Classifications

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  • 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
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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 disorders and conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such disorders and conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
• isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
• isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
• a "secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
• the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
• polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
• the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• a "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:X or the cDNA contained within the clone deposited with the ATCC.
• the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
• a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
• the full length sequence identified as SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
• a representative clone containing all or most of the sequence for SEQ ID NO:X was deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.
• the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
• the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
• a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC.
• “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
• 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).
• salt concentrations e.g. 5X SSC.
• Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
• the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• polynucleotide which hybridizes only to polyA-f sequences (such as any 3' terminal polyA-f tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
• polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
• the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
• the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
• polypeptides may be branched , for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
• SEQ ID NO:X refers to a polynucleotide sequence while “SEQ ID NO:Y” refers to a polypeptide sequence, both sequences identified by an integer specified in Table 1.
• a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)
• proteins and translated DNA sequences contain regions where the amino acid composition is highly biased toward a small subset of the available residues.
• membrane spanning domains and signal peptides typically contain long stretches where Leucine (L), Valine (V), Alanine (A), and Isoleucine (I) predominate.
• Poly-Adenosine tracts (poly A) at the end of cDNAs appear in forward translations as poly-Lysine (poly-K) and poly- Phenylalanine (poly-F) when the reverse complement is translated. These regions are often referred to as "low complexity" regions.
• Such regions can cause database similarity search programs such as BLAST to find high-scoring sequence matches that do not imply true homology.
• the problem is exacerbated by the fact that most weight matrices (used to score the alignments generated by BLAST) give a match between any of a group of hydrophobic amino acids (LN and I) that are commonly found in certain low complexity regions almost as high a score as for exact matches.
• BLASTX.2 version 2.0a5MP-WashU
• filters two filters which "mask” the low complexity regions in a particular sequence. These filters parse the sequence for such regions, and create a new sequence in which the amino acids in the low complexity region have been replaced with the character "X". This is then used as the input sequence (sometimes referred to herein as "Query” and/or "Q") to the BLASTX program. While this regime helps to ensure that high-scoring matches represent true homology, there is a negative consequence in that the BLASTX program uses the query sequence that has been masked by the filters to draw alignments.
• a stretch of "X"s in an alignment shown in the following application does not necessarily indicate that either the underlying D ⁇ A sequence or the translated protein sequence is unknown or uncertain. Nor is the presence of such stretches meant to indicate that the sequence is identical or not identical to the sequence disclosed in the alignment 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.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1246 of SEQ ID NO: 11, b is an integer of 15 to 1260, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 1 1 , 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. gil535586 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " proline rich protein [Medicago sativa]."
• proline rich protein [Medicago sativa].”
• a partial alignment demonstrating the observed homology is shown immediately below.
• PROTEIN PRECURSOR. Length 228 Minus Strand HSPs :
• the segment of gil535586 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 107. 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 as SEQ ID NO. 108 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 60 as residues: Arg-21 to Asp-33, Pro-41 to Gln-56, Asp-67 to Gly-75.
• 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: 12 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 1004 of SEQ ID NO: 12
• b is an integer of 15 to 1018, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 12, and where b is greater than or equal to a + 14.
• the 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 1330401 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " T27F7.1 gene product [Caenorhabditis elegans]."
• T27F7.1 gene product [Caenorhabditis elegans]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• segment of gil 1330401 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 109.
• Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 1 10 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Stratagene ovarian cancer (#937219); Human Chronic Synovitis; Human Heart; Bone Marrow Stromal Cell, untreated; Human Adrenal Gland Tumor; Human Whole Six Week Old Embryo; Stratagene lung (#937210); Smooth muscle, serum treated; Soares melanocyte 2NbHM; Anergic T-cell; Smooth muscle.control; Monocyte activated; Human Cerebellum; Soares infant brain 1NIB; Human Colon Cancer; Prostate; H. Adipose, subtracted; Larynx normal #10 261-273; Human Colon, subtraction; Human Cerebellum, subtracted; H.
• Atrophic Endometrium Human Pancreatic Carcinoma; human colon cancer; Soares retina N2b4HR; Smooth Muscle- HASTE normalized; Stomach cancer (human),re-excision; Synovial IL-l TNF stimulated; Smooth muscle, ILlb induced; healing groin wound, 7.5 hours post incision; Human Stomach,re-excision; Human Frontal Cortex, Schizophrenia; Human Osteoclastoma, re-excision; NCI_CGAP_GCB 1 ; Human endometrial stromal cells; Synovial hypoxia; Soares_senescent_fibroblasts_NbHSF; Human Ovary; H.
• a-b is any integer between 1 to 1978 of SEQ ID NO: 13
• b is an integer of 15 to 1992
• both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 13
• 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. gil4105341 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "surface antigen [Hepatitis B virus]."
• surface antigen [Hepatitis B virus]. A partial alignment demonstrating the observed homology is shown immediately below.
• the segment of gil4105341 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 111.
• Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 112 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Jurkat T-cell Gl phase; Human Eosinophils.
• 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 1122 of SEQ ID NO: 14, b is an integer of 15 to 1136, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.
• the 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 1224131 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " breast cancer suppressor element Ishmael Upper RP2 [Homo sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
• segment of gil 1224131 that is shown as "Sbjct" above is set out as sequence 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 as SEQ ID NO. 114 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Soares_pregnant_uterus_NbHPU Human Amygdala; Human Fetal Brain, normalized 5002 IF; Human Fetal Brain, normalized A5002F; Human White Fat; Human Frontal Cortex, Schizophrenia; Human Osteoclastoma, re-excision; Ulcerative Colitis; Soares_NhHMPu_Sl; Human Thymus Stromal Cells; Bone Marrow Stromal Cell, untreated; H. Frontal cortex,epileptic,re-excision; 12 Week Old Early Stage Human; Human Eosinophils; Human Bone Marrow, treated; Spleen, Chronic lymphocytic leukemia; Nine Week Old Early Stage Human.
• 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 2391 of SEQ ID NO: 15, b is an integer of 15 to 2405, 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.
• cytochrome c oxidase subunit CIX precursor (E.C. 1.9.3.1) [Bos taurus]."
• cytochrome c oxidase subunit CIX precursor (E.C. 1.9.3.1) [Bos taurus].”
• cytochrome c oxidase subunit CIX precursor (E.C. 1.9.3.1) [Bos taurus].”
• a partial alignment demonstrating the observed homology is shown immediately below. >gi 1162901 cytochrome c oxidase subunit CIX precursor (E.C.
• polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 116 which corresponds to the Query 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: 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 2923 of SEQ ID NO: 16, b is an integer of 15 to 2937, 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.
• 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. gil938294 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " reverse transcriptase [Volvox carteri]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• the segment of gil938294 that is shown as "Sbjct" above is set out as sequence 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 as SEQ ID NO. 118 which corresponds to the Query 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: 17 amino acid sequences
• amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1695 of SEQ ID NO: 17, b is an integer of 15 to 1709, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 17, 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 1871 of SEQ ID NO: 18, b is an integer of 15 to 1885, 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.
