tumor, re-excision; Soares breast 2NbHBst; CHME Cell Line,treated 5 hrs; Colon Tumor; Col
  • Leukocytes normalized cot 5B; Human Eosinophils; Human promyelocyte; Soares_NSF_F8_9W_OT_PA_P_Sl ; Soares_parathyroid_tumor_NbHPA; Soares_fetal_liver_spleen_lNFLS_S 1 ; Stratagene lung carcinoma 937218; Human Amygdala Depression, re-excision; Human Pancreatic Langerhans; Bone Cancer; Human Kidney; Human epithelioid sarcoma; Human (Caco-2) cell line, adenocarcinoma, colon, remake; Human Colon, subtraction; metastatic squamous cell lung carcinoma, poorly differentiated; Human Aortic Endothelium; Human colon carcinoma (HCC) cell line, remake; Hodgkin's Lymphoma I; Adipocytes,re-excision; NCI_CG AP_Ut3 ; NCI_CGAP_Col4; NCI
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO: 104 as residues: Asn-36 to Tyr-44. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:55 Some of these sequences are related to SEQ ID NO:55 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 1560 of SEQ ID NO:55, b is an integer of 15 to 1574, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a + 14.
  • the segments of gnllPIDId 1033268 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 179 and SEQ ID NO. 181 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 180 and/or SEQ ID NO. 182 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
  • Atrophic Endometrium Atrophic Endometrium; Stratagene corneal stroma (#937222); Human Neutrophils, Activated, re-excision; NTERA2 teratocarcinoma cell line+retinoic acid (14 days); Breast Cancer cell line, MDA 36; Soares_fetal_lung_NbHL19W; H.
  • Kidney Cortex subtracted; Human Colon, re-excision; Synovial hypoxia; Brain Frontal Cortex, re- excision; KMH2; L428; 12 Week Old Early Stage Human, II; Human Uterine Cancer; Stromal cell TF274; Bone Marrow Stromal Cell, untreated; Human Adrenal Gland Tumor; Macrophage-oxLDL, re-excision; Human Ovarian Cancer Reexcision; Resting T-Cell Library ,11; Human Placenta; Bone marrow; Endothelial-induced; Anergic T-cell; Soares_fetal_lung_NbHL19W; HUMAN B CELL LYMPHOMA; Human Bone Marrow, treated; Bone Marrow Cell Line (RS4,11); Human Endometrial Tumor; Hodgkin's Lymphoma II and Soares fetal liver spleen INFLS.
  • Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:56 Some of these sequences are related to SEQ ID NO:56 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 2721 of SEQ ID NO:56, b is an integer of 15 to 2735, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:56, 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: 106 as residues: Ser- 17 to Gly-22, Leu-34 to Ala-42. Polynucleotides encoding said polypeptides are also provided.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:57 Some of these sequences are related to SEQ ID NO:57 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.
  • polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2717 of SEQ ID NO:57, b is an integer of 15 to 2731, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:57, and where b is greater than or equal to a + 14.
  • HUMAN B CELL LYMPHOMA Spleen, Chronic lymphocytic leukemia; Activated T-cell(12h)/Thiouridine-re-excision; Primary Dendritic Cells, lib 1.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:58 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:58 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 1454 of SEQ ID NO:58, b is an integer of 15 to 1468, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:58, and where b is greater than or equal to a + 14.
  • the segments of gil 1872200 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 183 and SEQ ID NO. 185 . Based on the structural similarity, these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 184 and/or SEQ ID NO. 186 which correspond to the "Q" sequences 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 1600 of SEQ ID NO:59
  • b is an integer of 15 to 1614
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:59
  • b is greater than or equal to a + 14.
  • Table 1 summarizes the information corresponding to each "Gene No.” described above.
  • the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous ("overlapping") sequences obtained from the "cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones.
  • the overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
  • the cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones corresponding to the same gene. "Vector” refers to the type of vector contained in the cDNA Clone ID.
  • Total NT Seq refers to the total number of nucleotides in the contig identified by "Gene No.”
  • the deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5' NT of Clone Seq.” and the "3' NT of Clone Seq.” of SEQ ID NO:X.
  • the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon.”
  • the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep.”
  • the translated amino acid sequence beginning with the methionine, is identified as "AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques.
  • the polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
  • the first and last amino acid position of SEQ ID NO: Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep.”
  • the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion.”
  • the amino acid position of SEQ ID NO:Y of the last amino acid in the open reading frame is identified as "Last AA of ORF.”
  • SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below.
  • SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention.
  • polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1.
  • DNA sequences generated by sequencing reactions can contain sequencing errors.
  • the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
  • the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence.
  • the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
  • the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1.
  • the nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits.
  • amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
  • the present invention also relates to the genes corresponding to SEQ ID NO: 1
  • the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
  • allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC.
  • 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.
  • the polypeptides of the invention can be prepared in any suitable manner.
  • 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.
  • the 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.
  • the polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.
  • the present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA contained in ATCC deposit Z.
  • the present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z.
  • Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.
  • Signal Sequences The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone.
  • Polynucleotides encoding the mature forms are also encompassed by the invention.
  • proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein.
  • cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • SEQ ID 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 ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.
  • the present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID 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).
  • a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide
  • Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
  • the present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NON, the polypeptide sequence encoded by the cD ⁇ A 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)). In a sequence alignment the query and subject sequences are both D ⁇ A sequences.
  • An R ⁇ A sequence can be compared by converting U's to T's.
  • the result of said global sequence alignment is in percent identity.
  • the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
  • 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 preferred 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.
  • 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 corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, 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. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
  • 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 He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, 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.
  • additional amino acids such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity.
  • 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.
  • Polynucleotide and Polypeptide Fragments 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.
  • These 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
  • polynucleotide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary strand thereto, or the cDNA contained
  • 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.
  • “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1- 60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus 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.
  • Other preferred polypeptide fragments are biologically active fragments.
  • Bioly 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.
  • the functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123.
  • physiological correlates of binding of a polypeptide of the invention to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of polypeptides of the invention and fragments, variants derivatives and analogs thereof to elicit related biological activity related to that of the polypeptide of the invention (either in vitro or in vivo).
  • Other methods will be known to the skilled artisan and are within the scope of the invention.
  • the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID 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.
  • epitope of a polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID 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.
  • epitope of a polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID NO:X
  • 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. USA 81:3998- 4002 (1983)).
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic. Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
  • Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
  • Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910- 914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
  • Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • a carrier protein such as an albumin
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • polypeptides of the present invention 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) resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • CH2, CH3 any combination thereof and portions thereof
  • IgG Fusion proteins that haye 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.
  • 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., Curr. 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, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ 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, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • 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.
  • 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-terminal 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.
  • the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity.
  • Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
  • Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions as described herein.
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 2 M, IO "2 M, 5 X IO 3 M, IO "3 M, 5 X IO “4 M, 10 "4 M, 5 X 10 5 M, IO 5 M, 5 X 10 ° M, 10 "6 M, 5 X IO "7 M, IO 7 M, 5 X IO “8 M, IO "8 M, 5 X 10 ° M, IO "9 M, 5 X IO 10 M, IO 10 M, 5 X IO 11 M, 10 " M, 5 X IO 12 M, 10 12 M, 5 X IO 13 M, IO 13 M, 5 X IO 14 M, IO 14 M, 5 X IO "15 M, or IO 15 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope 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
  • receptor activation can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • antibodies are provided that inhibit 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.
  • 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 antibodies which activate the receptor.
  • antibodies may 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.
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, 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. Additionally, the derivative may contain one or more non-classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “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 carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and a framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 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 incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination.
  • homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • this technology for producing human antibodies see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995).
  • 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 ID 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.
  • a suitable source e.g., an antibody cDNA
  • Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
  • the 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.
  • Recombinant expression of 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
  • an expression vector containing a polynucleotide that encodes the antibody requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • 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,
  • yeast e.g., Saccharomyces, Pichia
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
  • telomeres e.g., TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • 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. Acad.
  • 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 correct 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.
  • breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D
  • normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
  • stable expression is preferred.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk-, hgprt- or aprt- cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture 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 an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule 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.
  • Patent 5,474,981 Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.
  • the present invention further includes compositions comprising the polypeptides 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.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
  • polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification.
  • One reported example describes chimeric proteins consisting 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. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
  • 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.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in 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.

