WO2000055171A1 - 50 human secreted proteins - Google Patents

50 human secreted proteins Download PDF

Info

Publication number
WO2000055171A1
WO2000055171A1 PCT/US2000/006043 US0006043W WO0055171A1 WO 2000055171 A1 WO2000055171 A1 WO 2000055171A1 US 0006043 W US0006043 W US 0006043W WO 0055171 A1 WO0055171 A1 WO 0055171A1
Authority
WO
WIPO (PCT)
Prior art keywords
human
seq
sequence
polypeptide
soares
Prior art date
Application number
PCT/US2000/006043
Other languages
French (fr)
Inventor
Craig A. Rosen
Steven M. Ruben
George Komatsoulis
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP00913810A priority Critical patent/EP1165587A4/en
Priority to JP2000605599A priority patent/JP2002538807A/en
Priority to CA002364549A priority patent/CA2364549A1/en
Priority to AU35181/00A priority patent/AU3518100A/en
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Publication of WO2000055171A1 publication Critical patent/WO2000055171A1/en
Priority to US10/105,299 priority patent/US7368527B2/en
Priority to US10/472,965 priority patent/US20070026454A1/en
Priority to US10/472,964 priority patent/US20070032414A1/en
Priority to US10/670,185 priority patent/US20070015162A1/en
Priority to US10/670,186 priority patent/US20070031842A1/en
Priority to US10/868,184 priority patent/US20070048818A1/en
Priority to US10/994,608 priority patent/US20070048297A1/en
Priority to US11/781,665 priority patent/US20070298491A1/en
Priority to US12/753,401 priority patent/US8410248B2/en
Priority to US13/848,789 priority patent/US20130203164A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

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 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.
  • polynucleotides of the invention comprise a poition of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron
  • the polynucleotides comp ⁇ sing coding sequences do not contain coding sequences of a genomic flanking gene (I e , 5' oi 3' to the gene of interest in the genome)
  • 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)
  • 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 Numbei The ATCC is located at 10801 Umveisity Boulevaid, Manassas Virginia 201 10-2209, USA
  • the ATCC deposit was made pursuant to the terms of the Budapest Treaty on the mtemational lecogmtion of the deposit of microorganisms for pin poses of patent piocedure
  • 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 withm the clone deposited with the ATCC "
  • 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 desc ⁇ bed 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 ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched , for example, as a result of ubiquitination and they may be cyclic with 01 without branching C ⁇ cl ⁇ c branched and
  • 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
  • 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 ammo acids in the low complexity region have been replaced with the character "X"
  • Query and/or "Q”
  • this regime helps to ensure that high-scoring matches represent true homology, there is a negative consequence in that the BLASTX piogram uses the query sequence that has been masked by the filters to diaw 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
  • the reference sequence(s) (sometimes referred to herein as "Subject”, “Sbjct", and/or "S) indicated in the specification, sequence table (Table 1), and/or the deposited clone is (are) the definitive embod ⁇ ment(s) of the present invention, and should not be construed as limiting the piesent invention to the partial sequence shown in an alignment, unless specifically noted otherwise herein
  • 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 ⁇ !288145 (all information available through the recited accession number is incorporated herein by reference) A partial alignment demonstrating the observed homology is shown immediately below
  • Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO 1 12 and/or SEQ ID NO 114 which correspond to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are. of course, removed) It has been discovered that this gene is expressed p ⁇ maiily in the following tissues/cDNA libraries Human Whole Six Week Old Embryo and to a lesser extent in Early Stage Human Lung, subtracted, Human Whole Brain #2 - Oligo dT > 1 5Kb, Jurkat T-Cell, S phase. Human Manic Depiession Tissue Human Fetal Kidney, human ovarian cancel.
  • EST sequences are publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 1 1 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 compiising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2506 of SEQ ID NO 1 1, b is an integer of 15 to 2520, where both a and b correspond to the positions of nucleot
  • Preferred 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 the sequence by the computer are, of course, removed).
  • Preferred epitopes include those comprising a sequence shown in SEQ ID NO 63 as residues Arg-24 to Asn 39, Se ⁇ -43 to Pro-61
  • 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 ⁇ l4106549 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "neuional-specific septin"
  • a partial alignment demonstrating the observed homology is shown immediately below.
  • Soares_pregnant_uterus_NbHPU Stratagene NT2 neuronal precursor 937230, Liver, Hepatoma, Human Activated Monocytes, Ulcerative Colitis, Human Fetal Brain, Soares breast 2NbHBst, T Cell helper I, Human Eosinophils, Dendritic cells, pooled, Colon Tumor II, Human Synovial Sarcoma, human tonsils, Hodgkin's Lymphoma II, Soares fetal liver spleen INFLS, K562 cells, Human left hemisphere, adult, Kidney cancel, Activated T-Cells, 12 hrs, subtracted, Brain Amygdala Depression, Human Thymus Tumor, subtracted, Werzmann Olfactory Epithelium, Human OB HOS treated (10 nM E2) fraction I, Human Ceiebellum subtracted Adipocytes ie excision, Human Pituitaiy.
  • SIHPRDKQ Sbjct 401 SIHPRDKQ 408
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO:20 amino acid sequences
  • amino acid sequences are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope 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 1977 of SEQ ID NO:20, b is an integer of 15 to 1991 , where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a + 14.
  • the segment of g ⁇ l29486 that is shown as "Sbjct" above is set out as sequence(s) 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 and described elsewhere herein. Assays for determining such activities are also known in the art some of which have been described elsewhere herein.
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO 28 is publicly available and accessible through sequence databases
  • a-b is any integer between 1 to 2353 of SEQ ID NO 28
  • b is an integer of 15 to 2367
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 28, and where b is greater than or equal to a + 14
  • polynucleotide sequences such as EST 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 i elated sequence would be cumbersome Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence desc ⁇ bed by the general formula of a-b, where a is any integer between 1 to 3773 of SEQ ID NO 31, b is an integer of 15 to 3787, 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 FEATURES OF PROTEIN ENCODED BY GENE NO: 22
  • polypeptides of the invention comp ⁇ se a polypeptide having the amino acid sequence set out as SEQ ID NO. 142 which corresponds to the Queiy sequence in the alignment shown above (gaps introduced in the sequence by the computer are. of couise. removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No. 22 in the Sequence listing).
  • polynucleotide sequences such as EST sequences
  • EST sequences are publicly available and accessible through sequence databases.
  • Some of ⁇ ese sequences are related to SEQ ID NO:32 and may have been publicly available prior to conception of the present invention.
  • such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
  • a-b is any integer between 1 to 2579 of SEQ ID NO:32
  • 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:32, 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 1906 of SEQ ID NO:33, b is an integer of 15 to 1920, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a + 14.
  • Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 85 as residues: Gln-1 to Asp-6.
  • 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. gnllPIDIel31 1091 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as "ubiquitin-conjugating enzyme".
  • ubiquitin-conjugating enzyme A partial alignment demonstrating the observed homology is shown immediately below. >gnl I PID j el311091 ubiquitin-conjugating enzyme [Mas musculus] >sp
  • the segment of gnllPIDIe 131 1091 that is shown as Sbjct" above is set out as SEQ ID NO 147 Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities, e g , ubiquitin conjugating enzyme activity Such activities are known in the ait and described elsewhere herein Assays for determining such activities are also known in the art some of which have been described elsewhere herein
  • T-Cell (12hs)/Th ⁇ ou ⁇ d ⁇ ne labelledEco, Soa ⁇ es_fetal_lung_NbHL19W, CD34 positive cells (Cord Blood), HUMAN B CELL LYMPHOMA, Human Testes, Osteoblasts, Human 8 Week Whole Embryo, Soares_mult ⁇ ple_scleros ⁇ s_2NbHMSP, Soares_pregnant_uterus_NbHPU, Human Adult Lymph Node, Rectum normal, Messangial cell, frac 1.
  • Activated T-Cells 8 hrs., WI 38 cells, Human Prostate BPH.
  • EGRl Early growth response 1
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO 39 is related to SEQ ID NO 39 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 2100 of SEQ ID NO 39
  • b is an integer of 15 to 21 14, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO.39. and where b is greater than or equal to a + 14.
  • Pieferred polypeptides of the invention comp ⁇ se a polypeptide having the amino acid sequence set out as SEQ ID NO 156 which co ⁇ esponds to the Queiy sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed)
  • 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.
  • Soares breast 2NbHBst and to a lesser extent in Soares_multiple_sclerosis_2NbHMSP, Bone Marrow Cell Line (RS4; 11), Human Aortic Endothelium, Human Adipose Tissue, re-excision, Soares_fetal_heart_NbHH19W, and Activated Human Neutrophil.
  • Piefe ⁇ ed epitopes include those compiising a sequence shown in SEQ ID NO 102 as ⁇ es ⁇ dues Se ⁇ -49 to Lys-59
  • polynucleotide sequences such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 52 and may have been publicly available pnoi to conception of the present invention Pieferably such related polynucleotides are specifically excluded from the scope of the piesent invention To list every related sequence would be cumbersome Accoidingly, preferably excluded from the present invention are one oi more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 827 of SEQ ID NO 52, b is an integei of 15 to 841 where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 52, and where b is greater than or equal to a + 14
  • polynucleotide sequences such as EST sequences
  • SEQ ID NO 53 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 952 of SEQ ID NO 53
  • b is an integer of 15 to 966
  • both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 53
  • b is greater than or equal to a + 14 FEATURES OF PROTEIN ENCODED BY GENE NO: 44
  • polynucleotide sequences such as EST sequences, are publicly available and accessible through sequence databases 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 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 825 of SEQ ID NO 56, b is an integer of 15 to 839, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 56, and where b is greater than or equal to a + 14
  • the computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-hmitmg example, the sequence accessible through the following database accession no gil l 710216 (all information available through the recited accession number is inco ⁇ orated herein by reference) which is described therein as ' unknown' A paitial alignment demonstrating the observed homology is shown immediately below
  • the cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No Z and Date " Some of the deposits contain multiple different clones corresponding to the same gene "Vector' refers to the type of vector contained in the cDNA Clone ID
  • SEQ ID NO X is useful foi designing nucleic acid hybudization probes that will detect nucleic acid sequences contained in SEQ ID NO X or the cDNA contained in the deposited clone These piobes 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 errois The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid
  • polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below) It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production
  • the 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.
  • 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 present invention provides secreted polypeptides having a sequence shown in SEQ ID NO Y which have an N-terminus beginning withm 5 residues (1 e , + or - 5 residues) of the predicted cleavage point Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secieted species
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ
  • 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.
  • 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.
  • deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
  • a preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence 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.
  • polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • polypeptide fragments of SEQ ID NO Y falling within conserved domains are specifically contemplated by the piesent invention Moreovei, poly nucleotides encoding these domams aie also contemplated
  • Antibodies of the present in ention may be described or specified in terms of the ep ⁇ tope(s) 01 po ⁇ t ⁇ on(s) of a poly peptide of the piesent invention w hich they recognize or specifically bind
  • the ep ⁇ tope(s) or polypeptide port ⁇ on(s ) may be specified as described herein, e g , by N-terminal and C-terminal positions, by size in contiguous amino acid lesidues, or listed in the Tables and Figuies Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded Therefoie, the present inv ention includes antibodies that specifically bind polypeptides of the present invention, and allows for
  • 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 CH 1 domain of the heavy chain
  • 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
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324, Mullinax et al , BioTechmques 12(6) 864-869 (1992), and Sawai et al , AJRI 34 26- 34 (1995), and Better et al , Science 240 1041-1043 (1988) (said references incorporated by reference in their entireties)
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promotei
  • the expiession of double-chained antibodies vectors encoding both the heavy and light chains may be co expressed in the host cell for expiession of the entne immunoglobulin molecule as detailed below
  • 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 entne inseit These exogenous translational control signals and initiation codons can be of a vanety of origins, both natuial and synthetic The efficiency of expiession may be enhanced by the inclusion of appiopriate transcription enhancer elements transcription teiminators etc (see Bittner et al , Methods in Enzymol 153 51-544 ( 1987))
  • 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
  • these techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (I e minimal residual disease (MRD) in acute leukemic patients) and "non-self cells in transplantations to prevent Graft-versus-Host Disease (GVHD)
  • I e minimal residual disease (MRD) in acute leukemic patients
  • GVHD Graft-versus-Host Disease
  • these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood
  • the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitio, then transplanted into the patient
  • direct in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors
  • indirect in which case, cells are first transformed with the nucleic acids in vitio, then transplanted into the patient
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection Usually, the method of transfer includes the transfer of a selectable marker to the cells The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene Those cells are then delivered to a patient
  • Cells mto which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, various stem or progenitor cells, in particular hematopoietic stem oi progenitor cells, e g , as obtained from bone marrow, umbilical cord blood, pe ⁇ pheial 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 Clime 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 approp ⁇ ate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity
  • 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/oi tissue sample can be determined utilizing techniques known to those of skill in the ait 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 otheiwise 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 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 controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC C ⁇ t. 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, supia, vol 2, pp 1 15-138 (1984))
  • 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.
  • 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.
  • 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 backgiound level indicates that the subject has a particular disease or disorder associated with aberrant expiession of the polypeptide of interest Background level can be determined by va ⁇ ous methods including, comparing the amount of labeled molecule detected to a standard value previously determined foi a particular system
  • 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
  • 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.
  • 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, 9131 1), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • 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 ⁇ bosome 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.
  • 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 Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated piocesses Thus, it is well known in the art that the N-teiminal methionine encoded by the translation initiation codon geneially is removed with high efficiency from any piotein after translation in all eukaryotic cells While the N terminal methionine on most proteins also is efficiently iemoved in
  • 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.
  • 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, homodimers 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 heterologous 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.
  • Trime ⁇ c polypeptides of the invention may offer the advantage of enhanced biological activity
  • Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form t ⁇ mers
  • One example is a leucine zipper derived from lung surfactant protem 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 de ⁇ ved from naturally occurring trime ⁇ c proteins may be employed in preparing trime ⁇ c polypeptides of the invention
  • multimers of the invention may be geneiated using techniques known in the ait to form one or more mter-molecule cioss-hnks between the cy steme 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 heiein incorporated by reference in its entirety)
  • polypeptides of the invention may be routinely modified by the addition of cysteine oi biotin to the C terminus oi 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 incorporated 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
  • 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 hgating 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
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO X Pnmeis can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes Only those hybrids containing the human gene corresponding to the SEQ ID NO X will yield an amplified fragment
  • 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.
  • 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),
  • reagents capable of identifying the souice of a particular tissue
  • Appropriate reagents can comp ⁇ se, 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 ohgomers 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 protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 1311, 1 12In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or mtraperitoneally) into the mammal.
  • an appropriate detectable imaging moiety such as a radioisotope (for example, 1311, 1 12In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S.W. Burchiel et al., "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 presence of a relatively high amount of ti nsc ⁇ pt in biopsied tissue fiom an individual may indicate a predisposition foi the development of the disease 01 may piovide a means for detecting the disease prior to the appearance of actual clinical symptoms
  • a more definitive diagnosis of this type may allow health pio professionals to employ pieventative measures or aggressive treatment earlier thereby preventing the development oi fuither progiession of the cancer
  • Another aspect of the present invention is to gene theiapy methods foi tieating disordei s, diseases and conditions
  • the gene therapy methods ielate 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 promotei and any othei genetic elements necessary for the expression of the polypeptide by the target tissue
  • Such gene therapy and dehveiy techniques are known in the art, see. foi example, WO90/11092, which is heiem incorporated by reference
  • Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccha ⁇ de matnx among the leticular fibeis of oigan tissues elastic tibeis in the walls of vessels 01 chambers collagen fibeis of fibrous tissues or that same matnx within connective tissue ensheathmg muscle cells or in the lacunae of bone It is similaily the space occupied by the plasma of the cnculation and the lymph fluid of the lymphatic channels Delivery to the interstitial space of muscle tissue is prefened for the reasons discussed below They may be convementlv delivered by injection into the tissues comprising these cells They aie preferably delivered to and expressed in persistent, non dividing cells which aie diffeientiated although delivery and expression may be achieved in non-diffeientiated or less completely differentiated cells, such as for example, stem cells of blood or skin fibroblasts In xtvo muscle cells aie particularly competent in their ability to take up and express polynucleotides
  • 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.
  • 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
  • DOPG/DOPC vesicles can be prepaied by drying 50 mg each of DOPG and DOPC under a sitesam of nitrogen gas into a sonication vial The sample is placed under a vacuum pump overnight and is hydiated the following day with deiomzed water The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 somcator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucle
  • the liposomes can comprise multilamellar vesicles (MLVs), small umlamellar vesicles (SUVs), oi large umlamellar vesicles (LUVs), with SUVs being preferred
  • MLVs multilamellar vesicles
  • SUVs small umlamellar vesicles
  • LUVs oi large umlamellar vesicles
  • SUVs being preferred
  • the various hposome-nucleic acid complexes are prepared using methods well known in the art See, e g .
  • MLVs containing nucleic acid can be prepared by depositing a thm film of phosphohpid 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 umlamellar 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 T ⁇ s/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
  • the ratio of DNA to liposomes will be from about 10: 1 to about 1: 10.
  • the ration will be from about 5: 1 to about 1 :5. More preferably, the ration will be about 3: 1 to about 1 :3. Still more preferably, the ratio will be about 1 : 1.
  • U.S. Patent NO: 5,676,954 (which is herein inco ⁇ orated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
  • WO 94/9469 (which are herein inco ⁇ orated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.
  • U.S. Patent Nos. 5,589,466, 5,693.622, 5,580,859, 5.703,055, and international publication NO: WO 94/9469 (which are herein incorporated 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+env Am 12, 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 ait. Such means include, but aie not limited to.
  • the letiovnal plasmid vector may be encapsulated into a liposome. or coupled to a lipid, and then administered to a host.
  • the producei cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention.
  • Such letroviral vectoi particles then may be employed, to transduce eukaryotic cells, eithei in vitro or in vivo. The transduced eukaryotic cells will expiess polypeptides of the invention.
  • 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 adenovnus (e g Ad3 Ad5 and Ad7) aie also useful in the piesent invention
  • 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
  • Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses
  • This method involves the activation of a gene which is piesent in the target cells but which is not normally expressed in the cells, or is expressed at a lower level than desired
  • Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the piomoter 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
  • 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.
  • hyperprohferative 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 (cential and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogemtal
  • the viral vector is defective and will not transform non-prohfeiating cells, only proliferating cells
  • the polynucleotides of the present invention inserted mto 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 oncogemc genes or antigens By repressing expression of the oncogenic genes " is intended the
  • cell prohferative 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.
  • the piesent invention is fuither dnected to antibody-based theiapies which involve administe ⁇ ng of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, piefei ably human patient foi tieatmg one 01 more of the desciibed disorders
  • Methods for pioducmg anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewheie herein
  • Such antibodies may be provided in phaimaceutically acceptable compositions as known in the ait or as desciibed 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) oi by effector cells (ADCC)
  • CDC complement
  • ADCC effector cells
  • tumor-specific cells such as tumor-associated macrophages
  • Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis dnectly, oi indirectly (See Witte L, et al , Cancer Metastasis Rev 17(2) 155-61 ( 1998), which is hereby incorporated by reference))
  • Polypeptides including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting prohferative cells or tissues through the induction of apoptosis
  • Said polypeptides may act either directly, or indirectly to induce apoptosis of prohferative cells and tissues, for example in the activation of a death- domain receptor, such as tumor necrosis factor (TNF) recepto ⁇ -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 incorporated by reference)
  • TNF tumor necrosis factor
  • TRAMP TNF-receptor-related apoptosis-mediated protein
  • TRAIL TNF-related apoptos
  • the invention provides a method of delivering compositions containing the polypeptides of the invention (e g , compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention
  • compositions containing the polypeptides of the invention e g , compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs
  • Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydiophihc, ionic and/or covalent interactions
  • Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the lmmunogenicity and/or antigemcity of
  • Cardiovascular disordeis also include heait disease, such as a ⁇ hythmias, carcinoid heait disease, high cardiac output low caidiac output, cardiac tamponade endocarditis (including bacterial) heait aneuiysm, caidiac arrest congestive heait failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heait hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypei trophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocaidial ischemia, pe ⁇ cardial effusion, pericarditis (including const ⁇ ctive and tuberculous), pneumope ⁇ cardium, postpe ⁇ cardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitai Syndrome, cardiovascular syphilis, and cardiovascular
  • Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, t ⁇ cuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, t ⁇ cuspid atresia, t ⁇ cuspid valve insufficiency, and t ⁇ cuspid valve stenosis
  • 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
  • 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.
  • 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 senous diseases aie dominated by abnormal neovascularization including solid tumoi growth and metastases, aith ⁇ tis, 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).
  • 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
  • neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization) comprising the step of administering to a patient a therapeutically effective amount of a compound (as desc ⁇ bed above) to the cornea, such that the formation of blood vessels is inhibited
  • 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 vascula ⁇ zed, 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 infections e g , trachoma, herpes simplex keratitis, leishmamasis and onchocercmsis
  • immunological processes e.g
  • the invention 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 admimsteied seveial times daily
  • anti-angiogenic compositions prepaied as desc ⁇ bed above, may also be admimsteied dnectly to the coinea
  • the anti-angiogenic composition is prepared with a muco- adhesive polymer which binds to cornea
  • the anti- angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy
  • Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns) In these instances the tieatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications
  • the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance
  • the preferred site of injection may vary with the 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 (l e , interspersed between the blood vessels and the normal cornea) In most cases this would involve pe ⁇ hmbic corneal injection to "protect" the cornea from the advancing blood vessels This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization
  • the material could be injected in the pe ⁇ limbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply
  • Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas In a sustained-release form injections might only be required 2-
  • prohferative 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 retma
  • this tieatment should be initiated prior to the acquisition of severe disease requiring photocoagulation
  • methods are provided foi treating letrolental 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, atheroscleiotic plaques, delayed wound healing, granulations, hemophihc joints, hypertrophic scars, nonunion fractuies, Osier- Weber syndrome, pyogemc 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 born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogemc granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), non
  • 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 aftei 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
  • agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas
  • 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
  • the polynucleotides, 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 Factor, retinoic acid and derivatives thereof, pachtaxel, Suramm, Tissue Inhibitor of Metalloprote ⁇ nase-1 , Tissue Inhibitor of Metalloprote ⁇ nase-2, Plasminogen Activator Inhibitor- 1, Plasminogen Activator Inh ⁇ b ⁇ tor-2, and various forms of the lighter "d group” transition metals
  • 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. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (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-.
  • cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as.