• segment of gil 193840 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 1 19.
• Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 120 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Anergic T-cell and to a lesser extent in Osteoarthritis (OA-4); Activated T-Cells, 24 hrs.; Breast Lymph node cDNA library; CD34 positive cells (cord blood),re-ex; Human Tonsils, Lib 2; Human Colon, re-excision; Human Thymus; Human Thymus; Human Eosinophils; NCI_CGAP_GCB 1 ; breast lymph node CDNA library; T Cell helper I; CD34 depleted Buffy Coat (Cord Blood), re- excision; T cell helper II; Hodgkin's Lymphoma II; Soares placenta Nb2HP.
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 19 is related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention.
• polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1064 of SEQ ID NO: 19, b is an integer of 15 to 1078. 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 segment of gill216306 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 121.
• Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 122 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Eosinophils H. Leukocytes, normalized cot 5A; Human Stomach.re-excision; Spleen metastic melanoma; Apoptotic T-cell; T-Cell PHA 24 hrs; Soares_pregnant_uterus_NbHPU; Macrophage (GM-CSF treated); Soares_fetal_liver_spleen_lNFLS_Sl; human tonsils; Monocyte activated; NCI_CGAP_GCB 1; T cell helper II; neutrophils control.
• Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention.
• polynucleotides are specifically excluded from the scope of the present invention.
• a-b is any integer between 1 to 1539 of SEQ ID NO:20
• b is an integer of 15 to 1553, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:20, 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 1698455 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " mariner transposase [Homo sapiens]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 124 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 69 as residues: Ser-11 to Ser-25, Ser-56 to Val-65.
• 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 1244 of SEQ ID NO:21, b is an integer of 15 to 1258, 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.
• 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 1743736 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " envelope glycoprotein, C2-V5 region [Human immunodeficiency virus type 1]."
• envelope glycoprotein, C2-V5 region Human immunodeficiency virus type 1].
• the segment of gil 1743736 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 125.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 126 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Thyroid; Human Epididymus.
• polynucleotide sequences such as EST sequences
• SEQ ID NO:22 amino acid sequences
• amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:22 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 332 of SEQ ID NO:22, b is an integer of 15 to 346, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:22, and where b is greater than or equal to a + 14.
• the segment of gill 141777 that is shown as "Sbjct" above is set out as sequence 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 as SEQ ID NO. 128 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen INFLS and to a lesser extent in Human Epididymus; 12 Week Old Early Stage Human, II; Soares_fetal_lung_NbHL19W; Soares_placenta_8to9weeks_2NbHP8to9W. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 527 of SEQ ID NO:23, b is an integer of 15 to 541, where both a and b co ⁇ espond 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. gnllPIDIe276397 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " erythroid isoform protein 4.1 [Sus scrofa]."
• erythroid isoform protein 4.1 [Sus scrofa]. A partial alignment demonstrating the observed homology is shown immediately below.
• ASNPVIAAQ CSKTNP S 104 ASNPVIAAQFCSKTNP 119
• the segment of gnllPIDIe276397 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 129. 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 130 which corresponds to the Query 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:24 Some of these sequences are related to SEQ ID NO:24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1805 of SEQ ID NO:24, b is an integer of 15 to 1819, 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 segment of gnllPIDIe264677 that is shown as "Sbjct" above is set out as sequence 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 as SEQ ID NO 132 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed)
• polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 25 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every related sequence would be cumbersome Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 450 of SEQ ID NO 25, b is an integer of 15 to 464, where both a and b co ⁇ espond 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 g ⁇ l533243 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " T cell secreted protein [Mus musculus] " A partial alignment demonstrating the observed homology is shown immediately below
• the segment of g ⁇ l533243 that is shown as "Sbjct" above is set out as sequence 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 134 which corresponds to the Query 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 439 of SEQ ID NO:26, b is an integer of 15 to 453, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a + 14.
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 75 as residues: His-23 to Leu-31, His-33 to Pro-41.
• GAS gamma activating sequence
• the Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells.
• activation of the Jak-STAT pathway can be used to indicate proteins involved in the proliferation and differentiation of cells.
• 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: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 3926 of SEQ ID NO:27, b is an integer of 15 to 3940, 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. gil 1710216 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " unknown [Homo sapiens]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• segment of gil 1710216 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 135.
• Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 136 which corresponds to the Query 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: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.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1633 of SEQ ID NO:28, b is an integer of 15 to 1647, where both a and b co ⁇ espond 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. gnllPIDIe225539 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " orf:PZA109 [Saccharomyces cerevisiae]."
• orf:PZA109 Sacharomyces cerevisiae.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 138 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Soares_parathyroid_tumor_NbHPA and to a lesser extent in Human Osteoclastoma; Early Stage Human Lung, subtracted; HEL cell line; Stratagene colon (#937204); H. Striatum Depression, subt; Human endometrial stromal cells; Synovial hypoxia; Human Uterine Cancer; Spinal cord; Human Adrenal Gland Tumor; H. Frontal cortex,epileptic,re-excision; Human Placenta; Human Amygdala; Soares infant brain 1NIB.
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 29 Some of these sequences are related to SEQ ID NO: 29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1484 of SEQ ID NO:29, b is an integer of 15 to 1498, 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.
• 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. gnllPIDIe325387 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " transcription factor [Homo sapiens]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• sequence SEQ ID NO. 139 The segment of gnllPIDIe325387 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 139. 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 140 which corresponds to the Query 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: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 1612 of SEQ ID NO:30, b is an integer of 15 to 1626, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO: 30, and where b is greater than or equal to a + 14.
• 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 655 of SEQ ID NO:31, b is an integer of 15 to 669, 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 segment of gil 1575353 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 141. 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 as SEQ ID NO. 142 which corresponds to the Query 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 Soares_total_fetus_Nb2HF8_9w; Stratagene colon (#937204); Stratagene HeLa cell s3 937216; healing groin wound, 7.5 hours post incision; Human endometrial stromal cells; Breast Cancer Cell line, angiogenic; Soares breast 2NbHBst; NCI_CGAP_GC4; NCI_CGAP _Thy 1 ; Colon Carcinoma; Human Synovial Sarcoma.
• Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 80 as residues: Lys-38 to Lys-46, Arg-83 to Thr-90.
• 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: 32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1011 of SEQ ID NO: 32, b is an integer of 15 to 1025, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO: 32, and where b is greater than or equal to a + 14.
• 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.
• a-b is any integer between 1 to 906 of SEQ ID NO:33
• b is an integer of 15 to 920, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:33, 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 801 of SEQ ID NO: 34, b is an integer of 15 to 815, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:34, and where b is greater than or equal to a + 14.
• Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 83 as residues: Pro-20 to Gly-26.
• 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 572 of SEQ ID NO:35, b is an integer of 15 to 586, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:35, and where b is greater than or equal to a + 14.