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Abstract

L'invention concerne des nouvelles protéines humaines sécrétées ainsi que des acides nucléiques isolés contenant les régions codantes des gènes codant de telles protéines. Elle concerne également des vecteurs, cellules hôtes et anticorps, ainsi que des procédés de recombinaison, destinés à produire des protéines humaines sécrétées. L'invention concerne encore des méthodes diagnostiques et thérapeutiques, utiles pour diagnostiquer et traiter des maladies et/ou états associés à ces nouvelles protéines humaines sécrétées.
EP00936426A 1999-06-11 2000-06-01 49 proteines humaines secretees Withdrawn EP1185700A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US13862899P 1999-06-11 1999-06-11
US138628P 1999-06-11
PCT/US2000/014926 WO2000077255A1 (fr) 1999-06-11 2000-06-01 49 proteines humaines secretees

Publications (1)

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EP1185700A1 true EP1185700A1 (fr) 2002-03-13

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EP00936426A Withdrawn EP1185700A1 (fr) 1999-06-11 2000-06-01 49 proteines humaines secretees

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EP (1) EP1185700A1 (fr)
JP (1) JP2003502057A (fr)
AU (1) AU5174300A (fr)
CA (1) CA2383048A1 (fr)
WO (1) WO2000077255A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2002077235A2 (fr) * 2000-12-21 2002-10-03 Incyte Genomics, Inc. Molecules de signalisation intracellulaire
EP1434783A4 (fr) * 2001-03-16 2006-06-07 Lilly Co Eli Proteines de mammiferes lp et reactifs associes
JP4399590B2 (ja) * 2003-12-08 2010-01-20 独立行政法人産業技術総合研究所 廃用性筋萎縮治療剤のスクリーニング方法

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JP2987818B2 (ja) * 1990-12-14 1999-12-06 塩野義製薬株式会社 Ｔｘａ２受容体およびそれをコードする遺伝子

Non-Patent Citations (1)

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Title
See references of WO0077255A1 *

Also Published As

Publication number Publication date
WO2000077255A1 (fr) 2000-12-21
JP2003502057A (ja) 2003-01-21
AU5174300A (en) 2001-01-02
CA2383048A1 (fr) 2000-12-21

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