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
  • Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas
  • leukemia including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloc
  • Diseases associated with increased apoptosis that could be treated or detected by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS, neurodegenerative disoiders (such as Alzheimer s disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retimtis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease), autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet' s disease Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e g
  • polynucleotides or polypeptides, and/or agonists or antagonists of the invention for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds
  • Polynucleotides 01 polypeptides as well as agonists or antagonists of the invention may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oial cavity wounds, diabetic ulceis, dermal ulceis, cubitus ulcers, arterial ulceis venous stasis ulceis, burns resulting trom heat exposure or chemicals, and other abnormal wound healing conditions such as uremia malnutrition, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation
  • polynucleotides or polypeptides, and/or agonists or antagonists of the invention could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithehalization from the wound bed
  • the following are a non- exhaustive list of grafts that polynucleotides or polypeptides agonists or antagonists of the invention, could be used to increase adherence to a wound bed autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair- Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hype ⁇ lastic graft, lamellar graft, mesh graft, mucosal
  • the polynucleotides 01 polypeptides, and/oi agonists 01 antagonists of the invention could also be used to reduce the side effects of gut toxicity that lesult from radiation, chemotherapy tieatments oi vnal infections
  • the polynucleotides or polypeptides, and/or agonists oi antagonists of the invention may have a cytoprotective effect on the small intestine mucosa
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention may also stimulate healing of mucositis (mouth ulcers) that lesult from chemotherapy and viral infections
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention could further be used in full regeneration of skm in full and partial thickness skin defects, including burns, (I e , repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithehalization of these lesions
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal
  • the polynucleotides oi polypeptides. and/or agonists or antagonists of the invention could be used to pievent and heal damage to the lungs due to various pathological states
  • a giowth factor such as the polynucleotides or polypeptides and/or agonists or antagonists of the invention, which could stimulate prohfeiation and diffeientiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chionic lung damage
  • emphysema which results in the piogressive loss of aveoh, and inhalation injuries, I e , resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using the polynucleotides or polypeptides and/or agonists or antagonists of the invention
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention could be effectively treated
  • the polynucleotides or polypeptides, and/or agonists or antagonists of the invention could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (I e , acetaminophen, carbon tetraholo ⁇ de and other hepatotoxins known in the art)
  • liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (I e , acetaminophen, carbon tetraholo ⁇ de and other hepatotoxins known in the art)
  • polynucleotides or polypeptides, and/or agonists or antagonists of the invention could be used treat or prevent the onset of diabetes melhtus In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function
  • Nervous system disorders which can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to. nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • compositions of the invention e.g., polypeptides, polynucleotides, and/or agonists or antagonists
  • Nervous system disorders include, but are not limited to. nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: ( 1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zo
  • the compositions of the invention are used to treat or prevent neural cell mjuiy associated with DCebral hypoxia
  • the polypeptides, polynucleotides, or agonists 01 antagonists of the invention are used to treat or pievent neural cell injury associated with cerebral ischemia
  • the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke
  • the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke
  • compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the spanval or differentiation of neurons
  • compositions of the invention which elicit any of the following effects may be useful according to the invention (1) increased survival time of neurons in culture, (2) increased sprouting of neurons in culture or in vivo, (3) increased production of a neuron-associated molecule in culture or in vivo, e g , choline acetyltransferase or acetylchohnesterase with respect to motor neurons, or (4) decreased symptoms of neuron dysfunction in vivo
  • increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al (J Neurosci 10.3507-3515 ( 1990)), increased sprouting of neurons may be detected by methods known in the art, such as
  • motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
  • disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar
  • a polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention.
  • viruses include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue. EBV, HIV.
  • Herpes Zoster Herpes Zoster. Mononegavirus (e.g., Paramyxoviridae. Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.. Influenza A, Influenza B, and parainfluenza). Papiloma virus. Papovaviridae. Parvoviridae. Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II. Lentivirus). and Togaviridae (e.g., Rubivirus).
  • Mononegavirus e.g., Paramyxoviridae. Morbillivirus, Rhabdoviridae
  • Orthomyxoviridae e.g... Influenza A, Influenza B, and parainfluenza
  • Papiloma virus Papovaviridae. Par
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g.. conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active. Delta), Japanese B encephalitis. Junin, Chikungunya. Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia.
  • polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat or detect any of these symptoms or diseases.
  • polynucleotides. polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B).
  • Actinomycetales e.g., Corynebacterium, Mycobacterium, Norcardia
  • Cryptococcus neoformans Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
  • Bacillaceae e.g., Anthrax, Clostridium
  • Bacteroidaceae Blastomycosis, Bordetella
  • Borrelia e.g., Borrelia burgdorferi
  • Brucellosis Candidiasis
  • Campylobacter e.g., Borrelia burgdorferi
  • Brucellosis Candidiasis
  • Vibrio cholerae Neisse ⁇ aceae (e g Acinetobactei Gonoirhea, Menigococcal), Meisseria meningitidis, Pasteuiellacea Infections (e g , Actinobacillus, Heamophilus (e g , Heamophilus influenza type B) Pasteurella) Pseudomonas Ricketts ceae, Chlamydiaceae, Syphilis Shigella spp Staphylococcal Meningiococcal, Pneumococcal and Streptococcal (e g , Streptococcus pneumoniae and Group B Streptococcus)
  • These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to bacteremia, endocarditis eye infections
  • gingivitis opportunistic infections e g , AIDS related infections
  • paronych piosthesis-related infections
  • Reiter's Disease respiratory tract infections, such as Whooping Cough or Empyema sepsis, Lyme Disease.
  • Cat-Scratch Disease Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e g , mengitis types A and B),
  • parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class Amebiasis. Babesiosis, Coccidiosis, Cryptospo ⁇ diosis, Dientamoebiasis, Dou ⁇ ne, Ectoparasitic, Gmrdiasis, Helminthiasis, Leishmaniasis, Theile ⁇ asis Toxoplasmosis, Trypanosomiasis, and T ⁇ chomonas and Sporozoans (e g , Plasmodium virax, Plasmodium falcipa ⁇ um, Plasmodium mala ⁇ ae and Plasmodium ovale) These parasites can cause a variety of diseases or symptoms, including, but not limited to Scabies, Trombicuhasis, eye infections, intestinal disease (e g , dysentery, gmrdiasis), liver
  • treatment using a polypeptide or polynucleotide and/or agonist oi antagonist of the piesent invention could eithei be by admmiste ⁇ ng an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and leturnmg the engineered cells to the patient (ex vivo therapy)
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease
  • Tissues that could be regenerated using the present invention include organs (e g , pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue
  • organs e g , pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vasculature including vascular and lymphatics
  • nervous hematopoietic
  • hematopoietic e.g vascular and lymphatics
  • a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal For example, increased tendon/ligament regeneration would quicken recovery time after damage
  • a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage
  • Specific diseases that could be treated include of tendinitis, ca ⁇ al tunnel syndrome, and other tendon or ligament defects.
  • a further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke).
  • a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hype ⁇ roliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells.
  • These chemotactic molecules can then be used to treat inflammation, infection, hype ⁇ roliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body.
  • chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location.
  • Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
  • a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic.
  • the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site).
  • the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
  • Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
  • the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtuies
  • the assay may also simplv compnse the steps of mixing a candidate compound with a solution containing a polypeptide measu ⁇ ng poly peptide/molecule activity oi binding and comparing the polypeptide/molecule activity 01 binding to a standard
  • an ELISA assay can measuie polypeptide level or activity in a sample (e g , biological sample) using a monoclonal or polyclonal antibody
  • the antibody can measuie polypeptide level oi activity by either binding directly or indirectly to the polypeptide or by competing with the polypeptide foi a substrate
  • alteration of polynucleotides and conesponding polypeptides of the invention may be achieved by DNA shuffling
  • DNA shuffling involves the assembly of two or moie DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide
  • the biological activity of the fiagments may include an impioved desned activity, or a decreased undesirable activity
  • this invention piovides a method of screening compounds to identify those which modulate the action of the polypeptide of the piesent invention
  • An example of such an assay comp ⁇ ses combining a mammalian fibroblast cell, a the polypeptide of the present invention the compound to be screened and 3[H] thymidine undei cell cultuie conditions where the fibroblast cell would normally proliferate
  • a control assay may be performed in the absence of the compound to be screened and compared to the amount of fibioblast proliferation in thesheetnce of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case
  • the amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the inco ⁇ oration of 3[H] thymidine
  • Both agonist and antagonist compounds may be identified by this procedure
  • a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with
  • the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of (a) incubating a candidate binding compound with the polypeptide, and (b) determining if binding has occu ⁇ ed Moieover, the invention includes a method of identifying agonists/antagonists compiising the steps of (a) incubating a candidate compound with the polypeptide (b) assaying a biological activity , and (b) determining if biological activity of the polypeptide has been alteied Also, one could identify molecules bind a polypeptide of the invention expe ⁇ mentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the
  • the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention
  • polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions
  • the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids
  • the invention provides a method for delivering a therapeutic protein into the targeted cell
  • the invention provides a method for delivering a single stranded nucleic acid (e g , antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
  • a single stranded nucleic acid e g , antisense or ribozymes
  • double stranded nucleic acid e.g., DNA that can integrate
  • toxin compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
  • Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
  • radioisotopes known in the art
  • compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
  • Drug Screening Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
  • One method of diug scieening utilizes eukaryotic oi prokaiyotic host cells which are stably tiansformed with recombinant nucleic acids expressing the polypeptide oi fragment Diugs aie scieened against such tiansformed cells in competitive binding assays
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on September 13, 1984, which is incorporated herein by reference herein Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface The peptide test compounds are reacted with polypeptides of the present invention and washed Bound polypeptides are then detected by methods well known in the art Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support
  • antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone.
  • antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988).
  • Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation.
  • Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241 :456 (1988); and Dervan et al., Science, 251: 1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRI site on the 5 end and a Hindlll site on the 3 end.
  • the pair of oligonucleotides is heated at 90°C for one minute and then annealed in 2X ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl/Hind III site of the retroviral vector PMV7 (WO 91/15580).
  • 2X ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
  • the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the antisense nucleic acid of the invention.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells.
  • Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells.
  • promoters can be inducible or constitutive.
  • Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 ( 1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.. 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contarn and still foim a stable duplex (oi triplex as the case may be)
  • One skilled in the ait can ascertain a tolerable degiee of mismatch by use of standaid procedures to deteimine the melting point of the hyb ⁇ dized complex
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybudization t ⁇ ggeied cross-linking agent, tianspoit agent hybudization t ⁇ ggeied cleavage agent, etc
  • the antisense oligonucleotide may comp ⁇ se at least one modified base moiety which is selected from the gioup including, but not limited to. 5-fluo ⁇ ourac ⁇ l, 5-bromourac ⁇ l, 5-chlorourac ⁇ l, 5- ⁇ odourac ⁇ l, hypoxanthine xantine 4-acetylcytos ⁇ ne, 5-(carboxyhyd ⁇ oxylmethyl) uracil 5-carboxymethylam ⁇ nomethyl-2-th ⁇ ou ⁇ d ⁇ ne, 5-carboxymethylammomethylurac ⁇ l dihydrouracil, beta-D-galactosylqueosine, inosine, N6- ⁇ sopentenyladen ⁇ ne, 1-methylguan ⁇ ne, 1-methyhnos ⁇ ne 2,2-d ⁇ methylguamne, 2-methyladen ⁇ ne, 2-methylguan ⁇ ne, 3-methylcytos ⁇ ne, 5-methylcytos ⁇ ne, N6-aden ⁇ ne, 7-methyl
  • the antisense oligonucleotide may also comp ⁇ se at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarab ⁇ nose, xylulose, and hexose
  • the antisense oligonucleotide is an a-anome ⁇ c oligonucleotide
  • An a-anome ⁇ c oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al , Nucl Acids Res , 15 6625-6641 (1987))
  • the oligonucleotide is a 2-0-methyl ⁇ bonucleot ⁇ de (Inoue et al , Nucl Acids Res , 15 6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al , FEBS Lett 215 327-330 (1987))
  • Polynucleotides of the invention may be synthesized by standaid methods known in the ait e g by use of an automated DNA synthesizei (such as aie commeicially available
  • the ribozymes of the invention can be composed of modified oligonucleotides (e g for improved stability, targeting, etc ) and should be delivered to cells which express the polynucleotides of the invention in vivo DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme undei the contiol of a strong constitutive promotei, such as, for example pol III 01 pol II promoter, so that transfected cells wrll produce sufficient quantities of the ribozyme to destioy endogenous messages and inhibit translation Since ribozymes unlike antisense molecules are catalytic, a lower intiacellular concentiation is iequned foi efficiency
  • Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the piesent invention on neoplastic cells and tissues, I e stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular giowth and piohferation, for example, in tumor foimation or growth
  • the antagonist/agonist may also be employed to prevent hyper-vasculai diseases, and prevent the proliferation of epithelial lens cells after extracapsulai cataract surgery Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty
  • the antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing
  • the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention
  • polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos
  • polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage
  • polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e g , cosmetic surgery)
  • polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabohsm. piocessing utilization, and storage of eneigy
  • Polypeptide or polynucleotides and/oi agonist or antagonists of the piesent invention may be used to change a mammal s mental state or physical state by influencing biorhythms, ca ⁇ cadic rhythms, depiession (including depiessive disorders), tendency for violence, tolerance for pain, reproductive capabilities

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Neurology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Cardiology (AREA)
  • Neurosurgery (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

Description

50 Human Secreted Proteins
Field ofthe Invention
This invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, uses of such polynucleotides and polypeptides. and their production.
Background ofthe Invention Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eucaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses "sorting signals," which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). 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. Here, 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. For example, 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.
Despite the great progress made in recent years, only a small number of genes encoding human secreted proteins have been identified. These secreted proteins include the commercially valuable human insulin, interferon, Factor VIII, human growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of the pervasive lole of secieted proteins in human physiology, a need exists for identifying and chaiacteπzing novel human secreted pioteins and the genes that encode them This knowledge will allow one to detect, to treat, and to pievent medical disordeis by using secreted pioteins 01 the genes that encode them
Summary ofthe Invention The piesent invention relates to novel polynucleotides and the encoded polypeptides Moreover, the present invention relates to vectois, 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 foi identifying binding partners of the polypeptides
Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain terms used throughout this specification
In the present invention, "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 For example, 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 The term "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 othei compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention In the present invention a secreted piotein refers to those proteins capable of being dnected to the ER secietory vesicles, or the extracellulai space as a result of a signal sequence as well as those pioteins released into the extracellular space without necessaπly containing a signal sequence If the secieted piotein is leleased into the extracellulai space, the secreted protem can undergo extracellular processing to produce a ' mature' protein Release into the extiacellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage
In specific embodiments, 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 In a further embodiment, polynucleotides of the invention comprise a poition of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron In another embodiment, the polynucleotides compπsing coding sequences do not contain coding sequences of a genomic flanking gene (I e , 5' oi 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)
As used herein, 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 For example, 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 Moreover, as used herein, a "polypeptide" refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined
In the present invention, 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 Numbei The ATCC is located at 10801 Umveisity Boulevaid, Manassas Virginia 201 10-2209, USA The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the mtemational lecogmtion of the deposit of microorganisms for pin poses of patent piocedure 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 withm the clone deposited with the ATCC "Stringent hybridization conditions" refers to an ovei night incubation at 42 degree C in a solution compiising 50% formamide, 5x SSC (750 mM NaCl, 75 mM tnsodium citrate), 50 mM sodium phosphate (pH 7 6), 5x Denhardt's solution, 10% dextran sulfate. and 20 μg/ml denatuied. sheared salmon sperm DNA, followed by washing the filters in 0 lx SSC at about 65 degiee C
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency), salt conditions, or temperature For example, lower stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl, 0 2M NaH,PO , 0 02M EDTA, pH 7 4), 0 5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA, followed by washes at 50 degree C with 1XSSPE, 0.1% SDS In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e g 5X SSC) Note that variations in the above conditions may be accomplished thiough the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatuied 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 Of coui se a polynucleotide which hybridizes only to polyA+ sequences (such as any 3 terminal polyA-t- tract of a cDNA shown in the sequence listing) 01 to a complementaiy stretch of T (01 U) lesidues would not be included in the definition of "polynucleotide, since such a polynucleotide would hybndize to any nucleic acid molecule containing a poly (A) stretch or the complement theieof (e g , practically any double-stranded cDNA clone geneiated using ohgo dT as a primer)
The polynucleotide of the present invention can be composed of any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA oi DNA For example, 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 mixtuie 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 In addition, the polynucleotide can be composed of tπple-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, tπtylated bases and unusual bases such as inosine A variety of modifications can be made to DNA and RNA, thus, "polynucleotide ' embraces chemically, enzymatically, or metabohcally 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 descπbed 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 ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched , for example, as a result of ubiquitination and they may be cyclic with 01 without branching C\clιc branched and branched cyclic polypeptides may lesult fiom posttranslation natuial piocesses 01 may be made by synthetic methods Modifications include acetylation acylation, ADP-πbosylation amidation covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deiivative, covalent attachment of a lipid 01 lipid derivative covalent attachment of phosphotidyhnositol cross-linking, cychzation, disulfide bond formation demethylation, foimation of covalent cross-links, formation of cysteine, formation of pyroglutamate formylation, gamma-carboxylation glycosylation GPI anchoi formation, hydroxylation, lodination, methylation, myristoylation oxidation, pegylation, proteolytic processing, phosphorylation, prenylation racemization, selenoylation sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993), POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Piess, New York, pgs 1-12 (1983), Seifter et al , Meth Enzymol 182 626-646 (1990), Rattan et al , Ann NY Acad Sci 663 48-62 (1992) )
"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 similai to the dose-dependence in a given activity as compared to the polypeptide of the present invention (l 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 )
Many proteins (and translated DNA sequences) contain regions where the ammo acid composition is highly biased toward a small subset of the available lesidues Foi example, membrane spanning domains and signal peptides (which are also membiane spanning) typically contain long stretches where Leucine (L), Vahne (V), Alanine (A), and Isoleucine (I) piedominate Poly Adenosine tracts (polyA) at the end of cDNAs appear m forwaid tianslations as poly-Lysme (poly-K) and poly- Phenylalanine (poly-F) when the reveise complement is tianslated These regions are often referred to as "low complexity ' regions
Such regions can cause database similarity search piograms 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 matπces (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
In order to compensate for this, BLASTX 2 (version 2 0a5MP-WashU) employs two filters ("seg" and "xnu") 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 ammo 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 piogram uses the query sequence that has been masked by the filters to diaw alignments
Thus, a stretch of "X"s in an alignment shown in the following application does not necessarily indicate that either the underlying DΝA 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 In all cases, the reference sequence(s) (sometimes referred to herein as "Subject", "Sbjct", and/or "S") indicated in the specification, sequence table (Table 1), and/or the deposited clone is (are) the definitive embodιment(s) of the present invention, and should not be construed as limiting the piesent invention to the partial sequence shown in an alignment, unless specifically noted otherwise herein
Polynucleotides and Polypeptides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
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 gι!288145 (all information available through the recited accession number is incorporated herein by reference) A partial alignment demonstrating the observed homology is shown immediately below
>gx I 288145 put ORF [Homo sapiens] >pιr 11380221138022 hypothetical protein - human
Length = 196
Plus Strand HSPs Score = 145 (51 0 bits) Expect = 1 3e-14 Sum P(2) = 1 3e-14 Identities = 31/45 (68%) Positives = 34/45 (75°o) Frame = +3