• the segment of gil2313687 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 143.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 144 which corresponds to the Query 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 232 of SEQ ID NO:36, b is an integer of 15 to 246, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:36, and where b is greater than or equal to a + 14.
• Kidney Pyramid subtracted; NCI_CGAP_Co3; NCI_CGAP_Ov2; NCI_CGAP_ColO; NCI_CGAP_Col2; NCI_CGAP_Kid5; NCI_CGAP_Kid6; NCI_CGAP_Pr23; Liver Tumour Met 5 Tu; Normal Prostate; human colon cancer; Human adult small intestine,re-excision; Human Pineal Gland; Healing groin wound - zero hr post-incision (control); Healing groin wound, 6.5 hours post incision; Human Manic Depression Tissue; Human
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 85 as residues: Met-35 to Lys-40.
• Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2579 of SEQ ID NO:37, b is an integer of 15 to 2593, 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.
• polynucleotide sequences such as EST sequences
• SEQ ID NO: 38 Some of these sequences are related to SEQ ID NO: 38 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 714 of SEQ ID NO:38, b is an integer of 15 to 728, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:38, and where b is greater than or equal to a + 14.
• Soares_pregnant_uterus_NbHPU and to a lesser extent in Soares breast 2NbHBst; Soares retina N2b4HR; Stratagene corneal stroma (#937222); Human Endometrial Tumor; Soares retina N2b5HR; Human Pineal Gland; Human endometrial stromal cells-treated with estradiol; Glioblastoma; Human Frontal Cortex, Schizophrenia; Synovial Fibroblasts (Ill/TNF), subt; Human umbilical vein endothelial cells, IL-4 induced; Pancreas Islet Cell Tumor; Colon Carcinoma; Human Osteoclastoma; Human 8 Week Whole Embryo; Nine Week Old Early Stage Human; Soares placenta Nb2HP.
• 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 1224 of SEQ ID NO:39, b is an integer of 15 to 1238, where both a and b co ⁇ espond 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. gil504522 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " ORF; putative [Homo sapiens].”
• ORF database accession no. gil504522
• ORF putative [Homo sapiens].
• the segment of gil504522 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 145.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 146 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Endometrial Tumor and to a lesser extent in Stratagene ovarian cancer (#937219); Human Adipose Tissue, re-excision. 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:40 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 1764 of SEQ ID NO:40
• b is an integer of 15 to 1778, where both a and b co ⁇ espond 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. gil3004664 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "misato [Drosophila melanogaster]."
• sequence homology is shown immediately below.
• the segment of gil3004664 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 147. 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 148 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 89 as residues: Glu- 17 to Ser-34.
• 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.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2797 of SEQ ID NO:41, b is an integer of 15 to 2811, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:41, and where b is greater than or equal to a + 14.
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 90 as residues: Glu-17 to Gly-22, Arg-29 to Phe-36.
• Many polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 845 of SEQ ID NO:42, b is an integer of 15 to 859, 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.
• 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. gnllPIDIe220189 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " reverse transcriptase [Arabidopsis thaliana]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• sequence SEQ ID NO. 149 The segment of gnllPIDIe220189 that is shown as "Sbjct" above is set out as sequence 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 as SEQ ID NO. 150 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares_NhHMPu_S 1 ; Soares infant brain 1NIB and to a lesser extent in Human Endometrial Tumor; Soares fetal liver spleen INFLS; Thyroid Tumour; Human Prostate; Human Adrenal Gland Tumor; Soares_pregnant_uterus_NbHPU.
• 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 946 of SEQ ID NO:43, b is an integer of 15 to 960, 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. gil522145 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " B-cell growth factor [Homo sapiens]."
• B-cell growth factor [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 as SEQ ID NO. 152 and/or SEQ ID NO. 154 which correspond to the Query sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 92 as residues: Pro-36 to Gln-48.
• polynucleotide sequences such as EST sequences
• SEQ ID NO:44 amino acid sequence sequences
• amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 622 of SEQ ID NO:44, b is an integer of 15 to 636, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:44, and where b is greater than or equal to a + 14.
• polynucleotide sequences such as EST sequences
• SEQ ID NO:45 amino acid sequence sequences
• amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1081 of SEQ ID NO:45, b is an integer of 15 to 1095, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:45, and where b is greater than or equal to a + 14.
• the segment of gil2791855 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 155. 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 156 which corresponds to the Query 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:46 amino acid sequences
• amino acid 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 499 of SEQ ID NO:46, b is an integer of 15 to 513, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a + 14.
• 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.
• a-b is any integer between 1 to 782 of SEQ ID NO:47, b is an integer of 15 to 796, 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.
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 96 as residues: Pro-49 to Pro-56.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 485 of SEQ ID NO:48, b is an integer of 15 to 499, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:48, and where b is greater than or equal to a + 14.
• the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no. gil2580578 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " ubiquitous TPR motif, Y isoform [Homo sapiens]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• the segments of gil2580578 that are shown as "Sbjct" above are set out as sequences SEQ ID NO. 157 and/or SEQ ID NO. 159.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 158 and/or SEQ ID NO. 160 which correspond to the Query sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Fetal Brain; neutrophils control.
• 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 774 of SEQ ID NO:49, b is an integer of 15 to 788, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:49, and where b is greater than or equal to a + 14.
• the segment of gil4164444 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 161. 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.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 162 which corresponds to the Query 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 1431 of SEQ ID NO:50, b is an integer of 15 to 1445, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:50, and where b is greater than or equal to a + 14.
• segment of gil 1710216 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 163.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 164 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in Human Fetal Brain.
• polynucleotide sequences such as EST sequences
• SEQ ID NO:51 amino acid sequences
• amino acid 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. 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 989 of SEQ ID NO:51, b is an integer of 15 to 1003, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:51, 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. gil288145 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " put. ORF [Homo sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 166 which corresponds to the Query 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 1163 of SEQ ID NO:52, b is an integer of 15 to 1177, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:52, 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 1710216 (all information available through the recited accession number is incorporated herein by reference) which is described therein as " unknown [Homo sapiens]."
• a partial alignment demonstrating the observed homology is shown immediately below.
• segment of gil 1710216 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 167.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 168 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• a-b is any integer between 1 to 1717 of SEQ ID NO:53
• b is an integer of 15 to 1731, 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.
• 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. gil3098268 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as " putative protein 2 [Fugu rubripes]."
• putative protein 2 [Fugu rubripes].
• the segment of gil3098268 that is shown as "Sbjct” above is set out as sequence SEQ ID NO. 169.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 170 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
• Soares_pregnant_uterus_NbHPU Human Uterine Cancer; Human Thymus; Soares breast 2NbHBst; Human Whole Six Week Old Embryo; Soares_NhHMPu_S 1 ; Pancreas Islet Cell Tumor; Soares_NhHMPu_S 1 ; Soares breast 3NbHBst; Human Testes Tumor; Human Fetal Lung III; Bone Marrow Cell Line (RS4,11); Keratinocyte; Human Cerebellum; Primary Dendritic Cells, lib 1; Human Fetal Kidney; H.