Query 2286 TGVQDQPGQHGETLSLQKIQKLTRHCGMCL*SQLLRRLTQENCLN 2420 +GVQD PGQHG+ SL KIQ+L H G CL SQLLRRL QEN LN Sbjct 6 SGVQDYPGQHGKIPSLLKIQELAGHGGRCLQSQLLRRLRQENHLN 50
Score = 84 (29 6 bits) Expect = 1 3e-14 Sum P(2) = 1 3e-14 Identities = 17/28 (60%) Positives = 18/28 (64%) Frame = +2 Query 2414 LEPGGGGCSELRLCHCTPAWATERDSVS 2497 L GG GCSE + C PAW TE DSVS Sb3ct 49 LNSGGRGCSEPKSHLCIPAWVTEGDSVS 76
The segments of gιl288145 that are shown as "Sbjct" above are set out as SEQ ID NO 1 1 1 and SEQ ID NO 113, respectively
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO 1 12 and/or SEQ ID NO 114 which correspond to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are. of course, removed) It has been discovered that this gene is expressed pπmaiily in the following tissues/cDNA libraries Human Whole Six Week Old Embryo and to a lesser extent in Early Stage Human Lung, subtracted, Human Whole Brain #2 - Oligo dT > 1 5Kb, Jurkat T-Cell, S phase. Human Manic Depiession Tissue Human Fetal Kidney, human ovarian cancel. Human Umbilical Vein Endothelial Cells, uninduced, Human Fetal Dura Matei , Stiatagene neuroepithehum (#937231), H Frontal cortex epileptic re-excision, Brain frontal cortex Aneigic T-cell, and Soaies infant btain 1NIB Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 1 1 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 compiising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2506 of SEQ ID NO 1 1, b is an integer of 15 to 2520, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 11. and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
The computer algorithm BLASTX has been used to determine that the tianslation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no gιl2650190 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "enoyl-CoA hydratase (fad-1)" A partial alignment demonstrating the observed homology is shown immediately below
>gι| 2650190 (AE001074) enoyl-CoA hydratase (fad-1) [Archaeoglobus fulgidus] >pιr|C69304 |C69304 enoyl-CoA hydratase (fad-1) homolog - Archaeoglobus fulgidus >sp | 029814 | 029814 ENOYL-COA HYDRATASE (FAD-1) Length = 256
Plus Strand HSPs
Score = 190 (66 9 bits) Expect = 7 8e-20 Sum P(2) = 7 8e-20 Identities = 47/156 (30%) Positives = 77/156 (49%) Frame = +3
Query 234 DNGIGILTLNNPSRMNAFSGVMMLQLLEKVIELENWTEGKGLIVRGAKNTFSSGSDLNAV 413
D I + TLN P ++NA ++L E + +E + LI+ G+ F++G+D+N + Sbjct 10 DGEIAVATLNRPEKLNALDTKTRMELAEVIEGIEE--VARVLIITGSGKAFAAGADINEL 67 Query 414 KSLGTPEDGMAVCMFMQNTLTRFMRLPLISVALVQGWALGGGAEFTTACDFRLMTPESKI 593 + + +R L + +A V G+ LGGG E ACD R+ + ++K
Sbjct: 68 LQRDAIK-AFEATKLGTDLFSRIEELEIPVIAAVNGYTLGGGCELAMACDIRIASEKAKF 126
Query: 594 RFVHKEMGIIPSWGGTTRLVEIIGSRQALK-VLSGAL 701 + IIP GGT RL ++G A K VL+G +
Sbjct: 127 GQPEINLAIIPGAGGTQRLPRLVGLGMAKKLVLTGEI 163
The segment of gil2650190 that is shown as "Sbjct" above is set out as SEQ ID NO. 1 15. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and 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. 116 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues: Soares placenta Nb2HP and to a lesser extent in Soares_NhHMPu_S 1 , Stratagene ovarian cancer (#937219), Soares melanocyte 2NbHM, Hodgkin's Lymphoma II, Messangial cell, frac 1 , Human Thyroid, Early Stage Human Lung, subtracted, Fetal Liver, subtraction II, Stratagene lung (#937210), Human Amygdala, Monocyte activated, Stratagene ovarian cancer (#937219), Keratinocyte, Colorectal Tumor, Human Adult Liver, subtracted, Al- CELL LINE, Human (HCC) cell line liver (mouse) metastasis, remake, Activated T-cells, Soares retina N2b4HR, Human adult small intestine,re-excision, NTERA2 teratocarcinoma cell line+retinoic acid ( 14 days), Human Pineal Gland, B Cell lymphoma, Soares_parathyroid_tumor_NbHPA, Synovial hypoxia, Human Ovary, T-Cell PHA 16 hrs, Apoptotic T-cell, 12 Week Old Early Stage Human, II, Spinal cord, Human Rhabdomyosarcoma, Human Adrenal Gland Tumor, NTERA2, control, Colon Tumor, 12 Week Old Early Stage Human, Smooth muscle, serum treated, H Macrophage (GM-CSF treated), re-excision, Soares_placenta_8to9weeks_2NbHP8to9W, Human Fetal Lung III, Human Testes, Reexcision, Human Fetal Heart, Human Osteoclastoma, Smooth uscle,control, Soares_fetal_heart_NbHH 19W, Soares_parathyroid_tumor_NbHPA, Human Bone Marrow, treated, Human Endometrial Tumor, Human 8 Week Whole Embryo, Soares fetal liver spleen 1NFLS, and Soares_placenta_8to9weeks_2NbHP8to9W. Pieferred epitopes include those compπsing a sequence shown in SEQ ID NO 62 as residues Lys-86 to Gly-93
Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 12 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides aie specifically excluded from the scope of the present invention To list every related sequence would be cumbersome Accordingly, preferably excluded from the piesent invention are one or more polynucleotides comprising a nucleotide sequence descπbed by the general formula of a-b, where a is any integer between 1 to 2137 of SEQ ID NO 12, b is an integer of 15 to 2151 , 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
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares adult bram N2b5HB55Y and to a lesser extent in Soares_fetalJιeaιt_NbHH19W, HUMAN B CELL LYMPHOMA. Soares_parathyroιd_tumor_NbHPA, Soares_pregnant_uterus_NbHPU, Human Endometπal Tumor. Soares fetal liver spleen 1NFLS, H Stπatum Depression, subt, Stratagene ovarian cancer (#937219), Soares_fetal_lung_NbHL19W, Soares_pregnant_uterus_NbHPU, Endothehal-induced, NCI_CGAP_Br2, NCI_CGAP_Pr2, NCI_CGAP_Kιd3, Soares_fetal_heart_NbHH19W, HUman Fetal Brain, normalized 100024F, Prostate-BPH subtracted II, Human colon cancer, metaticized to liver, subtraction, Bone Cancer, Human epithelioid sarcoma, Human OB HOS control fraction I, Human Colon, Adιpocytes,re-excιsιon, HSC172 cells, Smooth muscle, control, re-excision, Soares retina N2b4HR, Soares_placenta_8to9weeks_2NbHP8to9W, H Whole Bram #2, re-excision, Human Hypothalamus, schizophrenia, re-excision, Stratagene hNT neuron (#937233), Human endometπal stromal cells, Spinal Cord, re-excision, human ovarian cancer, T-Cell PHA 24 hrs, Human Ovarian Cancer Reexcision, Human
Hypothalmus,Schιzophrenιa, Soares_fetal_heart_NbHH19W, Human Adipose, Human Rhabdomyosarcoma. Human Chondrosarcoma, Human Fetal Brain, Human Liver, normal, Panci eas Islet Cell Tumoi Human Gall Bladdei, PC3 Prostate cell line, Human Testes Tumoi, H Maciophage (GM-CSF tieated), le excision, Normal colon, Primary Dendntic cells,fιac 2 NCI_CGAP_Lu5, NCI__CGAP_Pι 1, NCI_CGAP_Col2, NCI_CGAP_GCB 1 , NCI_CGAP_Leι2 NCI_CGAP_Bιn23 Soaιes_pιegnant_uterus_NbHPU Colon Normal III, Soaιes_senescent_fιbroblasts_NbHSF, Human Microvascular Endothelial Cells, tract A, Smooth muscle control, and Soaies placenta Nb2HP
Preferred epitopes include those comprising a sequence shown in SEQ ID NO 63 as residues Arg-24 to Asn 39, Seι-43 to Pro-61
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 13 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 681 of SEQ ID NO 13, b is an integer of 15 to 695, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 13, and where b is gieater than or equal to a + 14
FEATURES OFPROTEINENCODEDBY GENENO: 4
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares placenta Nb2HP and to a lesser extent in Human Cerebellum, Soares_fetal_lιver_spleen_lNFLS_S l, Soares infant brain 1MB. Soares adult brain N2b4HB55 Y, NCI_CGAP_GCB 1 , Stratagene HeLa cell s3 937216 Human Substantia Nigra, Human Cerebellum, subtracted, Human Pituitary, subtracted, Soares retina N2b4HR, Soares_senescent__fιbroblasts_NbHSF, Human Whole Brain #2 - Oligo dT > 1 5Kb, Stratagene colon (#937204), NCI_CGAP_Kιd5, NCI_CGAP_Pι22. Human Heart, Soares_pregnant_uterus_NbHPU, Soares breast 2NbHBst, Soares_fetal_heart_NbHH 19W, Soares melanocyte 2NbHM, Human Placenta, Human Fetal Heart, Monocyte activated, Keratinocyte, Soares_testιs_NHT, Soares ovary tumor NbHOT, Human Tongue, frac 1, Human Colon, subtraction, H cerebellum, Enzyme subtracted, H. Epididiymus. cauda, Breast Lymph node cDNA library, Messangial cell, frac 2, Human Normal Breast, HEL cell line, Stratagene placenta (#937225), human corpus colosum, Soaresjfetal_lung_NbHL19W, H. Kidney Cortex, subtracted, Human Adipose Tissue, re-excision, Human Osteosarcoma, Myoloid Progenitor Cell Line, NCI_CGAP_GCB 1 , Soares_NhHMPu_S 1 , Brain Frontal Cortex, re-excision, Human Infant Brain, Human Chronic Synovitis, T-Cell PHA 16 hrs, Human Primary Breast Cancer Reexcision, Human Pancreas Tumor, NCI_CGAP_AA1 , NCI_CGAP_Co9, NCI_CGAP_Kid3, NCI_CGAP_Brn25, Human Adult Testes, Large Inserts, Reexcision, Soares_pineal_gland_N3HPG, Human Hypothalmus, Schizophrenia, Human Pancreas Tumor; Reexcision, Liver, Hepatoma, Soares adult brain N2b5HB55Y, Bone Marrow Stromal Cell, untreated. Human Fetal Brain, Rejected Kidney, lib 4, H. Frontal cortex,epileptic;re-excision, Barstead spleen HPLRB2, Gessler Wilms tumor, Soares_testis_NHT, Soares_pineal_gland_N3HPG, Soares_total_fetus_Nb2HF8_9w, Soares_parathyroid_tumor_NbHPA, Soares_fetal_liver_spleen_lNFLS_S 1 , Soares_senescent_fibroblasts_NbHSF, Smooth muscle, serum induced,re-exc, Soares breast 3NbHBst, Adipocytes, Dendritic cells, pooled, Human Amygdala, Soares_fetal_lung_NbHL19W, Soares_fetal_heart_NbHH19W, Human Microvascular Endothelial Cells, fract. A, Soares fetal liver spleen 1NFLS.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope ofthe 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 2296 of SEQ ID NO: 14, b is an integer of 15 to 2310, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO: 14, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in NCI_CGAP_GCB 1 , Macrophage-oxLDL, Human Hypothalmus Schizophrenia Colon Noimal II, Lung Caicinoma A549 TNFalpha activated, Bone Cancel H ceiebellum, Enzyme subti acted. Healing groin wound 6 5 hours post incision, Piostate BPH, Human Adult Small Intestine, Human Prostate L428 T-Cell PHA 24 his. Human Hippocampus, Human Gall Bladdei Human Eosmophils, Bone maπow, Hodgkin's Lymphoma II. Human Cerebellum
Preferred epitopes include those comprising a sequence shown in SEQ ID NO 65 as residues Ala- 16 to Val-35
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 15 and may have been publicly available pπoi to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every i elated 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 1430 of SEQ ID NO 15, b is an integer of 15 to 1444, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares_multιple_sclerosιs_2NbHMSP, Soares retina N2b4HR and to a lesser extent in Morton Fetal Cochlea, Soares_pregnant_uterus_NbHPU, Human Substantia Nigra, Human Hypothalmus,Schιzophrenιa, Human Amygdala, Human
Amygdala,re-excιsιon, H Whole Brain #2, re-excision, Human Infant Brain, Spinal cord, 12 Week Old Early Stage Human, Soares melanocyte 2NbHM, Human Testes, Reexcision, Human Testes, Soares placenta Nb2HP, Soares infant bram 1MB, Hypothalamus, Human Hippocampus, subtracted. Human Aortic Endothelium, Human Cerebellum, subtracted, Frontal lobe,dementιa.re-excιsιon, Human
Hypothalamus,schιzophrenιa, re-excision, human corpus colosum, Ghoblastoma, Human Ovary, Soares_NhHMPu_S 1 , Soares_total_fetus_Nb2HF8_9w, Spinal Cord, re-excision, Human Brain. Striatum, L428, HUMAN JURKAT MEMBRANE BOUND POLYSOMES, Human Hippocampus. Human Fetal Brain, Human Liver, normal, Human Cerebellum, and Soares fetal liver spleen 1NFLS.
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: 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. 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 1236 of SEQ ID NO: 16. b is an integer of 15 to 1250, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
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. gil2618733 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "CC3". A partial alignment demonstrating the observed homology is shown immediately below.
>gi|2618733 CC3 [Homo sapiens] >sp | 015383 | 015383 CC3 (CC3). Length = 242
Plus Strand HSPs :
Score = 979 (344.6 bits), Expect = 1.9e-109, Sum P(2) = 1.9e-109 Identities = 187/189 (98%), Positives = 187/189 (98%), Frame = +2
Query : 212 KLTFDEEAYKNVNQEWDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL 391
KLTFDEEAYKNVNQEWDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL Sbj ct : 54 KLTFDEEAYKNVNQEWDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL 113
Query : 392 KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD 571
KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD Sbjct : 114 KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD 173 Query: 572 RQESRPGEWLVRKFFGΞLPDSWASGHSVPWTWRAMLNNWRPRDKQMELLENKAIHDL 751
RQESRPGEWLVRK FGSLPDSWA GHSVPWTWRAMLNNWRPRDKQMELLENKAIHDL Sbjct : 174 RQESRPGEWL KLFGSLPDS ARGHSVPWT RAMLNNWRPRDKQMELLENKAIHDL 233 Query 752 GKAHGSLKP 778
GKAHGSLKP Sbjct 234 GKAHGSLKP 242
Score = 129 (45 4 bits) Expect = 1 9e 109, Sum P(2) = 1 9e-109 Identities = 27/29 (93%) Positives = 27/29 (93%) Frame = +3
Query 126 GRHGETGRVLLKEILEQGLFSKVTLIGRR 212
G GETGRVLLKEILEQGLFSKVTLIGRR Sbjct 25 GASGETGRVLLKEILEQGLFSKVTLIGRR 53
Score = 127 (44 7 bits), Expect = 3 le-109, Sum P(2) = 3 le-109 Identities = 31/56 (55%) Positives = 37/56 (66%), Frame = +1
Query 55 MAETEALSKLREDFRMQNKSVFILGATEKPAECS*RKSWSRACFPKSRSLAGEAHL 222
MAETEALSKLREDFR QNKSVFILGA+ + ++ + F K +L G L Sb3ct 1 MAETEALSKLREDFR QNKSVFILGASGETGRVLLKEILEQGLFSKV-TLIGRRKL 55 The segments of gιl2618733 that are shown as "Sbjct" above are set out as SEQ ID NO 117, SEQ ID NO 1 19. and SEQ ID NO 121, lespectively Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and 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 1 18, SEQ ID NO. 120, or SEQ ID NO. 122 which correspond to the Query sequence in the alignments shown above (gaps introduced in the sequence by the computer are, of course, removed) It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries. Stratagene NT2 neuronal precursor 937230 and to a lesser extent in Soares fetal liver spleen INFLS, Stratagene liver (#937224), Stratagene endothelial cell 937223, Stratagene pancreas (#937208), Stratagene colon (#937204), Hepatocellular Tumor, Synovial IL-l/TNF stimulated, Human endometπal stromal cells, Liver, Hepatoma, Human T-Cell Lymphoma, Hodgkin's Lymphoma II, and Keratinocytes
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To hst every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides compiising a nucleotide sequence desciibed by the geneial formula of a-b. where a is any integer between 1 to 1363 of SEQ ID NO 17. b is an integer of 15 to 1377, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO.17, and where b is gieater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
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. gιl4106549 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "neuional-specific septin" A partial alignment demonstrating the observed homology is shown immediately below.
>gι 14106549 (AF104411) neuronal-specifIC septin 3 [Mus musculus] >sp|G4106549 |G4106549 NEURONAL-SPECIFIC SEPTIN 3
Length = 465
Plus Strand HSPs Score = 1118 (393.6 bits), Expect = 2 9e-115, Sum P(2) = 2 9e-115 Identities = 217/319 (68%), Positives = 256/319 (80%), Frame = +2
Query: 1166 VGDMADTPRDA-GLKQAPASRNEKAPVDFGYVGIDSILEQMRRKAMKQGFEFNIMWGQS 1342 + ++ PR + P S N GY+GID+I+EQMR+K MK GF+FNIMWGQS Sbjct. 1 MSELVPEPRPKPAVPMKPVSINSNL LGYIGIDTIIEQMRKKTMKTGFDFNIMWGQS 57
Query 1343 GLGKSTLINTLFKSKISRKSVQPTSEERIPKTIEIKSITHDIEEKGVRMKLTVIDTPGFG 1522
GLGKSTL+NTLFKS++SRK+ EE+IPKT+EIK+I H IEE GV+MKLTVIDTPGFG Sbjct- 58 GLGKSTLVNTLFKSQVΞRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFG 117
Query 1523 DHINNENCWQPIMKFINDQYEKYLQEEVNINRKKRIPDTRVHCCLYFIPATGHSLRPLDI 1702
D INNENC +PI K+IN+QYEK+L+EEVNI RKKPIPDTRVHCCLYFI TGHSLRPLD+ Sbjct. 118 DQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFIΞPTGHSLRPLDL 177 Query: 1703 EFMKRLSKV IVPVIAKADTLTLEERVHFKQRITADLLSNGIDVYPQKEFDEDSEDRLV 1882 EFMK LSKWNI+PVIAKADT+TLEE+ F L S GI YPQKEFDED ED+ Sbjct: 178 EFMKHLSKWNIIPVIAKADTMTLEEKSEF LPSEGI--YPQKEFDEDLEDKTE 228
Query: 1883 NEKFR-EMIPFAWGSDHEYQVNGKRILGRKTK GTIEVENTTHCEFAYLRDLLIRTHMQ 2059 N+K R E +PFAWGSD EYQVNGKR+LGRKT G IEVEN HCEFA LRD +IRTH+Q
Sbjct 229 NDKIRQESMPFAWGSDKEYQVNGKRVLGRKTP GIIEVENLNHCEFALLRDFVIRTHLQ 288
Query 2060 NIKDITSSIHFEAYRVKRLNE 2122 ++K++T +IH+E YR KRLN+ Sbjct 289 DLKEVTHNIHYETYRAKRLND 309
The segment of gιl4106549 that is shown as "Sbjct" above is set out as sequence SEQ ID NO. 123. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and desciibed elsewhere heiein Assays for deteimimng such activities aie also known in the ait some of which have been desciibed elsewheie heiein
Pieferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO 124 which coπesponds to the Queiy sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed)
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Stratagene pancreas (#937208) and Breast Lymph node cDNA library, and to a lesser extent in Soares infant brain 1NIB, Primary Dendritic Cells, lib 1 , Human Ceiebellum, Human Fetal Kidney, Soaιes_fetal_heart_NbHH19W, Soares melanocyte 2NbHM, Soares_fetal_heart__NbHH19W. Human Fetal Heart, Spleen, Chronic lymphocytic leukemia. Kerat ocyte, Messangial cell, frac 2, Jurkat T-Cell, S phase, Breast Cancer Cell line, angiogenic. Human Thymus, Human Osteoblasts II, Human Pancreas Tumor, Human Rhabdomyosarcoma, Human Chondrosarcoma, Rejected Kidney, lib 4, Human adult testis, large inserts, Soares_senescent_fιbroblasts_NbHSF, Human Adult Pulmonary,re-excιsιon, HUMAN B CELL LYMPHOMA, Stratagene pancreas (#937208), Bone Marrow Cell Line (RS4.11), Activated T-Cells, 12hrs, differentially expressed, Soares ovary tumor NbHOT, Human OB MG63 tieated ( 10 nM E2) fraction I, H. Epididiymus, caput & corpus, H cerebellum, Enzyme subtracted, Stratagene colon (#937204), Stratagene pancreas (#937208), Smooth Muscle- HASTE normalized,
Stratagene ovary (#937217), Soares adult bram N2b4HB55Y, pBMC stimulated w/ poly I/C, Breast Cancel cell line, MDA 36, Soares_senescent_fιbroblasts_NbHSF, Human endometπal stromal cells-treated with progesterone, Human Osteosarcoma, Stratagene fetal spleen (#937205), HUMAN JURKAT MEMBRANE BOUND POLYSOMES, Human Adult Testes, Large Inserts, Reexcision, T-Cell PHA 24 hrs,
Soares_pregnant_uterus_NbHPU, Stratagene NT2 neuronal precursor 937230, Liver, Hepatoma, Human Activated Monocytes, Ulcerative Colitis, Human Fetal Brain, Soares breast 2NbHBst, T Cell helper I, Human Eosinophils, Dendritic cells, pooled, Colon Tumor II, Human Synovial Sarcoma, human tonsils, Hodgkin's Lymphoma II, Soares fetal liver spleen INFLS, K562 cells, Human left hemisphere, adult, Kidney cancel, Activated T-Cells, 12 hrs, subtracted, Brain Amygdala Depression, Human Thymus Tumor, subtracted, Werzmann Olfactory Epithelium, Human OB HOS treated (10 nM E2) fraction I, Human Ceiebellum subtracted Adipocytes ie excision, Human Pituitaiy. subti acted, Human Fetal Bone, Human Pnmaty Bieast Cancel Human Thyioid H Epididiymus, cauda, Bieast Lymph node cDNA hbiary. Human Skin Tumoi, Human adult small intestine, re-excision Cem cells cyclohexamide tieated, Human Hypothalamus, schizophrenia, re excision, Synovial IL-l/TNF stimulated,
Soaιes_fetal_lung_NbHL19W, H Kidney Coitex, subtracted, Human Stomach. reexcision, Stiatagene neuroepithe um (#937231), Healing groin wound, 6 5 houis post incision, Stratagene neuioepithehum (#937231), Human endometπal stromal cells, Jurkat T-cell Gl phase, Prostate BPH, CD34 depleted Buffy Coat (Cord Blood), T-Cell PHA 16 hrs, Stratagene NT2 neuronal precursor 937230, Human Brain, Striatum, L428, Human Primary Breast Cancer Reexcision Macrophage-oxLDL, Stratagene pancreas (#937208), Human Adipose, Synovial Fibroblasts (contiol), Stratagene pancreas (#937208), Human Thymus, Hemangiopeπcytoma, Human Whole Six Week Old Embryo, Pancreas Islet Cell Tumor, PC3 Prostate cell line, Resting T-Cell Library ,11, Human T-Cell Lymphoma, Soares_fetal_lung_NbHL19W, Bram frontal cortex, Soares_fetal_lung_NbHL19W,
Human Fetal Lung III, Human Neutrophil, Activated, Stratagene NT2 neuronal precursor 937230, Activated T-Cell (12hs) Thιouπdme labelledEco, Colon Normal III, Anergic T- cell, Smooth muscle,control, Monocyte activated, Soares_pregnant_uterus_NbHPU, and T cell helper II Preferred epitopes include those comprising a sequence shown in SEQ ID NO
68 as residues Ser-21 to Tφ-32
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 18 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every related sequence would be cumbersome 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 4137 of SEQ ID NO 18, b is an integer of 15 to 4151, 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. FEATURES OF PROTEIN ENCODED BY GENE NO: 9
It has been discoveied that this gene is expressed primarily in the following tissues Human Manic Depiession Tissue, Stiomal cell TF274 and Bone maπow Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 19 and may have been publicly available pπoi to conception of the present invention Preferably, such related polynucleotides aie 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 compπsing a nucleotide sequence described by the general formula of a b, where a is any integer between 1 to 1050 of SEQ ID NO 19, b is an integer of 15 to 1064, 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
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
The computer algorithm BLASTX has been used to determine that the translation product of this gene shaies sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no gill 373055 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "siahc-acid O-acetylesterase" A partial alignment demonstrating the observed homology is shown immediately below
>gι|l373055 sialic-acid O-acetylesterase [Mus musculus] >sp | Q61044 | Q61044 YOLK SAC PROTEIN 2 (SIALIC-ACID O-ACETYLESTERASE) Length = 541
Plus Strand HSPs
Score = 576 (202 8 bits) Expect = 3 9e-lll Sum P(2) = 3 9e-lll Identities = 102/128 (79%) Positives = 108 128 (84%) Frame = +2
Query 2 SVLWNAMIHPLCNMTLKGW YQGESNINYNTDLYNCTFPALIED RETFHRGSQGQTER 181 SVLWNAMIHPL NMTLKGWWYQGESN +YN DLY C FP LIED R+TFH GSQGQT+R
Sbjct 281 SVL NAMIHPLQNMTLKGW YQGESNADYNRDLYTCMFPELIEDWRQTFHYGSQGQTDR 340
Query 182 FFPFGLVQXXXXXXXXXXXXGFPQIR HQTADFGYVPNPKMPNTFMAVAMDLCDRDSPFG 361 FFPFG VQ GFP+IR HQTADFG+VPNPKMPNTFMAVA+DLCDRDSPFG Sbjct 341 FFPFGFVQLSSYMLKNSSDYGFPEIRWHQTADFGHVPNPKMPNTFMAVAIDLCDRDSPFG 400 Query: 362 SIHPRDKQ 385
SIHPRDKQ Sbjct: 401 SIHPRDKQ 408
Score = 548 (192.9 bits), Expect = 3.9e-lll, Sum P(2) = 3.9e-lll Identities = 100/133 (75%), Positives = 115/133 (86%), Frame = +3
Query: 384 RTVAYRLHLGARALAYGEXNLTFEGPLPEKIELLAHKGLLNLTYYQQIQVQKKDNKIFEI 563 +TVAYRLHLGARA+AYGE NLTF+GPLP+KIELLA GLLNLTY Q+IQVQ +DNK FEI
Sbjct: 408 QTVAYRLHLGARAVAYGEKNLTFQGPLPKKIELLASNGLLNLTYDQEIQVQMQDNKTFEI 467
Query: 564 SCCSDHRCK LPASMNTVSTQSLTLAIDSCHGTWALRYAWTT PCEYKQCPLYHPSSAL 743 SCCSD CKWLPA +NT STQ+L L +++C GTWA+RYAWTTWPCEYKQC +YH ΞS L Sbjct: 468 SCCSDRHCKWLPAPVNTFSTQTLILDLNACLGTWAVRYAWTT PCEYKQCAVYHTSSML 527
Query: 744 PAPPFIAFITDQG 782
PAPPFIA 1+ +G Sbjct: 528 PAPPFIAQISHRG 540
The segments of gill 373055 that are shown as "Sbjct" above are set out as SEQ ID
NO. 125 and SEQ ID NO. 127, respectively. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities; e.g., sialyl O-acetylesterase activity. Such activities are known in the art and 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. 126 and/or SEQ ID NO. 128 which correspond to the Query sequence in the alignments shown above (gaps introduced in the sequence by the computer are, of course, removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No. 10 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following tissues: Soares fetal liver spleen INFLS and to a lesser extent in Soares_parathyroid_tumor_NbHPA, Soares adult brain N2b5HB55Y, NCI_CGAP_Lu5, NCI_CGAP_Kid5, Stratagene placenta (#937225), Soares_fetal_liver_spleen_lNFLS_S 1 , Soares_senescent_fibroblasts_NbHSF, Bone Marrow Stromal Cell, untreated, Soares breast 2NbHBst, NTERA2, control, Soares_NhHMPu_S 1 , Soares_pregnant_uterus_NbHPU, NCI_CGAP_Co3, NCI_CGAP_Co9, Soares melanocyte 2NbHM, Soares_multiple_sclerosis_2NbHMSP, Soares_multiple_sclerosis_2NbHMSP, Human Amygdala, Human Kidney Tumor, Bone marrow stroma,treated, Human Cardiomyopathy, subtracted, Human Thyroid, Synovial Fibroblasts (Ill/TNF), subt, Human Bone Marrow, re-excision, Human fetal heart, Lambda ZAP Express, NCI_CGAP_GC4, NCI_CGAP_Kid3, NCI_CGAP_Kid6, NCI_CGAP_Pr22, Human Umbilical Vein Endothelial Cells, uninduced. Human Uterine Cancer, Human Fetal Brain, Human Adrenal Gland Tumor, Smooth muscle, serum treated, NCI_CGAP_GCB 1. Colon Normal II, Normal colon, Soares_parathyroid_tumor_NbHPA, Colon Normal III, Human Osteoclastoma, Human Cerebellum, and in Soares infant brain 1NIB.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
It has been discovered that this gene is expressed primarily in the following tissues: human leiomyeloid carcinoma tissue and in neutrophils.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1977 of SEQ ID NO:20, b is an integer of 15 to 1991 , where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
It has been discovered that this gene is expressed primarily in the following tissues and cDNA libraries: Osteoblasts and to a lesser extent in H. Atrophic Endometrium,
Prostate, Soares_NhHMPu_Sl, STROMAL -OSTEOCLASTOMA. Soares_NhHMPu_Sl , Ea.hy.926 cell line, Human Umbilical Vein Endothelial cells, frac B, re-excision, Human Uterus, normal, Human adult small intestine.re-excision, Human Synovium, Human endometrial stromal cells-treated with estradiol, Human Stomach:re-excision, Human Adult Small Intestine, Human Fetal Kidney, Human umbilical vein endothelial cells, IL-4 induced, Soares_fetal_heart_NbHH19W, Synovial Fibroblasts (control), PERM TF274, Bone Marrow Stromal Cell, untreated. H. Frontal cortex,epileptic;re-excision, 2^
Hepatocellulai Tumoi , le-excision Adipocytes, Endothehal-induced, Soaιes_fetal_heaιt_NbHH 19W, Nine Week Old Early Stage Human, Soares placenta Nb2HP
Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie 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 Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, wheie a is any integer between 1 to 3083 of SEQ ID NO 22, b is an integer of 15 to 3097, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 22, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
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 gιl2734083 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "stat-hke protein" A partial alignment demonstrating the observed homology is shown immediately below
>gι 12734083 stat-like protein [Homo sapiens] Length = 710
Plus Strand HSPs
Score = 962 (338 6 bits) Expect = 3 7e-96 P = 3 7e-96 Identities = 184/184 (100%) Positives = 184/184 (100%) Frame = +2
Query 104 NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE 283
NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE
Sbjct 508 NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE 567
Query 284 SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECRVRFLSFLAVGRDVHTFAFIMAA 463
SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECR FLSFLAVGRDVHTFAFIMAA
Sbjct 568 SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECRVRFLSFLAVGRDVHTFAFIMAA 627 Query 464 GPASFCCHMFWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW 643 GPASFCCHMFWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW
Sbjct 628 GPASFCCH FWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW 687 Query: 644 TVRR 655
TVRP Sbjct: 688 TVRR 691
Score = 111 (39.1 bits), Expect = 0.019, P = 0.019
Identities = 40/104 (38%), Positives = 48/104 (46%), Frame = +1
Query: 400 AFPLLPGRGQRCPHVCIHHGCRPSLLLLPHVLVRAQCCQPLRGCAGCVHASLPEVSGC-P 576 AF + G C H+ C P+ L V+A C LR C+ A + C P
Sbjct: 622 AFIMAAGPASFCCHM FWCEPNAASLSEA-VQAACM--LR-YQKCLDARΞQAΞTSCLP 674
Query: 577 FPGLHLLPPSTPC*VCGTACRVDCPQGVQSLWGSLKPKRLGAHTP 711 P P + G R +GVQSLWGSLKPKRLGAHTP Sbjct: 675 AP PAESVARRVGWTVR RGVQSLWGSLKPKRLGAHTP 710
The segments of gil2734083 that are shown as "Sbjct" above are set out as sequence SEQ ID NO. 129 and SEQ ID NO. 131, respectively. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and described elsewhere herein. Assays for determining such activities are also known in the art some of which have been described elsewhere herein.
It has been discovered that this gene is expressed primarily in the following tissues and cDNA libraries: Human Cerebellum and to a lesser extent in Soares adult brain N2b4HB55Y, Early Stage Human Brain, Soares infant brain 1MB,
Soares_pregnant_uterus_NbHPU, Human Infant Brain, Human Brain, Striatum, H. Kidney Cortex, subtracted, Human Whole Brain #2 - Oligo dT > 1.5Kb, Human Hippocampus, breast lymph node CDNA library, Monocyte activated, Human Hippocampus, subtracted, H. Normalized Fetal Liver, II, Human Adult Retina, Human Pituitary, subtracted, H. Epididiymus, caput & coφus, H. cerebellum, Enzyme subtracted. Human Pineal Gland, H. Whole Brain #2, re-excision, Human Synovium, Soares_total_fetusJMb2HF8_9w, Alzheimers, spongy change, Amniotic Cells - Primary Culture, Human endometrial stromal cells. Jurkat T-Cell, S phase, Synovial Fibroblasts (Ill/TNF), subt. wilm's tumor, Human Manic Depression Tissue, Brain Frontal Cortex, re-excision, Human Umbilical Vein Endothelial Cells, uninduced, Human Hypothalmus, Schizophrenia, Ulcerative
Colitis, Hemangiopericytoma, Human Fetal Brain, H. Frontal cortex.epileptic;re-excision, Resting T-Cell Library I, Human Substantia Nigra, Brain frontal cortex, Soares_multiple_sclerosis_2NbHMSP, Soares melanocyte 2NbHM, Anergic T-cell, Human Microvascular Endothelial Cells, tract. A, Smooth muscle. control. Activated T- cell(12h)Thiouridine-re-excision, Neutrophils IL-1 and LPS induced, and Human Testes. 2
Pieferred epitopes include those compiising a sequence shown in SEQ ID NO 73 as residues Ser 15 to Tip 24, Pro-89 to Ser-96
Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 23 and may ha\e been publicly available pnor to conception of the piesent invention Preferably, such i elated polynucleotides are specifically excluded from the scope of the piesent invention To list eveiy related sequence would be cumbersome Accordingly, preferably excluded from the present invention are one or moie polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1 161 of SEQ ID NO 23, b is an integer of 15 to 1175, wheie both a and b coπespond to the positions of nucleotide residues shown in SEQ ID NO 23, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
The computer algorithm BLASTX has been used to determine that the translation product of this gene shaies sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no gιl29486 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "94 kDa B-raf protein" A partial alignment demonstrating the obseived homology is shown immediately below
>gι|29486 94 kDa B-raf protein [Homo sapiens] >sp | Q13878 | Q13878 94 KDA B-RAF PROTEIN (FRAGMENT) Length = 200
Plus Strand HSPs
Score = 168 (59 1 bits) Expect = 1 8e-ll P = 1 8e-ll Identities = 32/32 (100%) Positives = 32/32 (100%) Frame = +2
Query 566 VPARCGVTVRDSLKKALMMRGLIPECCAVYRI 661
VPARCGVTVRDSLKKALMMRGLIPECCAVYRI Sbjct 169 VPARCGVTVRDSLKKALMMRGLIPECCAVYRI 200
The segment of gιl29486 that is shown as "Sbjct" above is set out as sequence(s) 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 and described elsewhere herein. Assays for determining such activities are also known in the art some of which have been described elsewhere herein.
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares infant brain 1MB and to a lesser extent in Brain- medulloblastoma, human ovarian cancer. 12 Week Old Early Stage Human, II, Rejected Kidney, lib 4.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1 105 of SEQ ID NO:24, b is an integer of 15 to 11 19, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15
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. gill 872200 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "alternatively spliced product using exon 13A". A partial alignment demonstrating the observed homology is shown immediately below.
>gi 11872200 alternatively spliced product using exon 13A [Homo sapiens] Length **** 666
Plus Strand HSPs:
Score = 203 (71.5 bits), Expect = 3.1e-13, P = 3.1e-13 Identities = 45/66 (68%), Positives = 46/66 (69%), Frame = +2
Query: 2 FSCLSLPSSWDYRRGPPCLDNFXIXH HVGQAGLELLTSGDLPASASQSAGIKRVS 166 FSCLSLP SWDYR PP NF HVGQAGLELLTSGDLPASASQSA I VS
Sbjct: 587 FSCLSLPRSWDYRHPPPRPANFEFLVETGFLHVGQAGLELLTSGDLPASASQSARITGVS 646
Query: 167 HHTRPK 184 H RP+ Sbj ct : 647 HRARPE 652
The segment of gill 872200 that is shown as "Sbjct" above is set out as sequence(s) 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 the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues: Hypothalamus, Human Rhabdomyosarcoma, and Human Whole Six Week Old Embryo. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope 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 921 of SEQ ID NO:25, b is an integer of 15 to 935, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:25, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Primary Dendritic Cells, lib 1 and to a lesser extent in Soares melanocyte 2NbHM, Soares placenta Nb2HP, Hodgkin's Lymphoma II, Soares_pregnant_uterus_NbHPU, Soares_pregnant_uterus_NbHPU, Colon Normal III, Human Eosinophils, Spleen, Chronic lymphocytic leukemia, B Cell lymphoma, Soares_fetal_heart_NbHH19W, NCI_CG AP_GCB 1 , Healing groin wound, 6.5 hours post incision, Apoptotic T-cell, Soares_pregnant_uterus_NbHPU, Endothelial cells-control, Soares_senescent_fibroblasts_NbHSF, HUMAN STOMACH, pBMC stimulated w/ poly I/C, T-Cell PHA 16 hrs, human ovarian cancer, T-Cell PHA 24 hrs, NTERA2, control, NCI_CGAP_GCB 1 , Hepatocellular Tumor; re-excision, Human Gall Bladder, Human T- Cell Lymphoma. Human Substantia Nigra, Soares breast 3NbHBst. Adipocytes. Human Fetal Heart, Human Adult Pulmonary:re-excision. Human Bone Marrow, treated, Soares_pregnant_uterus_NbHPU, Human Thymus, Human kidney Cortex, subtracted, Human Eosinophils, Human Adult Skeletal Muscle, Palate carcinoma, Rectum normal, Pharynx Carcinoma, Osteoclastoma-normalized B, Larynx Tumor. CD34+ cell, I, frac II, Soares_multiple_sclerosis_2NbHMSP, CD34+cells, II, FRACTION 2, Human Leukocytes, LNCAP untreated, Human Gall Bladder, fraction II, Human Adult Spleen, Human Cardiomyopathy, subtracted, Human Fetal Spleen, Healing Abdomen wound;70&90 min post incision, Activated T-cells, HSA 172 Cells, Messangial cell, frac 2, Healing groin wound - zero hr post-incision (control). Ovarian Tumor 10-3-95. Pancreas Tumor PCA4 Tu, Human Hypothalamus, schizophrenia, re-excision, Human Synovium, Soares_fetal_lung_NbHL19W, H Female Bladder, Adult, Human Stomach;re- excision, Human Adipose Tissue, re-excision, Human Osteosarcoma, Human Osteoclastoma, re-excision, Jurkat T-cell Gl phase, Brain Frontal Cortex, re-excision, KMH2, Stratagene endothelial cell 937223, Spinal Cord, re-excision, Mo7e Cell Line GM-CSF treated (1 ng/ml), Human Bone Marrow, re-excision, Human Osteoblasts II, 12 Week Old Early Stage Human, II, HUMAN JURKAT MEMBRANE BOUND POLYSOMES, Human Heart, Human Ovarian Cancer Reexcision. Human umbilical vein endothelial cells, IL-4 induced, Human Activated Monocytes, Human Chondrosarcoma, Epithelial-TNFa and INF induced, Stratagene colon (#937204), Stratagene fibroblast (#937212), H. Frontal cortex,epileptic;re-excision, Resting T-Cell Library ,I 12 Week Old Early Stage Human, Soares_multiple_sclerosis_2NbHMSP, Colon Carcinoma, Colon Tumor II, Normal colon, Neutrophils control; re-excision, Anergic T-cell, Human Osteoclastoma, Human Microvascular Endothelial Cells, fract. A. Smooth muscle,control, Monocyte activated, HUMAN B CELL LYMPHOMA, Bone Marrow Cell Line (RS4;1 1), Human Endometrial Tumor, Soares_fetal_lung_NbHL19W, neutrophils control, Nine Week Old Early Stage Human, Soares fetal liver spleen INFLS.
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:26 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 4035 of SEQ ID NO:26, b is an integer of 15 to 4049, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:26, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 17 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares_placenta_8to9weeks_2NbHP8to9W and to a lesser extent in Smooth muscle, ILlb induced, Stratagene placenta (#937225), Soares_pregnant_uterus_NbHPU, Soares_placenta_8to9weeks_2NbHP8to9W, Human Placenta, Endothelial cells-control, and Soares fetal liver/spleen INFLS. 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. 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 2053 of SEQ ID NO:27, b is an integer of 15 to 2067, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in Soares fetal liver spleen INFLS, Soares_multiple_sclerosis_2NbHMSP, Soares infant brain 1MB, Soares_fetal_heart_NbHH19W, Nasal polyps, Morton Fetal Cochlea, A 1 -CELL LINE, LI Cell line, human colon cancer, Smooth muscle-ILb induced, HSA 172 Cells, Stratagene neuroepithelium (#937231 ), NTERA2 teratocarcinoma cell line+retinoic acid (14 days), Morton Fetal, Human Chiomc Synovitis, HUMAN JURKAT MEMBRANE BOUND POLYSOMES. Bone Manow Stiomal Cell, untieated, Hemangiopeπcytoma, Maciophage-oxLDL, re-excision, Human Placenta Human Testes Tumoi, Human Placenta, Human Fetal Heait Human Testes, Human 8 Week Whole Embiyo, Human Cerebellum
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 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 hst 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 2353 of SEQ ID NO 28, b is an integer of 15 to 2367, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 28, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 19
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares placenta Nb2HP and to a lesser extent in Soares fetal liver spleen INFLS, H Kidney Medulla, re-excision, Smooth muscle, serum treated, Soares ovary tumor NbHOT, H Frontal Cortex, Epileptic, Morton Fetal Cochlea, Synovial hypoxia, Bone Marrow Stromal Cell, untreated, Soares_fetal_heart_NbHH19W, Soares_pregnant_uterus_NbHPU, Biain frontal cortex, Soares melanocyte 2NbHM, Human Adult Pulmonary ,re-excιsιon, Endothehal-induced, NCI_CGAP_GCB 1 ,
Soares_NhHMPu_Sl, Human Fetal Bram, normalized A5002F, WI 38 cells, Colorectal Tumor, Human Aortic Endothelium. Aorta endothelial cells + TNF-a, Human Lung, Human Soleus, Human Adult Heart,re-excιsιon, Human Epididymus, Synovial IL-l/TNF stimulated, Human endometπal stromal cells, Jurkat T-cell Gl phase, Myoloid Progenitor Cell Line, Synovial Fibroblasts (II 1/TNF), subt, KMH2, Human Thymus, Human umbilical vein endothelial cells, IL-4 induced, Human Rhabdomyosarcoma, Synovial Fibroblasts (control), Human Thymus, Stratagene endothelial cell 937223, Hemangiopeπcytoma, H Fiontal coitex.epilepticae-exαsion. T Cell helper I, Adφocytes, Human Fetal Lung III, Soares_multιple_scleιosιs_2NbHMSP, Human Placenta, Human Fetal Heait, Activated T-Cell ( 12hs)/Thιouπdιne labelledEco, Human Microvasculai Endothelial Cells, fract A, Monocyte activated. HUMAN B CELL LYMPHOMA, Spleen, Chronic lymphocytic leukemia, Human 8 Week Whole Embryo.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 79 as residues- Gln-24 to Thr-35
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: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. 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 3223 of SEQ ID NO.29, b is an integer of 15 to 3237, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 20
The computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-hmitmg example, the sequence accessible through the following database accession no. gιl458692 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "homologous to mouse gene PC326". A partial alignment demonstrating the observed homology is shown immediately below.
>gι 1458692 homologous to mouse gene PC326 GenBank Accession Number M.95564 [Homo sapiens] >sp | Q12839 | Q12839 (H326) Length = 597
Plus Strand HSPs
Score = 270 (95.0 bits), Expect = 1 Oe-48, Sum P(3) = 1 0e-48 Identities = 58/129 (44%), Positives = 87/129 (67%), Frame = +1
Query. 2344 YKGHRNSRTMIKEANF G--ANFVMSGSDCGHIFIWDRHTAEHLMLLEADNH-WNCLQP 2514 YKGHRN+ T +K NF+G + FV+SGSDCGHIF+ ++ + + + +Ξ D WNCL+P Sbjct: 428 YKGHRNNAT-VKGVNFYGPKSEFWSGSDCGHIFLWEKSSCQIIQFHΞGDKGGWNCLEP 486
Query: 2515 HPFDPILASSGIDYDIKIWSPLEESRIFNRKLADEVITRNELMLEE-TRNTITVPASFML 2691 HP P+LA+SG+D+D+KIW+P E+ L D VI +N+ +E -r + + Ξ ML
Sbjct: 487 HPHLPVLATSGLDHDVKIWAPTAEASTELTGLKD-VIKKNKRERDEDSLHQTDLFDSHML 545
Query: 2692 RMLASLNHIRADR 2730 L ++H+R R Sbjct: 546 FL--MHHLRQRR 556
The segment of gil458692 that is shown as "Sbjct" above is set out as SEQ ID NO. 137. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and 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. 138 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the 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 Testes and to a lesser extent in Soares fetal liver spleen INFLS, Brain Frontal Cortex, re-excision, Human Fibrosarcoma, Human Cardiomyopathy, subtracted, Soares_pregnant_uterus_NbHPU, Stratagene endothelial cell 937223, Human Substantia Nigra, Human Bone Marrow, treated, Hodgkin's Lymphoma II, Primary Dendritic Cells, lib 1, Soares infant brain 1MB, Human Cardiomyopathy, diff exp, Human Pituitary, Human Fetal Brain, Human Pituitary, subtracted, Human Synovium, Alzheimers, spongy change, Synovial hypoxia-RSF subtracted. Human Frontal Cortex, Schizophrenia, Human endometrial stromal cells. Human Colon; re-excision, Jurkat T-cell Gl phase, 12 Week Old Early Stage Human, II, Human Fetal Dura Mater, Stromal cell TF274, Synovial Fibroblasts (control), Hemangiopericytoma. Human Whole Six Week Old Embryo, Human adult testis, large inserts, Human Gall Bladder, PC3 Prostate cell line, 12 Week Old Early Stage Human, Smooth muscle, serum treated. Colon Normal II, Soares melanocyte 2NbHM, Soares_multiple_sclerosis_2NbHMSP, Human Fetal Heart. Endothelial cells-control, Human Osteoclastoma, Monocyte activated,
HUMAN B CELL LYMPHOMA, Bone Marrow Cell Line (RS4.1 1), Human Endometrial Tumor, Soaιes_parathyιoιd_tumor_NbHPA. Soaιes_multιple_sclerosιs_2NbHMSP, and Stratagene HeLa cell s3 937216
Preferred epitopes include those compiising a sequence shown in SEQ ID NO 80 as residues Asp- 106 to Glu- 126 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 30 and may have been publicly available pπoi to conception of the present invention Pieferably, 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 descπbed by the general formula of a-b, where a is any integei between 1 to 3194 of SEQ ID NO 30, b is an integer of 15 to 3208, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 30, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 21
The computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no gnllPIDId 1021677 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "KIAA0381 " A partial alignment demonstrating the observed homology is shown immediately below
>gnl|PID|dl021677 (AB002379) KIAA0381 [Homo sapiens] >s | D1021677 | D1021677 KIAA0381 PROTEIN (FRAGMENT) Length = 864
Plus Strand HSPs
Score = 598 (210 5 bits) Expect = 7 3e-55 P = 7 3e-55 Identities = 131/203 (64%) Positives = 141/203 (69%), Frame = +1
Query 328 LPSVSPYSLAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS 507 LP + +LAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS
Sbjct 662 LPEAAKVNLAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS 721
Query 508 ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQDXXXXXXXXXXX 687 ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQD Sbjct 722 ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQDLEAMRRRKEEE 781 Query 688 XXXXXXXXXXXXQRERDKAGN*PSLXXXXXXXXXXXXXXN*WIPRAPG-I«HEGLMQAQA 864
QRER++ + + G + + L + +
Sbjct 782 ERRARMEAMLKEQRERERWQRQRKVLAAGSSLEEGGEFDDLVSALRSGEVFDKDLCKLKR 841
Query 865 HRKRSGSQALEVTRERAINRLNY 933
RKRSGSQALEVTRERAINRLNY Sbjct 842 SRKRSGSQALEVTRERAINRLNY 864 The segment of gnllPlDId 1021677 that is shown as "Sbjct" above is set out as SEQ ID NO 139
Prefened 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 the sequence by the computer are, of course, removed, and residues shown as "X" can be lesolved by looking to the sequences corresponding to Gene No 21 in the Sequence listing)
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares infant bram 1MB and to a lesser extent in human corpus colosum, Human Infant Brain, Human Hippocampus, Soares adult brain N2b5HB55Y, Human Substantia Nigra, Human Fetal Heart, Soares fetal liver spleen INFLS, H Adipose Tissue, Human Fetal Bone, H Epididiymus, caput & corpus, Soares adult brain N2b4HB55Y, Human Epididymus, Soares_multιple_sclerosιs_2NbHMSP, H Kidney Cortex, subtracted, Human Whole Brain #2 - Oligo dT > 1 5Kb, Human Adult Small Intestine, Human Osteoblasts II, Spinal coid, Rejected Kidney, lib 4, Colon Normal II, human tonsils, Soares_multιple_sclerosιs_2NbHMSP, Human Amygdala
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 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 i elated sequence would be cumbersome Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence descπbed by the general formula of a-b, where a is any integer between 1 to 3773 of SEQ ID NO 31, b is an integer of 15 to 3787, 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 FEATURES OF PROTEIN ENCODED BY GENE NO: 22
The computei algorithm BLASTX has been used to determine that the tianslation product of this gene shaies sequence homology with, as a non-limiting example, the sequence accessible thiough the following database accession no gιl2734081 (all information available thiough the recited accession number is incoφorated heiein by refeience) which is desciibed therein as similai to oxysterol-binding protein" A partial alignment demonstrating the observed homology is shown immediately below
>gι 12734081 (AF000195) similar to oxysterol-binding proteins [Caenorhabditis elegans] >sp | 001686 | 001686 C32F10 1 PROTEIN Length = 676
Plus Strand HSPs
Score = 839 (295 3 bits) Expect = 4 6e-83 P = 4 6e-83 Identities = 170/365 (46%) Positives = 220/365 (60%) Frame = +2
Query 185 VMEDQRSIILHLISQLKLGMDLTKWLPTFILEKRSLLEMYADFMAHPDLLLAITAGATP 364
V +D S+++HL+SQ+ +GMDLTKV LPTFILE+RSLLEMYADF AHPDL +A + Sbjct 308 VQQDHGSVLMHLLSQVSVGMDLTKVTLPTFILERRSLLEMYADFFAHPDLFIATNGNDSA 367 Query 365 EERVICFVEYYLTAFHEGRKGALAKKPYNPIIGETFHCS EVPKDRVKPKRTASRSPASC 544
E+R+I V YYL +F+ RK +AKKPYN I+GETF C + VP + K+T S P Sbjct 368 EKRMIAWRYYLNSFYAARKSGVAKKPYNAILGETFRCRYSVPNSSLSGKKTES-GP 423 Query 545 HEHPMADDPSKSYKLRFVAEQVSHHPPISCFYCECEEKRLCVNTHV TKSKFMGMSVGVS 724
P +D+ +L F+AEQVSHHPPIS FY E + + N H++TKS F+G+S+GV+ Sbjct 424 --WPGSDEK QLTFIAEQVSHHPPISAFYAEMPSEGISFNAHIYTKSSFLGLSIGVA 477 Query 725 MIGEGVLRLLEHGEE-YVFTLPSAYARSILTIPWVELGGKVSINCAKTGYSATVIFHTKP 901
IGEG+L L +G+E Y T PS Y RSI++ PW E GGKV + C KTGY A + F TKP Sbjct 478 SIGEGILTLHNYGDEQYTITFPSGYGRSIMSTP FEFGGKVKVECEKTGYRADIEFLTKP 537 Query 902 FYGGKVHRVTAEVKHNPTNTIVCKAHGE NGTLEFTYNNGETKV-IDTTTLPVYPKKIRP 1078
F+GGK HR+ + + V GEWNGT+ NGE V +D P K+ P Sbjct 538 FFGGKPHRIQGSICRDGAKKPVLTLRGEWNGTMYAKTQNGEEYVFVDVKAKPEVKKECVP 597 Query 1079 LEKQGPMESRNL REVTRYLRLGDIDAATEQKRHLXXXXXXXXXXXXNLRTP KPKYF-I 1255 + +QG ESR L R VT L I AT KR + P F Sbjct 598 VMQQGKRESRRL RHVTAALLRNRIQTATTSKRFIEQRQRKEAKERLESGEKWTPLLFDT 657 Query 1256 QEGDG- VY 1279 + DG +Y Sbjct 658 TDKDGEWMY 666
The segment of gιl2734081 that is shown as "Sbjct" above is set out as SEQ ID NO 141 Based on the structural similarity these homologous polypeptides aie expected to share at least some biological activities Such activities are known in the art and 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 compπse a polypeptide having the amino acid sequence set out as SEQ ID NO. 142 which corresponds to the Queiy sequence in the alignment shown above (gaps introduced in the sequence by the computer are. of couise. removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No. 22 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in Soares_fetal_lιver_spleen_lNFLS_S l , Human T-Cell Lymphoma. Spleen, Chronic lymphocytic leukemia, Human Testes, Soares fetal liver spleen INFLS, Soares_total__fetus_Nb2HF8_9w, Pharynx Carcinoma, H. Epididiymus, caput & coφus, Human T-cell lymphoma;re-excιsιon, STROMAL -OSTEOCLASTOMA. Human Adult Small Intestine, Human Prostate, Soares_fetal_heart_NbHH19W, Soares_placenta_8to9weeks_2NbHP8to9W, H. Kidney Medulla, re-excision, Human Thymus, Stromal cell TF274, Stratagene colon (#937204), Stratagene NT2 neuronal precursor 937230, Human Adipose, Bone Marrow Stromal Cell, untreated, Colon Tumor, 12 Week Old Early Stage Human, Human Placenta, Human Fetal Lung III, Human Testes, Reexcision, Human Fetal Heart, Soares_placenta_8to9weeks_2NbHP8to9W, Endothehal- induced, HUMAN B CELL LYMPHOMA, and Human Endometrial Tumor.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 82 as residues: Met-1 to Trp-9.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of \ese 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. 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 2579 of SEQ ID NO:32, 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:32, and where b is greater than or equal to a + 14. FEATURES OF PROTEIN ENCODED BY GENE NO: 23
The computer algoiifhm BLASTX has been used to determine that the translation product of this gene shaies sequence homology with, as a non-limiting example, the sequence accessible thiough the following database accession no gιl3170458 (all information available through the recited accession numbei is incoφorated herein by reference) which is described therein as "diphthamide biosynthesis proteιn-2" A partial alignment demonstiating the obsetved homology is shown immediately below
>gι I 3170458 (AF053003) diphthamide biosynthesis proteιn-2 DPH2 [Homo sapiens] >sp I 060623 I 060623 DIPHTHAMIDE BIOSYNTHESIS PROTEIN-2
Length = 489
Minus Strand HSPs Score = 1433 (504 4 bits) Expect = 5 le-146 P - 5 le-146
Identities = 275/336 (81%) Positives = 275/336 (81%) Frame = -1
Query 1887 EPACAHALEALATLLRPRYLDLLVSSPAFPQPVGSLSPEPMPLERFGRRFPLAPGRRLEE 1708 EPACAHALEALA LLRPRYLDLLVSSPAFPQPVGSLSPEPMP ERFGRRFPLAPGRRLEE Sbjct 154 EPACAHALEALAXLLRPRYLDLLVSSPAFPQPVGSLSPEPMPXERFGRRFPLAPGRRLEE 213
Query 1707 YGAFYVGGSKAXXXXXXXXXXXXXXXG APGQPFSSCCPDTGKTQXXXXXXXXXXXXXXX 1528