• RS4,11 Bone Marrow Cell Line
• hypothalamus frac A
• LNCAP + o.3nM R1881 Whole 6 Week Old Embryo; Human Cerebellum, subtracted; Human Primary Breast Cancer; Human Lung; Human adult small intestine,re-excision; Messangial cell, frac 2; H.
• Meningima Ml; Human Manic Depression Tissue; Prostate BPH; Human Chronic Synovitis; Soares_multiple_sclerosis_2NbHMSP; T-Cell PHA 16 hrs; Breast Cancer Cell line, angiogenic; Human Primary Breast Cancer Reexcision; Human Pancreas Tumor; Human Adult Testes, Large Inserts, Reexcision; Human Adipose; Ulcerative Colitis; Human Thymus Stromal Cells; Bone Ma ⁇ ow Stromal Cell, untreated; Human Adrenal Gland Tumor; Rejected Kidney, lib 4; Human Liver, normal; NCI_CGAP_GCB 1 ; 12 Week Old Early Stage Human; Colon Normal II; Early Stage Human Brain; H Macrophage (GM-CSF treated), re-excision; Colon Tumor II; Stratagene neuroepithelium (#937231); Spleen, Chronic lymphocytic leukemia; Soares_fetal_lung_NbHL19W; Hodgkin's Lymphom
• 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 1084 of SEQ ID NO:54, b is an integer of 15 to 1098, where both a and b co ⁇ espond 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. gil485096 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as a C. elegans protein.
• polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 172 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen INFLS and to a lesser extent in Soares_fetal_heart_NbHH19W; Soares infant brain 1MB;
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 103 as residues: Ala-94 to Pro- 103.
• 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 1108 of SEQ ID NO:55, b is an integer of 15 to 1122, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a + 14.
• PROTEIN I/II ZINC-METALLOTHIONEIN CLASS II
• ⁇ SUB 2-81 ⁇ Length 81 Plus Strand HSPs:
• polypeptide having the amino acid sequence set out as SEQ ID NO. 174 which corresponds to the Query 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: 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 1803 of SEQ ID NO:56, b is an integer of 15 to 1817, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:56, and where b is greater than or equal to a + 14.
• 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 815 of SEQ ID NO:57, b is an integer of 15 to 829, 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.
• 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. gil2688381 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "B. burgdorferi predicted coding region BB0470 [Borrelia burgdorferi].”
• a partial alignment demonstrating the observed homology is shown immediately below. >gi 12688381 (AE001151) B.
• Prefe ⁇ ed polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO. 176 which corresponds to the Query sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Fetal Brain.
• Prefe ⁇ ed epitopes include those comprising a sequence shown in SEQ ID NO. 106 as residues: Asn-21 to Gly-28.
• 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 873 of SEQ ID NO:58, b is an integer of 15 to 887, 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.
• 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 co ⁇ esponding deposit number listed in "ATCC Deposit No:Z and Date.” Some of the deposits contain multiple different clones corresponding to the same gene. "Vector” refers to the type of vector contained in the cDNA Clone ID.
• Total NT Seq refers to the total number of nucleotides in the contig identified by "Gene No.”
• the deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5' NT of Clone Seq.” and the "3' NT of Clone Seq.” of SEQ ID NO:X.
• the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon.”
• the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep.”
• the translated amino acid sequence beginning with the methionine, is identified as "AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques.
• the polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
• SEQ ID NO:Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep.”
• the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion.”
• 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. Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing e ⁇ ors.
• the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
• the e ⁇ oneously 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 co ⁇ esponding to SEQ ID
• the co ⁇ esponding 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 co ⁇ esponding 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.
• polypeptides of the invention can be prepared in any suitable manner.
• Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
• polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
• a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
• Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein.
• the present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA contained in ATCC deposit Z.
• the present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z.
• Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention.
• the present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ 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.
• 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. Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res.
• the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are inco ⁇ orated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1.
• 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.
• These polypeptides, and the polynucleotides encoding such polypeptides are contemplated by the present invention.
• the signal sequence identified by the above analysis may not necessarily predict the naturally occu ⁇ ing signal sequence.
• the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
• the predicted signal sequence will be capable of directing the secreted protein to the ER.
• the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as desribed below).
• a mammalian cell e.g., COS cells, as desribed below.
• polynucleotide and Polypeptide Variants The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X, the complementary strand thereto, and/or the cDNA sequence contained in a deposited clone.
• the present invention also encompasses variants of the polypeptide sequence disclosed in SEQ 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).
• Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these
• the present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).
• nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
• nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• the query sequence may be an entire sequence shown inTable 1, the ORF (open reading frame), or any fragment specified as described herein.
• nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs.
• a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)).
• a sequence alignment the query and subject sequences are both DNA sequences.
• An RNA sequence can be compared by converting U's to T's.
• the percent identity is co ⁇ ected 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 co ⁇ ected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
• a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
• the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
• the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
• a 90 base subject sequence is compared with a 100 base query sequence.
• deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
• percent identity calculated by FASTDB is not manually corrected.
• bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually co ⁇ ected for. No other manual co ⁇ ections are to made for the purposes of the present invention.
• polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention 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.
• up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
• alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• 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 NON) or to the amino acid sequence encoded by cD ⁇ A 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 refe ⁇ ed 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 percent identity is co ⁇ ected by calculating the number of residues of the query sequence that are ⁇ - and C-terminal of the subject sequence, which are not matched/aligned with a co ⁇ esponding 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 a ⁇ ive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
• 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 co ⁇ ected.
• the variants may contain alterations in the coding regions, non-coding regions, or both.
• Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
• variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also prefe ⁇ ed.
• 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 occu ⁇ ing variants may be produced by mutagenesis techniques or by direct synthesis. Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
• one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
• Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (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. As the authors state, these two strategies have revealed that proteins are su ⁇ risingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein.
• amino acid residues For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, 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 inventio- include (i) substitutions with one or more of the non-conserved amino acid resir 1 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.
• substitutions with one or more of the non-conserved amino acid resir 1 where the substituted amino acid residues may or may not be one encoded by the genetic code or substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptid
• polypeptide variants are deemed to be within the scope of those skilled in the art from the teachings herein.
• 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.
• 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 prefe ⁇ ed.
• 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.
• 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.
• polypeptide fragment refers to an amino acid sequence which is a portion of that contained in SEQ ID NON or encoded by the cD ⁇ A 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. In this context "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.
• Prefe ⁇ ed polypeptide fragments include the secreted protein as well as the mature form. Further prefe ⁇ ed 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.
• 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.
• any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form.
• any combination of the above amino and carboxy terminus deletions are preferred.
• 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 ⁇ O:Y falling within conserved domains are specifically contemplated by the present invention.
• polynucleotides encoding these domains are also contemplated.