YGAFYVGGSKA GWAPGQPFSSCCPDTGKTQ
Sbjct 214 YGAFYVGGSKAΞPDPDLDPDLSRLLLGWAPGQPFSSCCPDTGKTQDEGARAGGLRARRRY 273
Query 1527 XXXXXXXXXWGLLAGTLGVAQHREALAHLRNLTQAAGKRXYVLALGRPTPAKLANFPEV 134£
WGLLAGT GVAQHREALAHLRNLTQAAGKR YVLALGRPTPAKLANFPEV Sbjct 274 XVERARDARWGLLAGTXGVAQHREALAHLRNLTQAAGKRSYVLALGRPTPAKLANFPEV 333 Query 1347 DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPA PPPGLAPHLTHYADLLPGSPF 1168 DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPA PPPGLAPHLTHYADLLPGSPF Sbjct 334 DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPA PPPGLAPHLTHYADLLPGSPF 393
Query 1167 HVALPPPESEL ETPDVSLITGDLRPPPA KSSNDHGSLALTPRPQXXXXXXXXXXXXXX 988 HVALPPPESEL ETPDVSLITGDLRPPPAWKSSNDHGSLAL PRPQ
Sbjct 394 HVALPPPESELWETPDVSLITGDLRPPPA KSSNDHGSLALNPRPQLXLAESSPAVSFLS 453
Query 987 XXX QGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG 880 QGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG Sbjct 454 SRS QGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG 489
The segment of gιl3170458 that is shown as "Sbjct" above is set out as SEQ ID NO 143 Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities Such activities are known in the art and 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 144 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No. 23 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following tissues and cDNA libraries: Activated T-cell( 12h)/Thiouridine-re-excision and to a lesser extent in NCI_CGAP_GCB 1 , Soares fetal liver spleen INFLS, Soares infant brain 1MB, Pharynx carcinoma. Human Testes Tumor, Activated T-Cell (12hs)/Thiouridine labelledEco, Soares_fetal_heart_NbHH19W, NCI_CGAP_Co3, Soares_fetal_lung_NbHL19W, Soares_testis_NHT, Soares ovary tumor NbHOT, Soares_total_fetus_Nb2HF8_9w, Soares_testis_NHT, Raji cells, cyclohexamide treated, subtracted, Human B Cell 8866, Human epithelioid sarcoma, Human Aortic Endothelium, Human Primary Breast Cancer;re-excision. L428, Human Fetal Kidney, HUMAN JURKAT MEMBRANE BOUND POLYSOMES, Human Chondrosarcoma, Human Thymus, Human Testes Tumor, re-excision, Human Adrenal Gland Tumor, human tonsils, Human Adult Pulmonary;re-excision, Endothelial-induced, Human Amygdala, Human Microvascular Endothelial Cells, fract. A, Human Bone Marrow, treated,
NCI_CGAP_GC5, NCI_CGAP_Lul, NCI_CGAP_Col2, NCI_CGAP_Prl2, NCI_CGAP_Brn23, Soares_fetal_heart_NbHH19W, Stratagene NT2 neuronal precursor 937230.
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: 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. 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 1906 of SEQ ID NO:33, b is an integer of 15 to 1920, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:33, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 24 It has been discoveied that this gene is expiessed pπmanly in the following tissues Activated T-Cell ( 12hs)/Thιouπdιne labelled Eco
Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID NO 34 and may have been publicly available pπoi to conception of the piesent invention Preferably, such related polynucleotides are specifically excluded fiom the scope of the present invention To list every related sequence would be cumbersome Accordingly, pieferably excluded fiom the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integei between 1 to 141 1 of SEQ ID NO 34, b is an integer of 15 to 1425, wheie both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 34. and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO : 25
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 gill 196433 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "unknown protein" A partial alignment demonstrating the observed homology is shown immediately below
>gι|H96433 unknown protein [Homo sapiens] >sp | Q14288 | Q14288 HYPOTHETICAL PROTEIN (FRAGMENT) Length = 641
Plus Strand HSPs
Score = 191 (67 2 bits) Expect = 1 Oe 22 Sum P(2) = 1 Oe-22 Identities = 41/79 (51%) Positives = 50/79 (63%) Frame = +3
Query 987 QKIFANYSSDKGLIFRTYKDLKQLSNNNKNIQF*KWAKEPNKHL*KPDASGQQVYEKKKC 1166
+KIFA YSSDKGLI R Y +LKQ+ N K AK+ N+H K D + + KKC Sbjct 403 EKIFATYSSDKGLISRIYNELKQIYKKKTNNPIKKWAKDMNRHFSKEDIYAAKKH-MKKC 461
Query 1167 *TPLIIREMQTNTTMRYYL 1223
+ L IREMQ TTMRY+L Sbjct 462 SSSLAIREMQIKTTMRYHL 480 The segment of gil l 196433 that is shown as "Sbjct" above is set out as SEQ ID NO. 145. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art and 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. 146 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed). It has been discovered that this gene is expressed primarily in human colon carcinoma.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 85 as residues: Gln-1 to Asp-6.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1242 of SEQ ID NO:35, b is an integer of 15 to 1256, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:35, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
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. gnllPIDIel31 1091 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "ubiquitin-conjugating enzyme". A partial alignment demonstrating the observed homology is shown immediately below. >gnl I PID j el311091 ubiquitin-conjugating enzyme [Mas musculus] >sp | 088738 | 088738 UBIQUITIN-CONJUGATING ENZYME Length 4845
Plus Strand HSPs
Score - 286 (100 7 bits) Expect = 9 8e-20 P = 9 8e-20 Identities = 66/156 (42%) Positives - 86/156 (55%) Frame = +2
Query 179 DTVDMTKIHALITGPFDTPYEGGFFLFVFRCPPDYPIHPPRVKLMTTGNNTVRFNPNFYR 358
+ +D+ K+ LITGP DTPY G F F P DYP PP V L TTG ++VRFNPN Y Sbjcπ 4592 ERLDIMKV--LITGPADTPYANGCFEFDVYFPQDYPSSPPLVNLETTGGHSVRFNPNLYN 4649 Query 359 NGKVCLSILGT TG PA--WSPAXXXXXXXXXXXXXM--TENPYHNEPGFEQERH-PG-- 514 +GKVCLSIL T G P +P + PY NEPG+E+ R P
Sbjct 4650 DGKVCLSILNT HGRPEEKUNPQTSSFLQVLVSVQSLILVAEPYFNEPGYERSRGTPSGT 4709
Query 515 -DSKNYNECIRHETIRVAVCDMMEGKCPCPEPLRGVMEKSF 634 S+ Y+ IR T++ A+ + + PC + V+ K F
Sbjct 4710 QSSREYDGNIRQATVKWAMLEQIRNPSPC FKEVIHKHF 4747
The segment of gnllPIDIe 131 1091 that is shown as Sbjct" above is set out as SEQ ID NO 147 Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities, e g , ubiquitin conjugating enzyme activity Such activities are known in the ait and 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 148 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed, and residues shown as "X" can be lesolved by looking to the sequences corresponding to Gene No 26 in the Sequence listing)
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares infant brain 1MB and to a lesser extent in Human
Microvascular Endothelial Cells, fract A, human tonsils, Primary Dendritic Cells, lib 1, Smooth muscle,control, Human Fetal Kidney, Soares fetal liver spleen INFLS, Soares placenta Nb2HP, Soares melanocyte 2NbHM, Activated T-cell(12h)/Thιouπdιne-re- excision, Soares_placenta_8to9weeks_2NbHP8to9W, Human Synovium, Smooth muscle, serum ιnduced,re-exc, Human Testes Tumor, Human Synovial Sarcoma, Monocyte activated, neutrophils control, Nine Week Old Early Stage Human, Larynx carcinoma II, LNCAP untreated. Hodgkin's Lymphoma I, HSC172 cells, Human Skin Tumor, Resting T-Cell, re-excision Soares_multιple_sclerosιs_2NbHMSP, Soares_fetal_lung_NbHL19W, pBMC stimulated w/ poly I/C. Human Osteoclastoma Stromal Cells - unamp fied. Human Fetal Duia Mater, Human Activated T-Cells, Macrophage-oxLDL. Stratagene HeLa cell s3 937216, Hemangiopeπcytoma. Human Fetal Brain, Soaιes_pregnant_uteιus_NbHPU. Human Whole Six Week Old Embryo, Stratagene lung (#937210). Soares_pregnant_uterus_NbHPU, NCI_CGAP_GCB 1 , Human Placenta, Soaιes_placenta_8to9weeks_2NbHP8to9W, Stratagene HeLa cell s3 937216, Primary Dendritic cells, fiac 2, Human Adult Pulmonary ;re-excιsιon. Activated T-Cell (12hs)/Thιouπdιne labelledEco, Soaιes_fetal_lung_NbHL19W, CD34 positive cells (Cord Blood), HUMAN B CELL LYMPHOMA, Human Testes, Osteoblasts, Human 8 Week Whole Embryo, Soares_multιple_sclerosιs_2NbHMSP, Soares_pregnant_uterus_NbHPU, Human Adult Lymph Node, Rectum normal, Messangial cell, frac 1. Activated T-Cells, 8 hrs., WI 38 cells, Human Prostate BPH. re-excision, CD34+cells, II, FRACTION 2, Human Prostate, subtracted, K562 + PMA (36 hrs),re-excιsιon, Human (Caco-2) cell line, adenocarcinoma, colon, remake, Morton Fetal Cochlea, Soares_placenta_8to9weeks_2NbHP8to9W, Human Aortic Endothelium, Human Primary Breast Canceπre-excision, Human Adult Retina, Human Umbilical Vein Endothelial Cells, fract. A, Healing Abdomen wound, 70&90 min post incision, Human Placenta, Human Neutrophils, Activated, re-excision, Smooth muscle, control, re-excision, Supt Cells, cyclohexamide treated, Smooth muscle-ILb induced, H. Epididiymus, cauda, Human adult small intestine, re-excision. Human Normal Breast, Ovarian Tumor 10-3-95, Human Epididymus, Human Hypothalamus, schizophrenia, re-excision, Synovial IL- 1/TNF stimulated, Amniotic Cells - Primary Culture, Human Stomach;re-excιsιon, Salivary Gland, Lib 2, HL-60, PMA 4H. re-excision, Human endometrial stromal cells, Jurkat T-cell Gl phase, Human Prostate. T-Cell PHA 16 hrs, Human Thymus, Stratagene fetal spleen (#937205), HUMAN JURKAT MEMBRANE BOUND POLYSOMES, Human Uterine Cancer, Human Primary Breast Cancer Reexcision, Human Pancreas Tumor, Human Ovarian Cancer Reexcision, Merkel Cells, Human Thymus Stromal Cells, Soares breast 2NbHBst, Rejected Kidney, lib 4, Macrophage (GM-CSF treated), Macrophage-oxLDL; re-excision. Smooth muscle, serum treated, Soares breast 3NbHBst, H Macrophage (GM-CSF treated), re-excision, Human Fetal Lung III, Human Testes, Reexcision, Human Neutrophil, Activated, and Human Cerebellum. Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible thiough sequence databases Some of these sequences aie related to SEQ ID NO 36 and may have been publicly available pπoi to conception of the piesent invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list eveiy related sequence would be cumbersome Accoidingly, preferably excluded fiom the present invention are one or moie polynucleotides comprising a nucleotide sequence described by the general formula of a-b, wheie a is any integer between 1 to 2844 of SEQ ID NO 36, b is an integer of 15 to 2858, where both a and b coπespond to the positions of nucleotide residues shown in SEQ ID NO 36, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 27
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 gιl2352277 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "MAP kinase" A partial alignment demonstrating the observed homology is shown immediately below
>gι I 2352277 (AF002715) MAP kinase kinase kinase [Homo sapiens] Length = 1607
Plus Strand HSPs
Score = 2801 (986 0 bits) Expect = 4 9e-291 P = 4 9e-291 Identities = 529/542 (97%) Positives = 529/542 (97%) Frame = +1
Query 10 ISFARKWMNYVLTKCESGRGTRPRWATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI 189 ISFARK MNYVLTKCESGRGTRPR ATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI
Sbjct 1067 ISFARKWMNYVLTKCESGRGTRPRWATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI 1126
Query 190 GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEG^STRSMPSDARSH 369 GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEGFSTRSMPSDARSH Sbjct 1127 GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEGFSTRSMPSDARSH 1186
Query 370 GSPXXXXXXXXXXXXXSRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS 549
GSP SRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS
Sbjct 1187 GSPAAAAAAAAAVAA-SRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS 1245
Query 550 LSRHSSPTEERDEPAYPRGDSSGSTRRS ELRTLISQSKDTASKLGPIEAIQKSVRLFEE 729
LSRHSSPTEERDEPAYPRGDSSGSTRRS ELRTLISQSKDTASKLGPIEAIQKSVRLFEE Sbjct 1246 LSRHSSPTEERDEPAYPRGDSSGSTRRSWELRTLISQSKDTASKLGPIEAIQKSVRLFEE 1305 Query 730 KRYREMRRKNIIGQVCDTPKSYDNVMHVGLRKVTFKWQRGNKIGEGQYGKVYTCISVDTG 909 KRYREMRRKNIIGQVCDTP-vSYDNVMHVGLRKVTFK QRGNKIGEGQYGKx/YTCISVDTG Sbjct 1306 KR/REMRRKNIIGQVCDTP^SYDNVMHVGLRKVTFK QRGNKIGEGQYGKVYTCISVDTG 13D5
Query 910 ELMAMKEIRFQPNDHKTIKΞT^DELKIFEGIKHPNLVRYFGVELHREEMYIFMEYCDEGT 1089 ELMAMKEIRFQPNDHKTIKΞTADELKIFEGIKHPNLVRYFGVELHREEMYIFMEYCDEGT
Sbjct 1366 ELMAMKEIRFQPNDHKTIϊ-ΞTADELKIFEGIKHPNLVRYFGVELHREEMYIFMEYCDEGT 1425
Query 1090 LEEVSRLGLQEHVIRLYSKQITIAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV 1269 LEEVSRLGLQEHVIRLYSKQITIAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV Sbjct 1426 LEEVSRLGLQEHVIRLYSKQITIAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV 1485
Query 1270 KLK AQTMPGEVNSTLGTAAYMAPEVITRAKGEGHGRAADIWSLGCWIEMVTGKRPWH 1449
KLKNNAQTMPGEWSTLGTriAYMAPEVITRAKGEGHGRAADIWSLGCVVIEMVTGKRPWH Sbjct 1486 KLKNNAQTMPGEWSTLGTΛAYMAPEVITRAKGEGHGRAADIWSLGCWIEMVTGKRPWH 1545
QUERY 1450 EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMR TASQLLDHSFVKVCTD 1629
EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMR TASQLLDHSFVKVCTD SBJCT 1546 EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMR TASQLLDHSFVKVCTD 1605 Query 1630 EE 1635 EE Sbjct 1606 EE 1607
The segment of gιl2352277 that is shown as "Sbjct" above is set out as SEQ ID NO 149 Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities Such activities are known in the art and described elsewhere herein Assays for determining such activities are also known in the ait some of which have been described elsewheie 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 the sequence by the computer are, of course, removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No 26 in the Sequence listing)
It has been discovered that this gene is expiessed primarily in the following tissues/cDNA libraries Soares_pregnant_uterus_NbHPU,
Soares_placenta_8to9weeks_2NbHP8to9W, Macrophage-oxLDL, re-excision, Human Testes and to a lesser extent in Human frontal cortex, Stratagene colon (#937204), Human Colon Carcinoma (HCC) cell line, Early Stage Human Lung, subtracted, Human endometrial stromal cells, Spleen metastic melanoma, Prostate BPH, Brain Frontal Cortex, re-excision, Human Activated Monocytes, Human Rhabdomyosarcoma, Human adult (K.Okubo). Colon Normal II. Soares breast 3NbHBst, Human Amygdala, HUMAN B CELL LYMPHOMA, Human Endometrial Tumor, Soares fetal liver spleen INFLS 4
Pieteπed epitopes include those comprising a sequence shown in SEQ ID NO 87 as lesidues Met-1 to Gly-9, Arg-26 to Gly-32, Hιs-44 to Aιg-56. Ser-68 to Tιp-97, Gin- 105 to Ala-1 10, Glu-127 to Ile-139, Thι- 146 to Asp-151 , Glu-173 to Lys-178, Gln-199 to Asp-21 1. Leu-310 to Gln-315. Ala-339 to Gly-345, Lys-385 to Asp-398 Many polynucleotide sequences, such as EST sequences, aie 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 eveiy related sequence would be cumbersome Accordingly, preferably excluded fiom the present invention are one or more polynucleotides comprising a nucleotide sequence descπbed by the general formula of a-b, where a is any integer between 1 to 2157 of SEQ ID NO 37, b is an integer of 15 to 2171, 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
FEATURES OF PROTEIN ENCODED BY GENE NO: 28
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 gnllPIDIe321606 (all information available through the recited accession number is mcoφorated herein by reference) which is described therein as "apoptosis specific protem" An alignment demonstrating the observed homology is shown immediately below
>gnl |PID| e321606 apoptosis specific protein [Homo sapiens] >sp | 060875 | 060875 APOPTOSIS SPECIFIC PROTEIN Length = 275
Plus Strand HSPs
Score = 1457 (512 9 bits) Expect = 1 5e-148 P = 1 5e-148 Identities = 267/275 (97%) Positives = 267/275 (97%) Frame = +1
Query 172 MTDDKDVLRDV FGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM 351 MTDDKDVLRDVWFGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM
Sbjct 1 MTDDKDVLRDVWFGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM 60
Query 352 RQEDISEIWFEYEGTPLKWHYPIGLLFDXXXXXXXXPWNITVHFKSFPEKDLLHCPSKDA 531 RQEDISEIWFEYEGTPLKWHYPIGLLFD PWNITVHFKSFPEKDLLHCPSKDA Sbjct 61 RQEDISEIWFEYEGTPLKWHYPIGLLFDLLASSSALPWNITVHFKSFPEKDLLHCPSKDA 120 Query 532 IEAHFMSCMkEADALKHKSQVINEMQKKDHkQLWMGLQNDRFDQFWAIN^kLMEYPAEEN 711
IE HFMSCMKEADALKroSQ INEMQkKDHkQLWMGLQNDRFDQFWAI r-.LMEYPAEEN Sbjct 121 IEAHFMSCMKEADALKHk QVINEMQKkDHKQLWMGLQNDRFDQFWAIN kLMEYPAEEN 180
Query 712 GFRYIPFRIYQTTTERPFIQkLFRPVAADGQLHTLGDLLKEVCPSAIDPΞDGEKKNQVMI 891
GFRYIPFRIYQTTTERPFIQKLFRPVAADGQLHTLGDLLKEVCPSAIDPΞDGEKKNQVMI Sbjct 181 GFRYIPFRIYQTTTERPFIQkL^RPVAADGQLHTLGDLLKEVCPSAIDPEDGEKKNQVMI 240 Query 892 HGIEPMLETPLQWLSEHLSYPDNFi.HISIIPQPTD 996 HGIEPMLETPLQWLSEHLS PDNFLHISIIPQPTD Sbjct 241 hGIEPMLETPLQWLSEHLSYDDNFLHISIIPQPTD 275
The segment of gnllPIDIe321606 that is shown as ' Sbjct" above is set out as SEQ ID NO 151 Based on the stiuctuial similarity these homologous polypeptides aie expected to share at least some biological activities, such as the ability to induce apoptosis Such activities aie known in the ait and described elsewhere herein Assays for determining such activities are also known in the art some of which have been desciibed elsewhere herein Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO 152 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed, and residues shown as "X" can be resolved by looking to the sequences corresponding to Gene No 26 in the Sequence listing) It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Stromal cell TF274, Activated T-cell(12h)/Thιouπdιne-re- excision, Soares infant bram 1MB and to a lesser extent in CD34+cells, II, Human Colon Carcinoma (HCC) cell line, Human T-cell lymphoma, re-excision, Cem cells cyclohexamide treated, Human Whole Brain, le-excision, HEL cell line, Human Osteoclastoma Stromal Cells - unamphfied, H Memngima, Ml,
Soares_fetal_lung_NbHL19W, H Kidney Medulla, re-excision, Stratagene fetal spleen (#937205), Human Hippocampus, NCI_CGAP_GCB 1 , Stratagene pancreas (#937208), H Frontal cortex,epιleptιc,re-excιsιon, Human Bone Marrow, treated, Human Testes, T cell helper II, Soares fetal liver spleen INFLS 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 38 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded fiom the scope of the present invention To list every related sequence would be cumbeisome Accordingly piefei bly excluded fiom the piesent invention are one or moie polynucleotides compiising a nucleotide sequence desciibed by the geneial foimula of a-b, where a is any integer between 1 to 1515 of SEQ ID NO 38. b is an integei of 15 to 1529, wheie both a and b coirespond to the positions of nucleotide residues shown in SEQ ID NO 38, and where b is greatei than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 29 The computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with as a non-hmitmg example, the sequence accessible through the following database accession no gιl861321 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "T24H7 3 gene product" A partial alignment demonstrating the observed homology is shown immediately below
>gι I 861321 T24H7 3 gene product [Caenorhabditis elegans] Length = 156 Plus Strand HSPs
Score = 168 (59 1 bits) Expect = 6 Oe-11 P = 6 Oe-11 Identities = 43/115 (37%) Positives = 68/115 (59%) Frame = +1 Query 157 DMKDMRLEAEAV--VNDVLFAVNNMFVSKSLRCADDVAYINVETKERNRYCLELTEAGLK 330 D++ LE EA+ V + FAVN + VS+ L + +INV T E + +C+ELT+ G + Sbjct 36 DVEKSTLEEEAMAAVRENAFAVNLIGVSEMLPRTSQLLFINVTTFENHTHCIELTQKGWR 95
Query 331 WGYA FDQVDDHLQTPYHETVYSLLDTLSPAYREAFGNALLQRLEALKRD 480 V F Q+D H T Y E++++LL +SP +RE FG+ L+ +L LK++
Sbjct 96 VASNRNDCMNGDFRQLDIH -TKYFEΞLHTLLMDISPLFRETFGSKLISKLSELKKE 150
The segment of gιl861321 that is shown as "Sbjct" above is set out as SEQ ID NO 153 Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities Such activities are known in the art and 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 154 which coriesponds to the Query sequence in the alignment shown above (gaps intioduced in the sequence by the computer aie, of couise, removed)
It has been discoveied that this gene is expiessed pπmaπly in the following tissues/cDNA libiaπes NCI_CGAP__Co9 and to a lessei extent in Soares_parathyιoιd_tumoι_NbHPA. Soaies infant brain INIB. Stiatagene ovarian cancer (#937219), NCI_CGAP_GCB 1. Soares_NhHMPu_S l . Soares melanocyte 2NbHM, NCI_CGAP_GCB 1 , Colon Normal III, Human Osteoclastoma, Human Testes. Soares fetal liver spleen INFLS, Primary Dendritic Cells, lib 1 , Human (HCC) cell line livei (mouse) metastasis, remake, Human Colon Carcinoma (HCC) cell line, Salivary Gland, Human adult small intestine, re-excision, Human Amygdala.re-excision, Ovarian Tumor 10-3-95, pBMC stimulated w/ poly I/C, Human Colon re-excision. Human Prostate, Human Hypothalmus, Schizophrenia, Human umbilical vein endothelial cells, IL-4 induced, Ulcerative Colitis, Human Adrenal Gland Tumor, Hepatocellular Tumor, reexcision, Pancreas Islet Cell Tumor, 12 Week Old Early Stage Human, Human Placenta, Normal colon, Bone maπow, Human Fetal Heart, CD34 depleted Buffy Coat (Cord Blood), re-excision, NCI_CGAP_Kιd3, Human Cerebellum
When tested against sensory neuron cell lines, supernatants removed from cells containing this gene activated the EGRl assay Thus, it is likely that this gene activates sensory neuron cells through a signal transduction pathway Early growth response 1 (EGRl) is a promotei associated with certain genes that induces various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation
Preferred epitopes include those comprising a sequence shown in SEQ ID NO 89 as residues Lys-28 to Thr-33
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 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 Accordingly, preferably excluded from the present invention are one or more polynucleotides compπsing a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2100 of SEQ ID NO 39, b is an integer of 15 to 21 14, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO.39. and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 30 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares_fetal_heaιt_NbHH 19W, Stratagene pancieas (#937208) and to a lesser extent in HCC cell line metastisis to liver, CD34+ cell. I, frac II, Breast Cancer cell line, MDA 36, and Human Primary Breast Cancer Reexcision.