• polypeptide fragments are biologically active fragments.
• Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
• the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity.
• a polypeptide demonstrating a "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein.
• Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
• polypeptides of the invention and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
• various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
• competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled.
• Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting.
• 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 NON, 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.
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
• 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.
• 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.
• Prefe ⁇ ed 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 prefe ⁇ ed 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.
• Prefe ⁇ ed 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).
• Prefe ⁇ ed 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 ca ⁇ ier 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 ca ⁇ ier.
• a ca ⁇ ier 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 ca ⁇ ier, 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 ca ⁇ iers 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 ca ⁇ ier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• the 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.
• Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4- polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial ba ⁇ ier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813).
• antigens e.g., insulin
• FcRn binding partner such as IgG or Fc fragments
• IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide.
• an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Cu ⁇ . Opinion Biotechnol.
• alteration of polynucleotides co ⁇ esponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
• polynucleotides of the invention may be altered by being subjected to random mutagenesis by e ⁇ or-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 heteroiogous 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 heteroiogous epitope, such as a heteroiogous polypeptide or solid support material.
• a heteroiogous epitope such as a heteroiogous polypeptide or solid support material.
• Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-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. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
• Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the co ⁇ esponding epitopes thereof.
• Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
• the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
• antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 "4 M, 10 “4 M, 5 X 10 '5 M, 10 "5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 10 M, 10 “10 M, 5 X 10 u M, 10" M, 5 X 10 12 M, 10 - 12 M, 5 X 10 "13 M, 10 13 M, 5 X 10 "14 M, 10 14 M, 5 X 10 "15 M, or 10 "15 M.
• the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
• the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
• Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
• antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
• the invention features both receptor-specific antibodies and ligand-specific antibodies.
• the invention also features receptor- specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.
• receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• 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.
• antibodies which bind the ligand and prevent binding of the ligand to the receptor are included in the invention.
• antibodies which activate the receptor 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.
• Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
• the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (inco ⁇ orated by reference herein in its entirety).
• the antibodies of the present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heteroiogous 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 heteroiogous polypeptides, drugs, radionuchdes, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
• 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 ca ⁇ ied 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 inco ⁇ orated by reference in their entireties).
• the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• the term “monoclonal antibody” 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
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
• Methods for producing chimeric antibodies are known in the art. See e.g., Mo ⁇ ison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are inco ⁇ orated herein by reference in their entirety.
• Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non- human species and a framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• framework residues in the human framework regions will be substituted with the co ⁇ esponding 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. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
• the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
• Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
• a selected non-human monoclonal antibody e.g., a mouse antibody
• antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
• antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a 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.
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be
• nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non- human antibody, as described supra.
• the framework regions may be naturally occu ⁇ ing or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol.
• the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
• Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
• Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
• Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
• the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
• an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
• a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
• the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
• Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• the expression vector is transfe ⁇ ed 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 heteroiogous promoter.
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
• host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO)
• CHO Chinese hamster ovary cells
• 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. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• 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 co ⁇ ect 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 prefe ⁇ ed.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the antibody molecule.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al., Cell 11 :223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sci. USA 78: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
• the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid 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 heteroiogous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention.
• antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S.
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
• the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• 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.
• polypeptides co ⁇ esponding 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.
• polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5
• Fc portions for the 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.
• peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment i l l
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• examples of bioluminescent materials include luciferase, luciferin, and aequorin; and
• suitable radioactive material include 1251, 1311, 11 lln or 99Tc.
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213BL
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin).
• antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
• the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an a
• VEGI See, International Publication No. WO 99/23105
• a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
• biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-l”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-l interleukin-1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is inco ⁇ orated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• the antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples.
• the translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types.
• Monoclonal antibodies directed against a specific epitope, or combination of epitopes will allow for the screening of cellular populations expressing the marker.
• Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al, Cell, 96:737-49 (1999)).
• hematological malignancies i.e. minimal residual disease (MRD) in acute leukemic patients
• MRD minimal residual disease
• GVHD Graft- versus-Host Disease
• these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
• the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
• Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIP A buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
• a lysis buffer such as RIP A buffer (1% NP-40 or Triton X- 100, 1% sodium
• the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
• One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
• immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS- PAGE depending on the molecular weight of the antigen), transfe ⁇ ing the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non- fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
• ELIS comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• ELIS ELIS
• a second antibody which recognizes the antibody of interest conjugated to a detectable compound conjugated to a detectable compound may be added to the well.
• the antibody may be coated to the well.
• a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
• ELISAs see, e.g., Ausubel et al, eds, 1994, Cu ⁇ ent Protocols in Molecular Biology, Vol.
• the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
• the affinity of the antibody of interest for a particular antigen and the binding off -rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassay s.
• the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
• Therapeutic Uses is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions.
• Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
• the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.
• the treatment and/or prevention of diseases, disorders, or conditions associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is prefe ⁇ ed.
• human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis. It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 "4 M, 10 '4 M, 5 X 10 "5 M, 10 "5 M, 5 X 10 '6 M, 10 “6 M, 5 X 10 "7 M, 10 “7 M, 5 X 10 “8 M, 10 '8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10 10 M, 10 10 M, 5 X 10" M, 10 " M, 5 X 10 12 M, 10 " n M, 5 X 10 '13 M, 10 " I3 M, 5 X 10 '14 M, 10 "14 M, 5 X 10 15 M, and 10 15 M.
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Roller and Smithies, Proc. Natl.
• the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
• Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- ca ⁇ ying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
• This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
• microparticle bombardment e.g., a gene gun; Biolistic, Dupont
• coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
• the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination
• viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
• a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the co ⁇ ect packaging of the viral genome and integration into the host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644- 651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Cu ⁇ . Opin. in Genetics and Devel. 3: 110-114 (1993).
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Cu ⁇ ent Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).
• Another approach to gene therapy involves transfe ⁇ ing a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transfe ⁇ ed gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
• introduction can be ca ⁇ ied out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth.
• the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• the resulting recombinant cells can be delivered to a patient by various methods known in the art.
• Recombinant blood cells e.g., hematopoietic stem or progenitor cells
• the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
• the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity
• the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
• the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
• the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• a compound of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor- mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
• Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
• the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
• the compound or composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
• polymeric materials can be used (see Medical Applications of
• a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable ca ⁇ ier.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• ca ⁇ ier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical ca ⁇ iers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• Water is a prefe ⁇ ed ca ⁇ ier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid ca ⁇ iers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and ca ⁇ iers such as triglycerides.
• Oral formulation can include standard ca ⁇ iers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical ca ⁇ iers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of ca ⁇ ier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• the compounds of the invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques.
• in vitro assays may optionally be employed to help identify optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
• the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Diagnosis and Imaging Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic pu ⁇ oses to detect, diagnose, or monitor diseases and/or disorders associated with the abe ⁇ ant expression and/or activity of a polypeptide of the invention.