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. 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 429 of SEQ ID NO:40, b is an integer of 15 to 443, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:40, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 31
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Primary Breast Cancer Reexcision. Soares fetal liver spleen INFLS and to a lesser extent in Human Osteoblasts II, Human (HCC) cell line liver (mouse) metastasis, remake, Human Lung Cancer;re-excιsion, Glioblastoma, Healing groin wound, 6.5 hours post incision, Rejected Kidney, lib 4, Human Whole Six Week Old Embryo, and Colon Tumor.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: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 SO
would be cumbersome Accoidingly, preferably excluded fiom the piesent invention are one oi more polynucleotides compπsing a nucleotide sequence desciibed by the general foimula of a b wheie a is any integei between 1 to 1301 of SEQ ID NO 41 , b is an integer of 15 to 1315 w heie both a and b coπespond to the positions of nucleotide residues shown in SEQ ID NO 41 , and where b is greater than oi equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 32
The computer algoπthm BLASTX has been used to determine that the translation product of this gene shares sequence homology with as a non-limit g example, the sequence accessible through the following database accession no gιl940151 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "TAFII31 " A partial alignment demonstrating the observed homology is shown immediately below
>gι I 940151 TAFII31 [Homo sapiens] >gι| 882393 TBP-associated factor TAFII31 [Homo sapiens] >gι| 841308 TAFII32 precursor [Homo sapiens] >pιr I 139141 I 139141 transcription factor TFIID 32K chain TAFII32 - human Length = 264
Plus Strand HSPs
Score = 1255 (441 8 bits) Expect = 3 7e-127 P = 3 7e-127 Identities = 249/249 (100%) Positives = 249/249 (100%) Frame = +1
Query 163 MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH 342
MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH Sbjct 1 MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH 60
Query 343 AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY 522
AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY Sbjct 61 AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY 120 Query 523 CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM 702 CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM Sbjct 121 CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM 180
Query 703 SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES 882 SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES
Sbjct 181 SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES 240
Query 883 SNALKRKRE 909 SNALKRKRE Sbjct 241 SNALKRKRE 249
The segment of gιl940151 that is shown as "Sbjct' above is set out as sequence(s) SEQ ID NO 155 Based on the structural similarity these homologous polypeptides are expected i
to shaie at least some biological activities Such activities aie know n in the ait and descπbed elsewheie heiein Assays loi detei mining such activities aie also known in the art some of which have been desciibed elsewhere heiein
Pieferred polypeptides of the invention compπse a polypeptide having the amino acid sequence set out as SEQ ID NO 156 which coπesponds to the Queiy sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed)
It has been discovered that this gene is expressed pπmaiily in the following tissues/cDNA hbiaπes Soaies fetal liver spleen INFLS and to a lesser extent in Soares infant brain INIB, Human Osteoclastoma, re-excision, Human Fetal Lung III,
Soares_senescent_fιbroblasts_NbHSF Soaies retina N2b4HR, Ran Cells, cyclohexamide treated, Soares_pregnant_uteιus_NbHPU, Stiatagene endothelial cell 937223, Stratagene ovarian cancer (#937219), Stratagene neuroepithehum NT2RAMI 937234, Soares_pregnant_uterus_NbHPU, Smooth muscle, serum treated, Human Testes, Reexcision, Bone marrow, Human Adult Pulmonary ,re-excιsιon, Soares_NhHMPu_Sl, Spleen, Chronic lymphocytic leukemia, Human Testes, Bone Marrow Cell Line (RS4,1 1), Human Adult Spleen, fractionll, Activated T-Cells, 4 hrs, subtracted, LNCAP untreated, Human (HCC) cell line liver (mouse) metastasis, remake, HSC172 cells, Frontal lobe,dementιa,re-excιsιon, Aorta endothelial cells + TNF-a, Human Thyroid, Eaily Stage Human Lung, subtracted, Human Quadriceps, B Cell lymphoma, STROMAL -
OSTEOCLASTOMA, Human endometnal stromal cells-treated with estradiol, Salivary Gland, Lib 2, Human Colon, re-excision, Soares_NhHMPu_S 1 , Human Heart, Human Adipose, Bone Marrow Stromal Cell, untreated, Rejected Kidney, lib 4, NTERA2, control, Hepatocellular Tumor, re-excision, Colon Tumor, Resting T-Cell Library ,11, Soares breast 3NbHBst, Soares melanocyte 2NbHM, Soares_placenta_8to9weeks_2NbHP8to9W, Human Osteoclastoma, NCI_CGAP_GCB 1 , Hodgkin's Lymphoma II, and Human Cerebellum
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 cumbeisome Accoidingly pieferably excluded fiom the piesent invention aie one or more polynucleotides compπsing a nucleotide sequence desciibed by the geneial formula of a-b. wheie a is any integei between 1 to 1 160 of SEQ ID NO 42. b is an integer of 15 to 1 174 wheie both a and b coirespond to the positions of nucleotide residues shown in SEQ ID NO 42, and wheie b is greater than oi equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 33
The computer algorithm BLASTX has been used to determine that the tianslation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no gnllPIDIe 1247368 (all information available through the lecited accession number is incoφorated herein by reference) which is described therein as "W09H1 5" A partial alignment demonstrating the observed homology is shown immediately below
>gnl |PID|el247368 W09H1 5 [Caenorhabditis elegans] Length = 344
Plus Strand HSPs
Score = 235 (82 7 bits) Expect = 3 7e-19 P = 3 7e-19 Identities = 48/96 (50%) Positives = 67/96 (69%) Frame = +2
Query 143 ARVRALVYGHHGDPAKWELKNLELAAVRGSD-VRVKMLAAPINPSDINMIQGNYGLLPE 319 A R LVY + +P + ++LK + +A +D V V+ +AAPINP+D+N IQG Y + P
Sbjct 15 ASTRQLVYEGYRNPPEAIQLKTVTIADKPSADQVLVQWIAAPINPADLNQIQGVYPVKPA 74
Query 320 LPAVGGNEGVAQWAVGSNVTGLKPGDWVIPANAGL 427 LPAVGGNEG +V++VGSNV+ +K GD VIP +GL Sbjct 75 LPAVGGNEGFGKVISVGSNVSSIKVGDHVIPDRSGL 110
The segment of gnllPIDIe 1247368 that is shown as "Sbjct" above is set out as SEQ ID NO 157.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out as SEQ ID NO 158 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the 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 Rhabdomyosarcoma and to a lesser extent in Stratagene S3
lung (#937210). Human adult testis. laige inseits. Human Synovial Sarcoma, Soares placenta Nb2HP. Soaies infant bram INIB, Soaies let a N2b4HR. Soaιes_NhHMPu_S l , Soares_total_fetus_Nb2HF8_9w. Human Chondiosarcoma,
Soares_pregnant_uteιus_NbHPU, Soares_senescent_fιbιoblasts_NbHSF, Soares fetal livei spleen INFLS. CD34+cells, II, HL-60. RA 4h, Subtracted, Ovanan Tumor 10-3-95,
NTERA2 + retmoic acid, 14 days, H Kidney Cortex, subtracted. Human Adipose Tissue, re-excision. Human Brain, Striatum, Apoptotic T-cell, Human Fetal Dura Mater, Stromal cell TF274, Human Hippocampus, Soares breast 2NbHBst, H. Frontal cortex,epιleptιc;re- excision, Pancreas Islet Cell Tumor, T Cell helpei I, Human T-Cell Lymphoma, Human Substantia Nigra, Soares breast 3NbHBst. Early Stage Human Brain, Human Fetal Lung III, Bone marrow, Human Fetal Heart, Endothe al-induced, Endothelial cells-control, Smooth muscle,control, Osteoblasts, Soares_parathyroιd_tumor_NbHPA, Keratinocyte, Nine Week Old Early Stage Human, and Human Cerebellum.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 93 as residues. Asn-131 to Met- 140.
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.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. 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 591 of SEQ ID NO:43, b is an integer of 15 to 605, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 34
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: HL-60, RA 4h, Subtracted, HUMAN STOMACH, HEL cell line. Human Testes Tumor, Soares melanocyte 2NbHM, Human Microvascular Endothelial Cells, fract. A, HUMAN B CELL LYMPHOMA, Soares_testιs_NHT, T cell helper II. Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible thiough sequence databases Some of these sequences are related to SEQ ID NO 44 and may have been publicly available pπoi to conception of the piesent invention Piefeiably, such i elated polynucleotides aie specifically excluded fiom the scope of the present invention To list every related sequence would be cumbeisome Accordingly, preferably excluded fiom the piesent invention are one oi moie polynucleotides compiising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 902 of SEQ ID NO 44, b is an integer of 15 to 916, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 44, and where b is greatei than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 35
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares adult brain N2b4HB55Y and to a lesser extent in Soares infant brain INIB, Early Stage Human Brain, Soares adult brain N2b5HB55Y, Soares_fetal_heart_NbHH19W, Soares fetal liver spleen INFLS, HL-60, PMA 4H, reexcision, wilm's tumor, Hemangiopeπcytoma, Human Fetal Bram. Soares breast 2NbHBst, H Frontal cortex,epιleptιc.re-excιsιon, Soares_senescent_fιbroblasts_NbHSF, Human Fetal Lung III, HUMAN B CELL LYMPHOMA, Stratagene endothelial cell
937223, Normal Prostate, Duodenum, HL-60, PMA 4H, Human Primary Breast Cancer, Human Tonsils, Lib 2, B Cell lymphoma. Human Synovium, Human Prostate Cancer, Stage C fraction, Human endometrial stromal cells-treated with progesterone, Myoloid Progenitor Cell Line, Human Prostate, Breast Cancer Cell line, angiogenic, Human Activated T-Cells. Human Hypothalmus. Schizophrenia, Human Thymus Stromal Cells, Human Thymus, Human Whole Six Week Old Embryo, Human T-Cell Lymphoma, Soares breast 3NbHBst, Soares_fetal_lung_NbHL19W, breast lymph node CDNA library, H Macrophage (GM-CSF treated), re-excision, Soares melanocyte 2NbHM, Human Synovial Sarcoma, human tonsils, Endothehal-induced, Spleen, Chronic lymphocytic leukemia, T cell helper II, Keratinocyte, Nine Week Old Early Stage Human, Human
Cerebellum, Stratagene neuroepithehum NT2RAMI 937234, Primary Dendritic Cells, lib 1 Many pol\ nucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences aie related to SEQ ID \0 45 and may have been publicly available pπoi to conception of the piesent invention Pieferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every lelated sequence would be cumbersome Accoidingly, pieferably excluded from the piesent invention are one or moie polynucleotides compπsing a nucleotide sequence described by the general formula of a-b, where a is any integei between 1 to 937 of SEQ ID NO 45, b is an integer of 15 to 951 , where both a and b coirespond to the positions of nucleotide residues shown in SEQ ID NO 45, and wheie b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 36
It has been discovered that this gene is expressed in the following cDNA library 7TM Receptor enriched, lib II
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 46 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every related sequence would be cumbersome Accoidingly, 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 494 of SEQ ID NO 46, b is an integer of 15 to 508, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 46, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 37
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries. Soares infant brain INIB and to a lesser extent in Soares fetal livei spleen INFLS, Soares_multιple_sclerosιs_2NbHMSP, Human Amygdala, re-excision, Bone Marrow Stromal Cell, untreated, Primary Dendritic cells,frac 2. Human Placenta, T cell helpei II Pπmaiy Dendntic Cells, lib 1 , Human Maciophage subtracted Human osteoaithntic fiaction II, Piostate. Human osteoaithπtis.fiaction I H Adipose Tissue. Human Adult Retina. Adipocytes.ie-excision, Human Thyroid, H cerebellum. Enzyme subtiacted, Eai ly Stage Human Lung, subti acted Soares letina N2b4HR, Human Pineal Gland. Messangial cell, frac 2, Human Colon Cancer.re-excision. pBMC stimulated w/ poly I/C, Alzheimeis. spongy change Amniotic Cells - Primary Cultuie, NTERA2 + retinoic acid 14 days, Soares_NhHMPu_S 1. Human Adipose Tissue, ie-excision KMH2, Human Infant Brain, T-Cell PHA 24 hrs, Human Activated Monocytes, Soares breast 2NbHBst, Human Adrenal Gland Tumor, Human Eosinophils, Soaιes_fetal_heart_NbHH19W, Brain frontal cortex, human tonsils, Human Neutrophil, Activated, Human Osteoclastoma Monocyte activated, Hodgkin's Lymphoma II, Stratagene lung carcinoma 937218. Stiatagene neuroepithelium NT2RAMI 937234, Osteoblasts, Human Cerebellum, and Soares placenta Nb2HP
Preferred epitopes include those compπsing a sequence shown in SEQ ID NO 97 as residues Leu-6 to Ser- 12
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 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 Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence descπbed by the general formula of a-b, where a is any integer between 1 to 1286 of SEQ ID NO 47, b is an integer of 15 to 1300, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 47, and where b is gieater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 38
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares adult bram N2b4HB55Y,
Soares_multιple_sclerosιs_2NbHMSP and to a lesser extent in H Adipose Tissue, Soares_multιple_sclerosιs_2NbHMSP Spinal Coid ie-excision, and Nine Week Old Early Stage Human
Prefened epitopes include those compiising a sequence shown in SEQ ID NO 98 as residues Gly-8 to Ala-13 Many polynucleotide sequences such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 48 and may have been publicly available pπoi to conception of the present invention Pieferably, such related polynucleotides are specifically excluded from the scope of the present invention To list eveiy 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 976 of SEQ ID NO 48, b is an integer of 15 to 990, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 48, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 39
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 gιl818*41 1 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as "tubulomterstitial nephritis antigen" A partial alignment demonstrating the observed homology is shown immediately below
>gι I 818411 tubulomterstitial nephritis antigen [Oryctolagus cuniculus]
>pιr I A57480 I A57480 tubulomterstitial nephritis antigen precursor - rabbit >sp | Q28625 | Q28625 TUBULOINTERSTITIAL NEPHRITIS ANTIGEN Length = 474 Plus Strand HSPs
Score = 605 (213 0 bits) Expect = 4 7e 98 Sum P(2) = 4 7e-98 Identities = 115/239 (48%) Positives = 151/239 (63%) Frame = +3 Query 729 NCAGS AFSTAAVASDRVSIHSLGHMTPVLSPQNLLSCDTHQQQGCRGGRLDGAW FLRR 908 NCA SWAFSTA+VA+DR++I S G T LSPQNL+SC + GC G +D AW +LR+ Sbjct 237 NCAASWAFSTASVAADRIAIQSNGRYTANLSPQNLISCCAKNRHGCNSGSIDRA YLRK 296
Query 909 EG CLTTATPSRAVNETRLALRPPV*CTAEP VGASARPHAH--CPNSYVNNNDIYQVTP 1082 G P + ++ C + HA CPN+ +N IYQ +P Sbjct: 297 RGLVSHACYP--LFKDQNISNNT CAMTSKADGRGKRHATRPCPNNIEKSNRIYQCSP 351
Query: 1083 VYRLGSNDKEIMKELMENGPVQALMEVHEDFFLYKGGIYSHTPVSLGRPERYRRHGTHSV 1262
YR+ SN+ EIMKE+M+NGPVQA+M+VHEDFF YK GIY H + E+YR+ TH+V Sbjct: 352 PYRVSSNETEIMKEIMQNGPVQAIMQVHEDFFHYKTGIYRHVISTNEESEKYRKLQTHAV 411
Query: 1263 KITGWGEETLPDGRTLKYKTAANS GPAWGERGHFRIVRGVNECDIESFVLGVWGRVGME 1442
K+TG G G+ K+V AANSV.'G +VGE G+FRI+RGVNE DIE +^ G++ Sbjct: 412 KLTG GTLKGARGQKEKFl-iAANSWGKS GENGYFRILRGVNESDIEKLIIAA GQLTSS 471
Query: 1443 D 1445
D Sbjct: 472 D 472 The segment of gil81841 1 that is shown as "Sbjct" above is set out as sequence(s) 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 and 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. 162 which corresponds to the Query sequence in the alignment shown above (gaps introduced in the sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Endothelial cells-control and to a lesser extent in Human
Microvascular Endothelia] Cells, fract. A, Keratinocyte, Soares placenta Nb2HP, H. Epididiymus, cauda. Human Adipose Tissue, re-excision, Human Umbilical Vein Endothelial Cells, uninduced, Epithelial-TNFa and INF induced, Soares breast 2NbHBst, Colon Carcinoma, Human Placenta, Endothelial-induced. Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
99 as residues: Gin- 19 to Glu-26, Ala-39 to Leu-49, Gly-53 to Thr-59, Tyr- 1 14 to Gln-133, Ala-205 to Thr-211.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 2171 of SEQ ID NO:49, b is an integer of 15 to 2185. where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:49, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 40
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Keratinocyte and to a lesser extent in
Soares_fetal_heart_NbHH19W, Healing groin wound; 7.5 hours post incision, Healing groin wound, 6.5 hours post incision, Healing Abdomen wound;70&90 min post incision, Soares breast 2NbHBst, Soares_fetal_heart_NbHH19W, Healing groin wound - zero hr post-incision (control), Epithelial-TNFa and INF induced, Soares breast 3NbHBst, human tonsils, Sinus piniformis Tumour, Pharynx Carcinoma, Tongue carcinoma, Human Tongue, frac 1, Tongue Normal, Larynx Normal, Human Tonsil, Lib 3, H. Epididiymus, caput & coφus, Amniotic Cells - Primary Culture, Human Adipose Tissue, re-excision, Human Thymus, Colon Carcinoma, Human Fetal Heart.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO. 100 as residues: Ser- 16 to Glu-31.
The tissue distribution and homology to [gene or gene family] suggests that the protein product of this clone would be useful for [take best guess(es) and explain rationale].
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:50 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 3138 of SEQ ID NO:50, b is an integer of 15 to 3152, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:50, and where b is greater than or equal to a + 14. FEATURES OF PROTEIN ENCODED BY GENE NO: 41
It has been discovered that this gene is expressed primarily in the following tissues and cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Soares placenta Nb2HP, Soares infant brain INIB, NCI_CGAP_GCB 1 , Stratagene neuroepithelium (#937231 ), Stratagene lung (#937210). Stratagene colon (#937204), Soares_pregnant_uterus_NbHPU, Primary Dendritic Cells, lib 1, Human osteoarthritic;fraction II, Brain Amygdala Depression, Human Whole 6 Week Old Embryo (II), subt, Human osteoarthritis;fraction I. stomach cancer (human), Soares_NhHMPu_Sl , Human Adipose Tissue, re-excision, wilm's tumor, L428, HUMAN JURKAT
MEMBRANE BOUND POLYSOMES. Stromal cell TF274, Pancreas Islet Cell Tumor, Soares_fetal_lung_NbHL19W, Soares_fetal_lung_NbHL19W, Bone marrow, Activated T-cell(12h)/Thiouridine-re-excision, Spleen, Chronic lymphocytic leukemia, Human Endometrial Tumor, Human 8 Week Whole Embryo. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:51 and may have been publicly available prior to conception of the present invention. 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 1388 of SEQ ID NO:51, b is an integer of 15 to 1402, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:51, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 42
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares breast 2NbHBst and to a lesser extent in Soares_multiple_sclerosis_2NbHMSP, Bone Marrow Cell Line (RS4; 11), Human Aortic Endothelium, Human Adipose Tissue, re-excision, Soares_fetal_heart_NbHH19W, and Activated Human Neutrophil. Piefeπed epitopes include those compiising a sequence shown in SEQ ID NO 102 as ιesιdues Seι-49 to Lys-59
Many polynucleotide sequences such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 52 and may have been publicly available pnoi to conception of the present invention Pieferably such related polynucleotides are specifically excluded from the scope of the piesent invention To list every related sequence would be cumbersome Accoidingly, preferably excluded from the present invention are one oi more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 827 of SEQ ID NO 52, b is an integei of 15 to 841 where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 52, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 43
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares retina N2b4HR, Soares infant brain INIB and to a lesser extent in Soares fetal liver spleen INFLS, Human Old Ovary, Soares retina N2b5HR, Human Epididymus, NCI_CGAP_Br2, NCI_CGAP_Co3, NCI_CG APJLu 1 , NCI_CGAP_Col 1, NCI_CGAP_Kιd5, Human Adipose Tissue, re-excision, Human Infant Brain, and Human Brain, Striatum
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 53 and may have been publicly available prior to conception of the present invention Preferably, such related polynucleotides are specifically excluded from the scope of the present invention To list every related sequence would be cumbersome 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 952 of SEQ ID NO 53, b is an integer of 15 to 966, 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 FEATURES OF PROTEIN ENCODED BY GENE NO: 44
It has been discovered that this gene is expressed primarily in 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: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. 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 991 of SEQ ID NO:54, b is an integer of 15 to 1005, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:54, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 45
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares breast 3NbHBst and to a lesser extent in Human Adipose, Soares breast 2NbHBst, breast lymph node CDNA library, Human Normal Breast, Human Synovium, Human Adipose Tissue, re-excision, Stratagene liver (#937224), Human Infant Adrenal Gland, Human colon cancer, metaticized to liver, subtraction, Human White* Fat, H. Adipose Tissue, Human Quadriceps, Human Soleus, Human Pituitary, subt IX, Human Chronic Synovitis, and Bone marrow. Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
105 as residues: Pro-45 to Gln-51.
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: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. Accordingly, preferably excluded from the present invention 6
are one 01 moie polynucleotides compiising a nucleotide sequence described by the general formula of a-b, wheie a is any integei between 1 to 450 of SEQ ID NO 55, b is an integer of 15 to 464, wheie both a and b conespond to the positions of nucleotide residues shown in SEQ ID NO 55 and where b is greatei than oi equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 46
It has been discoveied that this gene is expressed primarily in Human Adipose tissue
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 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 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 825 of SEQ ID NO 56, b is an integer of 15 to 839, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 56, and where b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 47
The computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-hmitmg example, the sequence accessible through the following database accession no gil l 710216 (all information available through the recited accession number is incoφorated herein by reference) which is described therein as ' unknown' A paitial alignment demonstrating the observed homology is shown immediately below
>gι I 1710216 unknown [Homo sapiens] >sp | Q99770 | Q99770 HYPOTHETICAL 15 4 KD PROTEIN Length = 139
Minus Strand HSPs
Score = 140 (49 3 bits) Expect = 4 Oe-18 Sum P(2) - 4 Oe-18 Identities = 31/45 (68%) Positives = 35/45 (77%) Frame = -3
Query 1043 RLECSGAVSAHCKLHFPGSHHSPASASRVAGI--TCARHHA LIFLY 909 RLECSG +SAHC L PGS +SP^SASRVAG TC R A LIF++ Sbjct 62 RLECSGTISAHCNLCLPGSSNSP-SASRVAGTAGTCRR--AQLIFVF 106
The segment of gil l 710216 that is shown as "Sbjct" above is set out as sequence(s) SEQ ID NO 163 Based on the stiuctural similarity these homologous polypeptides aie expected to shaie at least some biological activities Such activities are known in the ait and descπbed elsewhere herein Assays for determining such activities are also known in the art some of which have been described elsewheie heiein
Preferred 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 the sequence by the computer aie, of course, removed) It has been discovered that this gene is expressed primarily m Human Adipose
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 57 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 compπsing a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1088 of SEQ ID NO 57, b is an integer of 15 to 1 102, 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
FEATURES OF PROTEIN ENCODED BY GENE NO: 48
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Supt Cells, cyclohexamide treated, Apoptotic T-cell, re-excision, and Human Adipose
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible thiough 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 cumbeisome Accordingly, pieferably excluded fiom the present invention aie one or moie polynucleotides compiising a nucleotide sequence desciibed by the general formula of a-b, wheie a is any integer between 1 to 1098 of SEQ ID NO 58, b is an integei of 15 to 1 1 12. wheie 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
FEATURES OF PROTEIN ENCODED BY GENE NO: 49
It has been discoveied that this gene is expressed pπmaπly in the following tissues Human Adipose and Human Placenta
When tested against sensory neuron cell lines, supernatants removed from cells containing this gene activated the EGRl assay Thus, it is likely that this gene activates sensory neuron cells through a signal transduction pathway Early growth response 1 (EGRl) is a promoter associated with certain genes that induces various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation 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 59 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 2517 of SEQ ID NO 59, b is an integer of 15 to 2531 , where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 59, and wheie b is greater than or equal to a + 14
FEATURES OF PROTEIN ENCODED BY GENE NO: 50
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries Soares infant brain INIB and to a lesser extent in Soares ovary tumor NbHOT, Soares adult brain N2b4HB55Y, G oblastoma, Hemangiopeπcytoma, Soares_fetal_heart_NbHH19W, Early Stage Human Brain, Soares_NhHMPu_S l , Human epithelioid saicoma. Human Adult Pulmonaiy, eosιnophιl-IL5 induced. Human Quadnceps Human Epididymus, Human Fetal Dm a Matei, Meikel Cells. Human Adipose Human Thymus, Soaies bieast 2NbHBst, Pnmaiy Dendiitic cells. fiac 2, Soares_total_fetus_Nb2HF8_9w, Hodgkin's Lymphoma II, Osteoblasts, Keratinocyte, Human Cerebellum. Soaιes_total_fetus_Nb2HF8_9w, and Pπmary Dendiitic Cells, lib Pieferred epitopes include those comprising a sequence shown in SEQ ID NO 1 10 as residues Pιo-9 to Thr- 14, Seι-37 to Trp-44, Gly-79 to Thr-85. Arg-88 to Lys- 139 Many polynucleotide sequences, such as EST sequences, aie publicly available and accessible through sequence databases Some of these sequences are related to SEQ ID NO 60 and may have been publicly available prior to conception of the present invention Pieferably, 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 piesent 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 653 of SEQ ID NO 60, b is an integer of 15 to 667, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO 60, and where b is greater than or equal to a + 14
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Table 1 summanzes the infoimation corresponding to each "Gene No ' desciibed above The nucleotide sequence identified as "NT SEQ ID NO X' was assembled from partially homologous ( overlapping ) sequences obtained fiom the "cDNA clone ID" identified in Table 1 and, in some cases, from additional related DNA clones The ovei lapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO X
The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No Z and Date " Some of the deposits contain multiple different clones corresponding to the same gene "Vector' refers to the type of vector contained in the cDNA Clone ID
"Total NT Seq " refers to the total number of nucleotides in the contig identified by "Gene No " The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as "5' NT of Clone Seq " and the "3' NT of Clone Seq " of SEQ ID NO X The nucleotide position of SEQ ID NO X of the putative start codon (methionine) is identified as "5' NT of Start Codon " Similarly , the nucleotide position of SEQ ID NO X of the predicted signal sequence is identified as "5' NT of First AA of Signal Pep "
The translated amino acid sequence, beginning with the methionine. is identified as "AA SEQ ID NO Y," although other reading frames can also be easily translated using known molecular biology techniques The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention
The first and last ammo acid position of SEQ ID NO Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep " The predicted first amino acid position of SEQ ID NO Y of the secreted portion is identified as "Predicted First AA of Secreted Portion " Finally, the amino acid position of SEQ ID NO Y of the last amino acid in the open reading frame is identified as "Last AA of ORF " SEQ ID NO X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently 7^
accurate and otheiwise suitable for a v anety of uses well known in the ait and described furthei below For instance SEQ ID NO X is useful foi designing nucleic acid hybudization probes that will detect nucleic acid sequences contained in SEQ ID NO X or the cDNA contained in the deposited clone These piobes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention Similarly, 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 errois The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence In these cases, the predicted amino acid sequence diveiges 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)
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention piovides 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
Moreover, the amino acid sequence of the protem encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence The present invention also relates to the genes corresponding to SEQ ID
NO X, SEQ ID NO Y, or the deposited clone The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein Such methods include preparing probes 01 pnmeis fiom the disclosed sequence and identifying or amplifying the corresponding gene from appiopπate souices of genomic material
Also provided in the piesent ιn\entιon aie allelic variants, orthologs. and/or species homologs Proceduies known in the att can be used to obtain full-length genes, allelic variants, splice variants full-length coding portions, orthologs. and/or species homologs of genes corresponding to SEQ ID NO X, SEQ ID NO Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC For example, allelic variants and/oi species homologs may be isolated and identified by making suitable probes or pnmeis from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/oi the desired homologue
The 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
The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below) It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification , such as multiple histidine residues, or an additional sequence for stability during recombinant production
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67 31-40 (1988) Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the secreted protein The present invention provides a polynucleotide comprising, or alternatively consisting of. the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA contained in ATCC deposit Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO: Y and/or a polypeptide sequence encoded by the cDNA contained in ATCC deposit Z are also encompassed by the invention. Signal Sequences The present invention also encompasses mature forms of the polypeptide having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. 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. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues -13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-809?- (von Heinje, supia ) However the two methods do not always produce the same piedicted cleavage poιnt(s) foi a given protein
In the present case, the deduced ammo acid sequence of the secreted polypeptide was analyzed by a computer piogram called SignalP (Henrik Nielsen et al , Prote 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 incorporated The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1
As one of ordinary skill would appieciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO Y which have an N-terminus beginning withm 5 residues (1 e , + or - 5 residues) of the predicted cleavage point Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secieted species These polypeptides and the polynucleotides encoding such polypeptides, are contemplated by the present invention Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER Nonetheless, 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 desπbed below) These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention
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 polynucleotides.
The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained in a deposited clone, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein).
By a 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. In other words, to obtain a 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.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245(1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identiy are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group Length=0, Cutoff Score= 1 , Gap
Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the lenght of the subject nucleotide sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. 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.
For example, 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%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether 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 cDΝA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245( 1990)). In a sequence alignment 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. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l , Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5. Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to Ν- or C- terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for Ν- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the Ν- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are Ν- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the Ν- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest Ν- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York ( 1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. Using known methods of protem engineeπng and lecombinant DNA technology variants may be geneiated to impiove 01 altei the characteristics of the polypeptides of the piesent invention Foi instance one or more ammo acids can be deleted from the N-terminus 01 C-terminus ot the secieted protein without substantial loss of biological function The authors of Ron et al . J Biol Chem 268 2984-2988 ( 1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, oi 27 amino-terminal ammo acid lesidues Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the cai boxy terminus of this protem (Dobeh et al , J Biotechnology 7 199-216 (1988) )
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein For example, Gayle and coworkers (J Biol Chem 268 22105 22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a They used random mutagenesis to generate over 3,500 individual IL-1 a mutants that averaged 2 5 amino acid changes per variant over the entire length of the molecule Multiple mutations were examined at every possible ammo acid position The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity] " (See, Abstract ) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus Whether a particular polypeptide lacking N- or C-termmal residues of a protein retains such immunogenic activities can readily be determined by routine methods descπbed herein and otherwise known in the art
Thus, the invention further includes polypeptide variants which show substantial biological activity Such 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 surprisingly 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. 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. Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, wheie the substituted ammo acid lesidues may 01 may not be one encoded by the genetic code, or (n) substitution with one oi moie of amino acid lesidues having a substituent group 01 (in) fusion of the matuie polypeptide with another compound, such as a compound to increase the stability and/oi 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, oi leader or secietory sequence, oi a sequence facilitating puπfication. Such variant polypeptides aie deemed to be withm the scope of those skilled in the art from the teachings herein For example, polypeptide variants containing ammo 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. (Pinckard et al , Clin Exp. Immunol. 2 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Cπt. Rev Therapeutic Drug Carrier Systems 10 307-377 (1993) )
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 ammo 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 Of course, in order of ever-increasing preference, 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. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention oi fragments thereof (e g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments 8s
The piesent invention is also directed to polynucleotide fiagments of the polynucleotides of the invention
In the piesent invention a polynucleotide fiagment ' refers to a shoit polynucleotide having a nucleic acid sequence which is a poition of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone, is a portion ot that shown in SEQ ID NO X or the complementary strand thereto, oi is a poition of a polynucleotide sequence encoding the polypeptide of SEQ ID NO Y The nucleotide fragments of the invention aie preferably at least about 15 nt, and more pieferably at least about 20 nt, still moie 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 In this context "about" includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein Of course, larger fragments (e g , 50, 150, 500, 600, 2000 nucleotides) are preferred
Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1 100, 1 101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851 - 1900, 1901 - 1950, 1951 -2000, or 2001 to the end of SEQ ID NO X, or the complementary strand thereto, or the cDNA contained in a deposited clone In this context "about" includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini Preferably, these fragments encode a polypeptide which has biological activity More preferably, these polynucleotides can be used as probes or primers as discussed herein Polynucleotides which hybridize to these nucleic acid molecules under stringent hybudization conditions oi lowei stiingency conditions aie also encompassed by the invention as aie polypeptides encoded by these polynucleotides
In the piesent invention, a ' polypeptide fiagment" refeis to an amino acid sequence which is a portion of that contained in SEQ ID NO Y oi encoded by the cDNA contained in a deposited clone Protein (polypeptide) fragments may be "free standing," 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 numbei 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 Moreover, 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 Preferred polypeptide fragments include the secreted protein as well as the mature form Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both For example, any number of amino acids, ranging from 1- 60, can be deleted from the ammo terminus of either the secreted polypeptide or the mature form Similarly, any number of ammo acids, ranging from 1-30. can be deleted from the carboxy terminus of the secreted protem or mature form Furthermore, any combination of the above amino and carboxy terminus deletions are preferred Similarly, polynucleotides encoding these polypeptide fragments are also preferred
Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions Polypeptide fragments of SEQ ID NO Y falling within conserved domains are specifically contemplated by the piesent invention Moreovei, poly nucleotides encoding these domams aie also contemplated
Other prefened polypeptide fiagments aie biologically activ e fragments Biologically activ e fiagments are those exhibiting activity similar but not necessanly identical to an activity of the polypeptide of the present invention The biological activity of the fi gments may include an improved desned activity or a decreased undesirable activity Polynucleotides encoding these polypeptide fragments are also encompassed by the invention
Preferably, the polynucleotide fragments of the inv ention encode a polypeptide which demonstrates a functional activity By 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 protem Such functional activities include, but are not limited to, biological activity, antigemcity [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 foi a polypeptide of the invention
The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods
For example, in one embodiment where one is assaying for the ability to bind or compete with full-length polypeptide of the invention for binding to an antibody of the polypeptide of the invention, 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, lmmunoradiometπc 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 lmmunoelectrophoresis assays, etc In one embodiment, antibody binding is detected by detecting a label on the primary antibody In another embodiment, the piimaiy antibody is detected by detecting binding of a secondary antibody oi leagent to the primary antibody In a further embodiment, the secondary antibody is labeled Many means aie known in the ait foi detecting binding in an immunoassay and aie within the scope of the piesent invention In another embodiment, wheie a ligand for a polypeptide of the invention identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimeπze is being evaluated, 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 blottmg See generally, Phizicky, E , et al , 1995, Microbiol Rev 59:94- 123. In another embodiment, physiological correlates of binding of a polypeptide of the invention to its substrates (signal transduction) can be assayed.
In addition, assays described herein (see Examples) and otherwise known in the art 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
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, oi 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 complementaiy strand under stringent hybudization conditions oi lowei stringency hybridization conditions defined supra
The term ' epitopes " as used heiein iefeis to poitions of a polypeptide having antigenic or immunogenic activity in an animal, prefeiably a mammal and most preferably in a human In a preferred embodiment the present invention encompasses a polypeptide compπsing an epitope as well as the polynucleotide encoding this polypeptide An "immunogenic epitope," as used herein is defined as a portion of a protem that elicits an antibody response in an animal, as determined by any method known in the ait, for example, by the methods for generating antibodies described infra (See, for example Geysen et al , Proc Natl Acad Sci USA
81 3998- 4002 (1983)) The term "antigenic epitope," as used herein, 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 descπbed herein Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens Antigenic epitopes need not necessarily be immunogenic
Fragments which function as epitopes may be produced by any conventional means (See, e g , Houghten, Proc Natl Acad Sci USA 82 5131-5135 (1985), further described in U S Patent No 4,631,21 1) In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7. more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14. at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids Preferred polypeptides compπsing immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes Antigenic epitopes can be used as the target molecules in immunoassays (See, foi instance Wilson et al Cell 37 767-778 (1984), Sutchffe et al , Science 219 660-666 ( 1983))
Similarly immunogenic epitopes can be used foi example, to induce antibodies according to methods ell known in the ait (See, for instance, Sutchffe et al , supi , Wilson et al , supra. Chow et al , Pioc Natl Acad Sci USA 82 910- 914, and Bittle et al , J Gen Vnol 66 2347-2354 ( 1985) Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five oi more of these immunogenic epitopes The polypeptides comprising one or more immunogenic epitopes may be presented foi eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), oi if the polypeptide is of sufficient length (at least about 25 ammo acids), the polypeptide may be presented without a carrier However, 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-beaπng 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 , Sutchffe et al , supra, Wilson et al , supra, and Bittle et al , J Gen Virol , 66 2347- 2354 (1985) If in vivo immunization is used, animals may be immunized with free peptide, however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid For instance, peptides containing cysteine residues may be coupled to a carrier usmg a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response Several 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 titei of anti-peptide antibodies in seium from an immunized animal may be incieased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art As one of skill in the ait will appreciate, and as discussed above, the polypeptides of the piesent invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences For example, the polypeptides of the present invention may be fused with the constant domain of lmmunoglobuhns (IgA, IgE, IgG, IgM), or poitions thereof (CH I, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeπc 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 barrier to the immune system has been demonstrated for antigens (e g , insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e g , PCT Publications WO 96/22024 and WO 99/04813) IgG Fusion proteins that have a disulfide-linked dimeπc structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeπc 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 For example, a system described by Janknecht et al allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al , 1991, Proc Natl Acad Sci USA 88 8972- 897) In this system, the gene of interest is subcloned into a vaccmia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues The tag serves as a matrix binding domain for the fusion protein Extracts from cells infected with the recombinant vaccinia virus are loaded onto Nι2+ nitπloacetic acid agaiose column and histidine-tagged proteins can be selectively eluted with lmidazole-contaimng buffeis
Additional fusion pioteins of the invention may be geneiated through the techniques of gene-shuffling, motif-shufflmg, exon-shuffling, and/or codon-shuffling (collectively ref ei red to as "DNA shuffling ) 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, 5,81 1 ,238, 5,830,721 , 5,834,252, and 5,837.458, and Patten et al , Curr Opinion Biotechnol 8 724-33 (1997), Harayama, Trends Biotechnol 16(2) 76 82 (1998), Hansson. et al , J Mol Biol 287 265-76 (1999), and Loienzo and Blasco, Biotechmques 24(2) 308- 13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety) In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc , of a polynucleotide encoding a polypeptide of the invention may be recombmed with one or more components, motifs, sections, parts, domains, fragments, etc of one or more heterologous molecules
Antibodies
Further 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) Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeπc antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression hbraiy, anti-idiotypic (anti-Id) antibodies (including e g , anti-Id antibodies to antibodies of the invention) and epitope-binding fi gments of any of the above The term antibody as used heiein, refeis to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules 1 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
Most preferably 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-lmked Fvs (sdFv) and fragments comprising either a VL or VH domain Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regιon(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 regιon(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 Preferably, the antibodies are human, murine (e g , mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken As used herein, "human ' antibodies include antibodies having the amino acid sequence of a human immunoglobulm 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, tπspecific or of greater multispecificity Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material See, e g , PCT publications WO 93/17715, WO 92/08802, WO 91/00360, WO 92/05793, Tutt, et al . J Immunol 147 60-69 (1991), U S Patent Nos 4,474,893, 4,714,681 , 4,925,648, 5,573,920, 5,601,819, Kostelny et al , J Immunol 148 1547-1553 (1992) Antibodies of the present in ention may be described or specified in terms of the epιtope(s) 01 poιtιon(s) of a poly peptide of the piesent invention w hich they recognize or specifically bind The epιtope(s) or polypeptide portιon(s ) may be specified as described herein, e g , by N-terminal and C-terminal positions, by size in contiguous amino acid lesidues, or listed in the Tables and Figuies Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded Therefoie, the present inv ention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same Antibodies of the present inv ention 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 w ith at least 95%, at least 90%. at least 85%, at least 80%, at least 75%, at least 707c 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 corresponding epitopes thereof Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%. less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combιnatιon(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein) Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 102 M. 102 M, 5 X 10 ' M, lO ' M^ X 104 M, 104 M, 5 X 10 ' M, 10 ' M, 5 X 106 M, 106M. 5 X 107 M, 107 M, 5 X 10 s M, 108 M, 5 X 109 9 s
M, 10 ; M, 5 X 10 I(, M, 10 I0 M. 5 X 10 " M 10 " M, 5 X 10 ' M lϋ p M 5 X 10 1 M, 10 ' M 5 X 10 i4 M 10 I4 M 5 X 10 " M oi 10 " M
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as detei mined by any method known in the art for detei mining competitive binding foi example, the immunoassays described herein In preferred embodiments 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 For example, the present invention includes antibodies which disrupt the receptor/hgand interactions with the polypeptides of the invention either partially or fully Preferrably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof The invention features both receptor-specific antibodies and hgand-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 For example, receptor activation can be determined by detecting the phosphorylation (e g , tyrosine or seπne/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra) In specific embodiments, 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-hgand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor Further included in the invention are antibodies which activate the receptor These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281 ; U.S. Patent No. 5,81 1,097; Deng et al.. Blood 92(6): 1981-1988 ( 1998); Chen et al.. Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 ( 1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170- 3179 (1998); Prat et al., J. Cell. Sci. 1 1 l(Pt2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2): 177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272( 17): 1 1295-1 1301 (1997); Taryman et al., Neuron 14(4):755-762 ( 1995); Muller et al., Structure 6(9): 1 153-1 167 (1998); Bartunek et al., Cytokine 8(1): 14-20 ( 1996) (which are all incorporated by reference herein in their entireties).