• the invention provides for the detection of abe ⁇ ant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of abe ⁇ ant expression.
• the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior
• Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al, J. Cell . Biol. 105:3087-3096 (1987)).
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with abe ⁇ ant expression of the polypeptide of interest.
• Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
• the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI). Kits
• kits that can be used in the above methods.
• a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
• the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
• the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
• kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
• a kit may include a control antibody that does not react with the polypeptide of interest.
• a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
• a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry).
• the kit may include a recombinantly produced or chemically synthesized polypeptide antigen.
• the polypeptide antigen of the kit may also be attached to a solid support.
• the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
• a kit may also include a non-attached reporter-labeled anti-human antibody.
• binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
• the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention.
• the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
• the antibody is attached to a solid support.
• the antibody may be a monoclonal antibody.
• the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
• the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
• the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
• the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
• the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adso ⁇ tion of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl. hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• the invention provides an assay system or kit for carrying out this diagnostic method.
• the kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface -bound anti-antigen antibody.
• any polypeptide of the present invention can be used to generate fusion proteins.
• the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
• Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
• secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
• domains that can be fused to polypeptides of the present invention include not only heteroiogous signal sequences, but also other heteroiogous functional regions.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
• polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• Fusion proteins having disulfide-linked dimeric structures can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• EP-A-O 464 533 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins, such as hIL-5 have been fused with Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
• the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• HA hemagglutinin
• the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
• the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
• Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, t ⁇ , phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE- 9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
• eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Other suitable vectors will be readily apparent to the skilled artisan.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
• a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
• HPLC high performance liquid chromatography
• Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
• a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
• the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
• polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
• N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heteroiogous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.
• endogenous genetic material e.g., coding sequence
• genetic material e.g., heteroiogous polynucleotide sequences
• heteroiogous control regions e.g., promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• heteroiogous control regions e.g., promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310: 105-111 (1984)).
• a polypeptide co ⁇ esponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro- amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (
• the invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be ca ⁇ ied out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
• the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the prefe ⁇ ed molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
• Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• the polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them.
• the polypeptides of the invention are monomers, dimers, trimers or tetramers.
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers. Multimers encompassed by the invention may be homomers or heteromers.
• homomer refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, co ⁇ esponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences.
• the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences).
• the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• the term heteromer refers to a multimer containing one or more heteroiogous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention.
• the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers of the invention such as, for example, homodimer s or homotrimers, are formed when polypeptides of the invention contact one another in solution.
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heteroiogous polypeptide sequence in a fusion protein of the invention) in solution.
• multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occu ⁇ ing) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• such covalent associations may involve one or more amino acid residues contained in the heteroiogous polypeptide sequence in a fusion protein of the invention.
• covalent associations are between the heteroiogous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
• the covalent associations are between the heteroiogous sequence contained in an Fc fusion protein of the invention (as described herein).
• covalent associations of fusion proteins of the invention are between heteroiogous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
• polypeptide linkers In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby inco ⁇ orated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
• Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
• Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240: 1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occu ⁇ ing peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby inco ⁇ orated by reference.
• Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
• Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.
• Prefe ⁇ ed leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.
• One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344: 191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby inco ⁇ orated by reference.
• Other peptides derived from naturally occu ⁇ ing trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
• proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide seuqence.
• associations proteins of the invention are associated by interactions between heteroiogous polypeptide sequence contained in Flag® fusion proteins of the invention and anti- Flag® antibody.
• the multimers of the invention may be generated using chemical techniques known in the art.
• polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers of the invention may be generated using genetic engineering techniques known in the art.
• polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be inco ⁇ orated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymo ⁇ hisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
• sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene co ⁇ esponding to the SEQ ID NO:X will yield an amplified fragment.
• somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments.
• Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
• FISH fluorescence in situ hybridization
• the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
• Prefe ⁇ ed polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
• Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease.
• Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) .
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
• the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
• the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject.
• the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the present invention and a suitable container.
• the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present invention, where each probe has one strand containing a 31'mer-end internal to the region.
• the probes may be useful as primers for polymerase chain reaction amplification.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• measuring the expression level of polynucleotide of the present invention is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample).
• the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder.
• a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA.
• biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• the method(s) provided above may prefe ⁇ ably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support.
• the support may be a "gene chip” or a "biological chip” as described in US Patents 5,837,832, 5,874,219, and 5,856,174.
• a gene chip with polynucleotides of the present invention attached may be used to identify polymo ⁇ hisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymo ⁇ hisms (i.e.
• the present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art.
• PNA peptide nucleic acids
• the use of PNAs would serve as the preferred form if the polynucleotides are inco ⁇ orated onto a solid support, or gene chip.
• a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems).
• PNAs phosphorus, phosphorus oxides, or deoxyribose derivatives
• PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does.
• PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding.
• single base mismatches can be determined with PNA DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15- mer duplex.
• the present invention is useful for detecting cancer in mammals.
• the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly prefe ⁇ ed are humans.
• Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types.
• a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA.
• Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);
• Inhibitors of Gene Expression CRC Press, Boca Raton, FL (1988).
• Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.
• Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease.
• Polynucleotides of the present invention are also useful in gene therapy.
• One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
• the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
• Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.
• the polynucleotides are also useful for identifying individuals from minute biological samples.
• the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
• RFLP restriction fragment length polymorphism
• an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
• This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
• the polynucleotides of the present invention can be used as additional DNA markers for RFLP.
• the polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome.
• sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
• DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,urine,fecal matter, etc.
• body fluids e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant,urine,fecal matter, etc.
• gene sequences amplified from polymo ⁇ hic loci such as DQa class II HLA gene, are used in forensic biology to identify individuals.
• polynucleotides of the present invention can be used as polymo ⁇ hic markers for forensic pu ⁇ oses.
• reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
• Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
• the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
• a polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
• protein expression in tissues can be studied with classical immunohistological methods.
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc)
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• proteins can also be detected in vivo by imaging.
• Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR.
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
• suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated into the antibody by labeling of nutrients for the relevant hybridoma.
• a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 1311, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal.
• a radioisotope for example, 1311, 112In, 99mTc
• a radio-opaque substance for example, parenterally, subcutaneously, or intraperitoneally
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)
• the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
• a diagnostic method of a disorder involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
• a more definitive diagnosis of this type may allow health professionals to employ preventative measures or
• polypeptides of the present invention can be used to treat disease.
• patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
• a desired response e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues.
• antibodies directed to a polypeptide of the present invention can also be used to treat disease.
• administration of an antibody directed to a polypeptide of the present invention can bind and reduce ove ⁇ roduction of the polypeptide.
• administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
• polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities. Gene Therapy Methods
• Another aspect of the present invention is to gene therapy methods for treating disorders, diseases and conditions.
• the gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention.
• This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue.
• Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein inco ⁇ orated by reference.
• cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
• a polynucleotide DNA or RNA
• Such methods are well-known in the art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216 (1993); Fe ⁇ antini et al., Cancer Research, 53: 107-1112 (1993); Fe ⁇ antini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J.
• the cells which are engineered are arterial cells.
• the arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
• the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).
• the polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
• the polynucleotide of the invention is delivered as a naked polynucleotide.
• naked polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like.
• the polynucleotides of the invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein inco ⁇ orated by reference.
• polynucleotide vector constructs of the invention used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication.
• Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen.
• Other suitable vectors will be readily apparent to the skilled artisan.
• Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heteroiogous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the He ⁇ es Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters.
• the promoter also may be the native promoter for the polynucleotides of the invention.
• one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
• the polynucleotide construct of the invention can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
• Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone.
• the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
• an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight.
• the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
• this dosage will vary according to the tissue site of injection.
• the appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues.
• parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose.
• naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
• the naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.
• the constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
• the polynucleotide constructs of the invention are complexed in a liposome preparation.
• Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
• cationic liposomes are particularly prefe ⁇ ed because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
• Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 (1987), which is herein inco ⁇ orated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein inco ⁇ orated by reference); and purified transcription factors (Debs et al., J. Biol. Chem.,
• Cationic liposomes are readily available.
• N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporated by reference).
• Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
• Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.
• DOTAP l,2-bis(oleoyloxy)-3-(trimethylammonio)propane liposomes.
• DOTMA liposomes Preparation of DOTMA liposomes is explained in the literature, see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is herein inco ⁇ orated by reference.
• anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.
• Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others.
• DOPC dioleoylphosphatidyl choline
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphoshatidyl ethanolamine
• DOPC dioleoylphosphatidyl choline
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphosphatidyl ethanolamine
• DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water.
• the sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC.
• negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
• Other methods are known and available to those of skill in the art.
• the liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being prefe ⁇ ed.
• MLVs multilamellar vesicles
• SUVs small unilamellar vesicles
• LUVs large unilamellar vesicles
• the various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology , 101:512-527 (1983), which is herein incorporated by reference.
• MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated.
• SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes.
• the material to be entrapped is added to a suspension of preformed MLVs and then sonicated.
• liposomes containing cationic lipids the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA.
• the liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA.
• SUVs find use with small nucleic acid fragments.
• LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca 2+ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim. Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun, 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl.
• the ratio of DNA to liposomes will be from about 10: 1 to about 1 : 10.
• the ration will be from about 5: 1 to about 1 :5. More preferably, the ration will be about 3: 1 to about 1:3. Still more preferably, the ratio will be about 1:1.
• U.S. Patent NO: 5,676,954 (which is herein inco ⁇ orated by reference) reports on the injection of genetic material, complexed with cationic liposomes ca ⁇ iers, into mice.
• WO 94/9469 (which are herein inco ⁇ orated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.
• U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein inco ⁇ orated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals.
• cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding polypeptides of the invention.
• Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
• the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
• packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT- 19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy , 1:5-14 (1990), which is inco ⁇ orated herein by reference in its entirety.
• the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation.
• the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
• the producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention.
• retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
• the transduced eukaryotic cells will express polypeptides of the invention.
• cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector.
• Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)).
• adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha- 1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434 (1991); Rosenfeld et al., Cell, 68: 143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
• Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell , 68: 143-155 (1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein incorporated by reference.
• the adenovirus vector Ad2 is useful and can be grown in human 293 cells.
• These cells contain the El region of adenovirus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector.
• Ad2 other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
• the adenoviruses used in the present invention are replication deficient.
• Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles.
• the resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells.
• Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or LI through L5.
• the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV).
• AAV adeno-associated virus
• AAVs are naturally occu ⁇ ing defective viruses that require helper viruses to produce infectious particles (Muzyczka, Cu ⁇ . Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
• an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration.
• the polynucleotide construct containing polynucleotides of the invention is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989).
• the recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
• helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
• packaging cells Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express the desired gene product.
• Another method of gene therapy involves operably associating heteroiogous control regions and endogenous polynucleotide sequences (e.g.
• Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein.
• the targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence.
• the targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
• the promoter and the targeting sequences can be amplified using PCR.
• the amplified promoter contains distinct restriction enzyme sites on the 5' and ⁇ ends.
• the X end of the first targeting sequence contains the same restriction enzyme site as the 5 ' end of the amplified promoter and the 5 ' end of the second targeting sequence contains the same restriction site as the 3 ' end of the amplified promoter.
• the amplified promoter and targeting sequences are digested and ligated together.
• the promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above.
• the P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
• the promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
• the polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins.
• Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
• the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein.
• the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5' end of the coding region.
• the signal sequence may be homologous or heteroiogous to the polynucleotide of interest and may be homologous or heteroiogous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art. Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect.
• biolistic injectors particle accelerators (i.e., "gene guns”
• gelfoam sponge depots other commercially available depot materials
• osmotic pumps e.g., Alza minipumps
• oral or suppositorial solid (tablet or pill) pharmaceutical formulations e.g., osmotic pumps
• decanting or topical applications during surgery e.g., direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of
• a prefe ⁇ ed method of local administration is by direct injection.
• a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
• Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
• Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound.
• a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
• compositions useful in systemic administration include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention.
• Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
• Prefe ⁇ ed methods of systemic administration include intravenous injection, aerosol, oral and percutaneous (topical) delivery.
• Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA , 189: 11277-11281 (1992), which is incorporated herein by reference).
• Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a ca ⁇ ier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such ca ⁇ iers, include plastic capsules or tablets, such as those known in the art.
• Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
• a lipophilic reagent e.g., DMSO
• Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
• Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Prefe ⁇ ed mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly
• polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
• the polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
• Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
• the etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
• immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia). ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
• blood protein disorders e.g. agammaglobulinemia, dysgammaglobulinemia.
• ataxia telangiectasia common variable immunodeficiency
• Digeorge Syndrome HIV infection
• HTLV-BLV infection leukocyte adhesion deficiency syndrome
• lymphopenia phagocyte bactericidal dysfunction
• SCIDs severe combined immunodeficiency
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation).
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or sca ⁇ ing.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful in treating or detecting autoimmune disorders.
• Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
• autoimmune disorders examples include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Pu ⁇ ura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Ba ⁇ e Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
• allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polynucleotides or polypeptides, or agonists or antagonists of the present invention.
• these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
• Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
• an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
• the administration of a polynucleotides or polypeptides, or agonists or antagonists of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells may be an effective therapy in preventing organ rejection or GVHD.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation.
• the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
• These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement- mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-l.)
• infection e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)
• ischemia-reperfusion injury e.g., endotoxin lethality, arthritis, complement- mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e
• a polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat or detect hype ⁇ roliferative disorders, including neoplasms.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.
• a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hype ⁇ roliferative disorder.
• hype ⁇ roliferative disorders can be treated.
• This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• decreasing an immune response may also be a method of treating hype ⁇ roliferative disorders, such as a chemotherapeutic agent.
• Examples of hype ⁇ roliferative disorders that can be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
• neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,
• hype ⁇ roliferative disorders can also be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention.
• hype ⁇ roliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, pu ⁇ ura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hype ⁇ roliferative disease, besides neoplasia, located in an organ system listed above.
• One prefe ⁇ ed embodiment utilizes polynucleotides of the present invention to inhibit abe ⁇ ant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
• the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.
• Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells.
• polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides.
• the DNA construct encoding the poynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more prefe ⁇ ably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby inco ⁇ orated by reference).
• the viral vector is defective and will not transform non-proliferating cells, only proliferating cells.
• the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product.
• an external stimulus i.e. magnetic, specific small molecule, chemical, or drug administration, etc.
• the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
• Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens.
• repressing expression of the oncogenic genes is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
• polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification.
• the polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A.
• vaccinia virus system Chokrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art.
• vaccinia virus system Chokrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art.
• retrovirus or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
• the polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site.
• the polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
• cell proliferative disease any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
• any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site.
• biologically inhibiting is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells.
• the biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
• the present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders.
• antibody-based therapies involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders.
• Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein.
• Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies. It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragements thereof.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 "6 M, 5X10 "7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 “9 M, 10 "9 M, 5X10 " 10 M, 10 10 M, 5X10"M, 10"M, 5X10 12 M, 10 12 M, 5X10 13 M, 10 13 M, 5X10 14 M, 10 " 14 M, 5X10 ,5 M, and 10 15 M.
• polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein.
• said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor- specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21): 1648-53 (1998), which is hereby inco ⁇ orated by reference).
• Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2): 155-61 (1998), which is hereby inco ⁇ orated by reference)).
• Polypeptides including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis.
• Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death- domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998), which is hereby inco ⁇ orated by reference).
• TNF tumor necrosis factor
• TRAMP TNF-receptor-related apoptosis-mediated protein
• TRAIL TNF-related apoptos
• said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat Res 400(l-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. Apr 24;11 1-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J Tissue React;20(l):3-15 (1998), which are all hereby inco ⁇ orated by reference).
• small molecule drugs or adjuviants such as apoptonin, galectins, thioredoxins, antiinflammatory proteins
• Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Cu ⁇ Top Microbiol Immunol 1998;231:125-41, which is hereby inco ⁇ orated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
• the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heteroiogous polypeptides, heteroiogous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention.
• compositions containing the polypeptides of the invention e.g., compositions containing polypeptides or polypeptide antibodes associated with heteroiogous polypeptides, heteroiogous nucleic acids, toxins, or prodrugs
• Polypeptides or polypeptide antibodes of the invention may be associated with with heteroiogous polypeptides, heteroiogous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
• Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
• proteins known to enhance the immune response e.g. chemokines
• Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
• Cardiovascular disorders include cardiovascular abnormalities, such as arterio- arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
• Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
• Cardiovascular disorders also include heart disease, such as a ⁇ hythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
• heart disease such as a ⁇ hythmias, carcinoi
• Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim- type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
• Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
• Klippel-Trenaunay-Weber Syndrome Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia
• Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma. subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
• Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.
• Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
• Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch pu ⁇ ura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• Polynucleotides or polypeptides, or agonists or antagonists of the invention are especially effective for the treatment of critical limb ischemia and coronary disease.
• Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art.
• Polypeptides of the invention may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides of the invention are described in more detail herein.
• angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
• a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al, Biotech. 9:630-634 (1991); Folkman et al, N Engl. J. Med., 333: 1757-1763 (1995); Auerbach et al, J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol 94:715-743 (1982); and Folkman et al, Science 221:719-725 (1983).
• the present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention.
• Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
• the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention.
• a polynucleotide, polypeptide, antagonists and/or agonist of the invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor.
• Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium. kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias.
• polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
• polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration.
• Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter.
• the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
• Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis.
• disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations
• methods for treating hypertrophic scars and keloids comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.
• polynucleotides, polypeptides, antagonists and/or agonists are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions.
• This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.
• the present invention also provides methods for treating neovascular diseases of the eye, including for example, comeal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
• neovascular diseases of the eye including for example, comeal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
• Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, comeal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al, Am. J. Ophthal ⁇ 5:704- 710 (1978) and Gartner et al, Surv. Ophthal. 22:291-312 (1978).
• neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization)
• corneal neovascularization including corneal graft neovascularization
• a compound as described above
• the cornea is a tissue which normally lacks blood vessels.
• capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus.
• the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates.
• corneal neovascularization A wide variety of disorders can result in corneal neovascularization, including for example, comeal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali bums, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
• comeal infections e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis
• immunological processes e.g., graft rejection and Stevens-Johnson's syndrome
• alkali bums e.g., trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
• saline may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form.
• the solution or suspension may be prepared in its pure form and administered several times daily.
• anti-angiogenic compositions prepared as described above, may also be administered directly to the cornea.
• the anti-angiogenic composition is prepared with a muco- adhesive polymer which binds to cornea.
• the anti- angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy.
• Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
• the compounds described above may be injected directly into the comeal stroma by an ophthalmologist under microscopic guidance.
• the prefe ⁇ ed site of injection may vary with the mo ⁇ hology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic comeal injection to "protect" the cornea from the advancing blood vessels. This method may also be utilized shortly after a comeal insult in order to prophylactically prevent corneal neovascularization.
• neovascular glaucoma comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma.
• the compound may be implanted by injection into the region of the anterior chamber angle.
• the compound may also be placed in any location such that the compound is continuously released into the aqueous humor.
• methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
• proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina.
• this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
• methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically, via intravitreous injection and/or via intraocular implants.
• disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures. Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• disorders and/or states which can be treated with be treated with the the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, solid tumors, blood bom tumors such as leukemias.
• tumor metastasis Kaposi's sarcoma
• benign tumors for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, comeal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis.
• an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method.
• Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
• Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be inco ⁇ orated into surgical sutures in order to prevent stitch granulomas.
• compositions in the form of, for example, a spray or film
• a compositions may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues.
• compositions e.g., in the form of a spray
• surgical meshes which have been coated with anti- angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized.
• a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti- angiogenic factor.
• methods for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recu ⁇ ence of cancer and the formation of new blood vessels at the site is inhibited.
• the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound).
• the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration.
• the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
• polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors.
• anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
• polypeptides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors.
• anti-angiogenic factors include: Anti-Invasive
• Lighter "d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
• vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes.
• Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate.
• Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
• tungsten and molybdenum complexes also include oxo complexes.
• Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes.
• Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid.
• Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide.
• Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes.
• Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates.
• Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid.
• Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate.
• Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
• anti-angiogenic factors include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res.
• SP- PG Sulphated Polysaccharide Peptidoglycan Complex
• the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate
• Staurosporine modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L- 3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.

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