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. For example, 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) (incoφorated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387. The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g.. by glycosylation, acetylation. pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation. formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various 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. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody refeis to an antibody that is denved fiom a single clone, including any eukaiyotic, piokaryotic oi phage clone, and not the method by which it is pioduced
Methods for producing and scieemng for specific antibodies using hybridoma technology are routine and well known in the ait and are discussed in detail in the Examples (e g , Example 16) In a non-limiting example, mice can be immunized with a polypeptide of the invention oi a cell expressing such peptide Once an immune response is detected, e g , antibodies specific foi the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC Hybridomas are selected and cloned by limited dilution The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones Accordingly, 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 For example, 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 CH 1 domain of the heavy chain
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them In a particular embodiment, such phage can be utilized to display antigen binding domains expiessed fiom a lepeitoire or combinatoπal antibody hbraiy (e g human or munne) Phage expressing an antigen binding domain that binds the antigen of inteiest can be selected 01 identified with antigen, e g using labeled antigen or antigen bound 01 captured to a solid surface oi bead Phage used in these methods are typically filamentous phage including fd and M13 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 Bπnkman et al , J Immunol Methods 182 41-50 ( 1995), Ames et al , J Immunol Methods
184 177-186 ( 1995), Kettleborough et al , Eui J Immunol 24 952-958 ( 1994). Persic et al , Gene 187 9-18 (1997), Burton et al , Advances in Immunology 57 191-280 (1994), PCT application No PCT/GB91/01 134, PCT publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and U S Patent Nos 5,698,426, 5.223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108, each of which is incorporated herein by reference in its entirety
As described in the above references, after phage selection, 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 For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324, Mullinax et al , BioTechmques 12(6) 864-869 (1992), and Sawai et al , AJRI 34 26- 34 (1995), and Better et al , Science 240 1041-1043 (1988) (said references incorporated by reference in their entireties)
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U S Patents 4,946,778 and 5,258,498, Huston et al , Methods in Enzymology 203 46-88 (1991), Shu et al , PNAS 90 7995-7999 (1993), and Skerra et al , Science 240 1038-1040 (1988) For some uses, including in vivo use of antibodies in humans and in vitio detection assays, it may be piefeiable to use chimeπc, humanized oi human antibodies A chimeric antibody is a molecule in which diffeient portions of the antibody are denved fiom different animal species, such as antibodies having a variable region denved from a murine monoclonal antibody and a human immunoglobulm constant legion Methods for producing chimeric antibodies are known in the ait See e g , Morrison, Science 229 1202 (1985), Oi et al , BioTechmques 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 mcorpoiated herein by reference in their entnety 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 Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding These framework substitutions are identified by methods well known in the art, e g , by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (See, e g , Queen et al , U S Patent No 5,585,089, Riechmann et al , Nature 332 323 ( 1988), which are incorporated herein by reference in their entireties ) 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), Studmcka 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)
Completely human antibodies are particulaily 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, 1 1 1 , 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 mcoiporated heiein by refeience in its entnety
Human antibodies can also be produced using tiansgemc mice which are incapable of expressing functional endogenous immunoglobulins, but which can expiess human immunoglobulin genes Foi example, the human heavy and light chain immunoglobulm gene complexes may be intioduced randomly or by homologous recombination into mouse embryonic stem cells Alternatively, 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 poition 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 Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int Rev Immunol 13 65-93 (1995) For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e g , PCT publications WO 98/24893, WO 92/01047, WO 96/34096, WO 96/33735, European Patent No 0 598 877, U S Patent Nos 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318. 5,885,793, 5,916,771 , and 5,939,598, which are incorporated by reference herein in their entirety In addition, companies such as Abgenix, Ine (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
Further, 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)). For example, 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. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such 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.
Polynucleotides Encoding Antibodies
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. For example, if the nucleotide sequence of the antibody is known, 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. Alternatively, 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 cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide 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. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A . Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, 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. Using routine lecombinant DNA techniques one 01 more of the CDRs may be inserted within framewoik legions e g . into human fiamewoik regions to humanize a non human antibody as desciibed supi The fiamework legions may be naturally occurπng 01 consensus fiamewoik legions, and pieferably human fiamework regions (see, e g , Chothιa et l J Mol Biol 278 457-479 ( 1998) for a listing of human framework regions) Preferably, the poly nucleotide geneiated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention Preferably as discussed supia 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 ammo acid substitutions or deletions of one or more variable region cysteine residues paiticipating in an intrachain disulfide bond to generate antibody molecules lacking one or more intiachain disulfide bonds Other alterations to the polynucleotide are encompassed by the piesent invention and within the skill of the art
In addition, techniques developed for the production of "chimeric antibodies' (Morrison et al , Proc Natl Acad Sci 81 851-855 (1984), Neuberger et al , Nature 312 604-608 ( 1984), Takeda et al , Natuie 314 452-454 ( 1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appiopriate biological activity can be used As described supra, a chimeπc antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region deπved from a murine mAb and a human immunoglobulin constant region, e g , humanized antibodies Alternatively, techniques described for the production of single chain antibodies (U S Patent No 4,946,778. Bird, Science 242 423- 42 (1988), Huston et al , Proc Natl Acad Sci USA 85 5879-5883 (1988), and Ward et al , Nature 334 544-54 ( 1989)) can be adapted to produce single chain 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)) Methods of Producing Antibodies
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once 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. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promotei In piefeπed embodiments foi the expiession of double-chained antibodies vectors encoding both the heavy and light chains may be co expressed in the host cell for expiession of the entne immunoglobulin molecule as detailed below
A vanety of host-expiession vector systems may be utilized to expiess the antibody molecules of the invention Such host-expression systems repiesent vehicles by which the coding sequences of inteiest may be produced and subsequently purified, but also represent cells which may, when transformed or tiansfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ These include but are not limited to micioorganisms such as bacteria (e g , E coli, B subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectois containing antibody coding sequences, yeast (e g , Saccharomyces Pichia) tiansformed 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 recombmant expression constructs containing promoters derived from the genome of mammalian cells (e g , metallothionein promoter) or from mammalian viruses (e g , the adenovirus late promoter, the vaccinia virus 7 5K promoter) Preferably, bacterial cells such as Escheπchia 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 For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al , Gene 45 101 (1986), Cockett et al , Bio/Technology 8 2 (1990)) In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, 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. 24:5503- 5509 (1989)): and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region 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. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entne inseit These exogenous translational control signals and initiation codons can be of a vanety of origins, both natuial and synthetic The efficiency of expiession may be enhanced by the inclusion of appiopriate transcription enhancer elements transcription teiminators etc (see Bittner et al , Methods in Enzymol 153 51-544 ( 1987))
In addition 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 piotein products may be important for the function of the piotein Different host cells have charactenstic and specific mechanisms for the posttranslational processing and modification of pioteins and gene products Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed To this end, 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 For long-term, high-yield production of recombinant proteins, stable expression is preferred For example, cell lines which stably express the antibody molecule may be engineered Rather than using expression vectors which contain viral origins of replication, 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
Following the introduction of the foreign DNA, 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 paiticulaily useful in scieemng and evaluation of compounds that interact directly oi mdnectly with the antibody molecule
A numbei of selection systems may be used, including but not limited to the herpes simplex vnus thymidme kinase (Wiglei et al , Cell 1 1 223 ( 1977)), hypoxanthme-guanine phosphoribosyltiansfeiase (Szybalska & Szybalski, Pioc Natl Acad Sci USA 48 202 ( 1992)), and adenine phosphonbosyltransferase (Lowy et al , Cell 22 817 ( 1980)) genes can be employed in tk-, hgpit- or aprt- cells, respectively Also, 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 mycophenohc acid (Mulligan & Berg, Proc Natl Acad Sci USA 78 2072 (1981)), neo, which confers resistance to the aminoglycoside G- 418 Clinical Pharmacy 12 488-505, Wu and Wu, Biotherapy 3 87-95 (1991), Tolstoshev, Ann Rev Pharmacol Toxicol 32 573-596 (1993), Mulligan, Science 260 926-932 ( 1993), and Morgan and Anderson, Ann Rev Biochem 62 191-217 (1993), May, 1993, TIB TECH 11(5) 155-215), and hygro, which confers resistance to hygromycin (Santerre et al , Gene 30 147 (1984)) Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al (eds ), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993),
Kπegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), and in Chapters 12 and 13, Dracopoh et al (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colberre-Garapin et al , J Mol Biol 150 1 (1981), which are incoφorated by reference herein in their entireties 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)) When a marker in the vector system expressing antibody is amphfiable, 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. Alternatively, 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.
Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it 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. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art. to facilitate purification. The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, 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., Harboi et al supia. and PCT publication WO 93/21232 EP 439 095, Naramura et al , Immunol Lett 39 91 99 ( 1994) U S Patent 5 474 981 , Gillies et al , PNAS 89 1428-1432 ( 1992), Fell et al , J Immunol 146 2446-2452( 1991 ) which aie incorporated by leference in then entπeties The present invention furthei includes compositions compπsing the polypeptides of the present invention fused or conjugated to antibody domains othei than the variable regions For example 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 piesent invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof The polypeptides may also be fused or conjugated to the above antibody portions to form multimeis For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions Higher multimenc forms can be made by fusing the polypeptides to portions of IgA and IgM Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art See, e g , U S Patent Nos 5.336.603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946, EP 307,434, EP 367,166, PCT publications WO 96/04388, WO 91/06570, Ashkenazi et al , Proc Natl Acad Sci USA 88 10535-10539 (1991), Zheng et al , J Immunol 154 5590-5600 (1995), and Vil et al , Proc Natl Acad Sci USA 89 11337- 11341(1992) (said references incorporated by reference in their entireties)
As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art Further, the polypeptides corresponding to SEQ ID NO Y may be fused or conjugated to the above antibody portions to facilitate purification One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptιde and various domains of the constant regions of the heavy or light chains of mammalian lmmunoglobulins (EP 394,827, Traunecker et al , Nature 331 84-86 (1988) The polypeptides of the present invention fused or conjugated to an antibody having disulfide linked dimeiic stiuctures (due to the IgG) may also be moie efficient in binding and neutiahzing othei molecules than the monomenc secreted piotein oi protein fiagment alone (Fountoulakis et al , J Biochem 270 3958-3964 ( 1995)) In many cases the Fc pait in a fusion protein is beneficial in therapy and diagnosis, and thus can result in foi example, improved pharmacokmetic propei ties (EP A 232,262) Alternatively, deleting the Fc part after the fusion piotein has been expressed, detected, and purified, would be desned For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations In diug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the puφose of high-throughput screening assays to identify antagonists of hIL-5 (See. Bennett et al , J Moleculai Recognition 8 52-58 (1995), Johanson et al , J Biol Chem 270 9459-9471 (1995)
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Ine , 9259 Eton Avenue, Chatsworth, CA, 9131 1). among others, many of which are commercially available As described in Gentz et al , Proc Natl Acad Sci USA 86 821-824 (1989), for instance, hexa- histidine provides foi convenient purification of the fusion protein Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinm 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 eithei directly to the antibody (or fragment theieof) 01 indirectly thiough an mteimediate (such as foi example a lmkei known in the ait) using techniques known in the ait See, foi example. U S Patent No 4,741 ,900 tor metal ions which can be conjugated to antibodies foi use as diagnostics according to the piesent invention Examples of suitable enzymes include hoiseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylchohnesteiase, examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotm, examples of suitable fluorescent materials include umbelhferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotπazinylamine fluorescein, dansyl chloride or phycoerythπn. an example of a luminescent material includes luminol, examples of bioluminescent materials include luciferase, lucifeπn, and aequoπn. and examples of suitable radioactive material include 1251, 1311, 11 lln or 99Tc
Further, 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, 213Bι A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells Examples include pachtaxol, cytochalasm B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincπstine, vinblastme, colchicin, doxorubicin. daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydiotestosterone, glucocorUcoids, procame, tetracaine, lidocame, propranolol, and puromycin and analogs or homologs thereof Therapeutic agents include, but are not limited to, antimetabohtes (e g , methotrexate, 6-mercaptopuπne, 6-thιoguanιne, cytarabine, 5-fluorouracil decarbazme), alkylating agents (e g , mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomanmtol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyc nes (e g , daunorubicin (formerly daunomyc ) and doxorubicin), antibiotics (e g , dactinomycm (formerly actinomycin), bleomycm, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e g , vincπstine and vinblastme) 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 For example, 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. β-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 97/3491 1), Fas Ligand (Takahashi et al., Int. Immunol, 6: 1567-1574 ( 1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss. Inc. 1985): Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thoφe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 1 19-58 (1982). Alternatively, 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 incoφorated herein by reference in its entirety. An antibody with or without a theiapeutic moiety conjugated to it administeied alone oi in combination with cytotoxic factor(s) and/oi cytokιne(s) can be used as a therapeutic
Immunophenotyping
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 maiker that is differentially expressed at various stages of diffeientiation 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))
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (I e minimal residual disease (MRD) in acute leukemic patients) and "non-self cells in transplantations to prevent Graft-versus-Host Disease (GVHD) Alternatively, 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
Assays For Antibody Binding 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, lmmunoradiometπc assays, fluorescent immunoassays, protein A immunoassays, to name but a few Such assays are routine and well known in the ait (see e g Ausubel et al eds 1994 Cuπent Piotocols in Moleculai Biology, Vol 1 , John Wiley & Sons Ine New Yoik which is incorporated by reference herein in its entnety) Exemplaiy immunoassays aie described bnefly below (but aie not intended by way of limitation) Immunopiecipitation piotocols generally compnse lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Tnton 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 piotein 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 protem G sepharose beads to the cell lysate incubating foi about an hour or more at 4° C, washing the beads in lysis buffei and resuspending the beads in SDS/sample buffer 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-cleaπng the cell lysate with sepharose beads) For further discussion regarding immunoprecipitation protocols see, e g , Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol 1 , John Wiley & Sons, Ine , 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), transferring the protem 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 One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology. Vol. 1 , John Wiley & Sons. Inc., New York at 10.8.1. ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 1 1.2.1. The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of 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 radioimmunoassays. In this case, 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 The present invention is furthei dnected to antibody-based theiapies which involve admimsteπng antibodies of the invention to an animal, piefeiably a mammal and most pieferably a human, patient for tieating one oi moie of the disclosed diseases, disorders. 01 conditions Therapeutic compounds of the inv ention include, but are not limited to antibodies of the invention (including fragments, analogs and derivatives thereof as descπbed herein) and nucleic acids encoding antibodies of the invention (including fiagments, analogs and derivatives thereof and anti-idiotypic antibodies as descπbed herein) The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders oi conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disordeis or conditions described herem The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein
A summary 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 descπbed in more detail below Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic puφoses without undue experimentation 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 pioducts of a species ongin or species leactivity (in the case of antibodies) that is the same species as that of the patient is pieteπed Thus, in a piefeπed embodiment, human antibodies, fiagments deπvatives analogs 01 nucleic acids aie administeied to a human patient for therapy or prophylaxis It is piefeπed to use high affinity and/oi potent in vivo inhibiting and/or neutiahzmg 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 fiagments 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 oi Kd less than 5 X 10 ' M, 10 ' M, 5 X lO ' M 10 ' M, 5 X 104 M, 104 M, 5 X 10 ' M, 10 ' M, 5 X 106 M, 106 M, 5 X 107 M, 107 M, 5 X 108 M, 108 M, 5 X 109 M, 109 M, 5 X 10 10 M, 10 10 M, 5 X 10 " M, 10 " M, 5 X 10 n M, 10 P M, 5 X 10 ' ' M, 10 π M, 5 X 10 14 M, 10 ,4 M. 5 X 10 " M, and 10 !l M
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid In this embodiment of the invention, 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
For general reviews of the methods of gene therapy, see Goldspiel et al , Clinical Pharmacy 12 488-505 (1993), Wu and Wu, Biotherapy 3 87-95 (1991), Tolstoshev, Ann Rev Pharmacol Toxicol 32 573-596 ( 1993), Mulligan, Science 260 926-932 (1993), and Morgan and Anderson, Ann Rev Biochem 62 191-217 (1993), May, TIBTECH 1 1(5) 155-215 (1993) Methods commonly known in the art of recombinant DNA technology which can be used aie described in Ausubel et al (eds ), Current Protocols in Molecular Biology, John Wiley & Sons. NY ( 1993), and Kriegler, Gene Transfer and Expiession A Laboiatory Manual, Stockton Press, NY (1990)
In a preferred aspect, the compound compnses nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody oi fragments or chimeπc pioteins or heavy or light chains thereof in a suitable host In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc Natl Acad Sci USA 86 8932-8935 (1989), Zijlstra et al , Nature 342 435-438 (1989) In specific embodiments, the expressed antibody molecule is a single chain antibody, alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitio, then transplanted into the patient These two approaches are known, respectively, as in vivo or ex vivo gene therapy
In a specific embodiment, 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 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e g , a gene gun, Biolistic, Dupont), or coating with 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 administenng 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 expiessing the receptors), etc. In another embodiment, nucleic acid-hgand complexes can be formed in which the ligand comprises a fusogenic vital peptide to disrupt endosomes. allowing the nucleic acid to avoid lysosomal degradation In yet another embodiment, the nucleic acid can be targeted in vivo foi cell specific uptake and expression, by taigetmg a specific receptor (see, e g , PCT Publications WO 92/06180, WO 92/22635. WO92/20316; WO93/14188, WO 93/20221) Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination
(Koller and Smithies, Pioc Natl. Acad Sci USA 86-8932-8935 (1989), Zijlstra et al . Nature 342.435-438 ( 1989))
In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al , Meth. Enzymol 217:581-599 (1993)) These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6.291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644- 651 (1994); Kiem et al., Blood 83: 1467-1473 (1994), Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epitheha. Adenoviruses naturally infect respiratory epitheha where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3 499-503 ( 1993) piesent a rev iew of adenovirus-based gene theiapy Bout et al , Human Gene Theiapy 5 3- 10 ( 1994) demonstrated the use of adenovirus vectors to tiansfer genes to the respiratory epitheha of rhesus monkeys Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al Science 252 431 -434 ( 1991 ) Rosenfeld et al , Cell 68 143- 155 ( 1992), Mastrangeh et al , J Clin Invest 91 225-234 (1993), PCT Publication WO94/12649, and Wang, et al , Gene Therapy 2 775-783 ( 1995) In a preferred embodiment, adenovirus vectors are used
Adeno-associated virus (AAV) has also been proposed foi use in gene therapy (Walsh et al , Proc Soc Exp Biol Med 204 289-300 ( 1993), U S Patent No 5,436, 146)
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection Usually, the method of transfer includes the transfer of a selectable marker to the cells The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene Those cells are then delivered to a patient
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, 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 to cells (see, e g , Loeffler and Behr, Meth Enzymol 217 599-618 (1993), Cohen et al , Meth
Enzymol 217 618-644 (1993), Cline, Pharmac Ther 29 69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted 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 lecombinant cells can be delivered to a patient by various methods known in the ait Recombinant blood cells (e.g , hematopoietic stem or piogenitor cells) aie preferably administeied intravenously The amount of cells envisioned foi use depends on the desned effect, patient state, etc , and can be determined by one skilled in the art
Cells mto which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, various stem or progenitor cells, in particular hematopoietic stem oi progenitor cells, e g , as obtained from bone marrow, umbilical cord blood, peπpheial blood, fetal liver, etc
In a preferred embodiment, the cell used for gene therapy is autologous to the patient. In an embodiment in which recombinant cells are used in gene therapy, 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. In a specific embodiment, 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 Clime Proc. 61 :771 (1986)).
In a specific embodiment, 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 appropπate 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. For example, 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/oi tissue sample can be determined utilizing techniques known to those of skill in the ait including, but not limited to, rosette formation assays and cell lysis assays In accordance with the invention 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 otheiwise administered a compound, and the effect of such compound upon the tissue sample is observed
Therapeutic/Prophylactic Administration and Composition
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 In a preferred aspect, 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
Various delivery systems are known and can be used to administer 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 In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including lntiaventπculai and intrathecal injection; intraventricular injection may be facilitated by an intraventricular cathetei . for example, attached to a reservoir, such as an Ommaya reservou Pulmonary admmistiation can also be employed, e g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound diessing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, 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.)
In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Cπt. Ref. Biomed Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J Med. 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton. Florida (1974); Controlled Drug Bioavailabihty, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 ( 1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71 : 105 (1989)). In yet another embodiment, 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, supia, vol 2, pp 1 15-138 (1984))
Other contiolled release systems are discussed in the ieview bv Langer (Science 249 1527 1533 (1990)) In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to piomote expression of its encoded piotein, 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 4,980,286), or by direct injection, or by use of microparticle bombardment (e g , a gene gun, Biolistic, Dupont), or coating with lipids or cell-surface leceptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to entei the nucleus (see e g , Johot et al , Proc Natl Acad Sci USA 88 1864-1868 ( 1991)), etc Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination
The present invention also provides pharmaceutical compositions Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U S Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like Water is a preferred carrier when the pharmaceutical composition is administered intravenously Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol. lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, 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. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, 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 aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness 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.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, 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. Generally, 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. Further, 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 puφoses to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, 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 aggressive treatment earlier thereby preventing the development or further progression of the cancer. 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 are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (1 12In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, 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 backgiound level indicates that the subject has a particular disease or disorder associated with aberrant expiession of the polypeptide of interest Background level can be determined by vaπous methods including, comparing the amount of labeled molecule detected to a standard value previously determined foi a particular system
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuπes 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 descπbed 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 Ine (1982)
Depending on several variables, including the type of label used and the mode of administration, 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
In an embodiment, 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.
In a specific embodiment, 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). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI). Kits
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, 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. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the 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).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a lecombinantly pioduced or chemically synthesized polypeptide antigen The polypeptide antigen of the kit may also be attached to a solid suppoit
In a more specific embodiment the detecting means of the above described kit includes a solid support to which said polypeptide antigen is attached Such a kit may also include a non-attached reporter-labeled anti-human antibody In this embodiment binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody
In an additional embodiment, 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 In one embodiment, the antibody is attached to a solid support In a specific embodiment, 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
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention 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 Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometπc, luminescent or coloπmetπc 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 adsoφtion 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 stieptavidin coated plates can be used in conjunction w ith biotinylated antιgen(s)
Thus the invention provides an assay system 01 kit for carrying out this diagnostic method The kit generally includes a support with surface- bound recombinant antigens, and a repoiter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody
Fusion Proteins Any polypeptide of the present invention can be used to generate fusion proteins For example, 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 Moreover, because 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
Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions The fusion does not necessarily need to be direct but may occur through linker sequences
Moreover, 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
Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, 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. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331 :84-86 (1988).)
Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 ( 1995).) Similarly, EP-A-O 464 533 (Canadian counteφart 2045869) 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. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the puφose 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).)
Moreover, the polypeptides of the present invention can be fused to marker . sequences, such as a peptide which facilitates purification of the fused polypeptide. In preferred embodiments, 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, 9131 1), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984).) Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention
Vectors. Host Cells, and Protein Production The present invention also relates to vectois 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 markei for propagation in a host Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a vims, 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, tφ, 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 πbosome 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. As indicated, the expression vectors will preferably include at least one selectable marker. Such 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 appropπate 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-descπbed host cells are known in the art
Among vectois preferred for use in bacteria include pQE70, pQE60 and pQE- 9, available from QIAGEN, Ine , pBluescπpt vectors, Phagescπpt vectors, pNH8A pNHlόa, pNH 18A, pNH46A, available fiom Stratagene Cloning Systems, Ine , and ptrc99a, pKK223 3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Ine Among preferred eukaryotic vectors are pWLNEO, pS V2C AT, pOG44, pXT 1 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 hpid-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 puπfied 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
Polypeptides of the present invention, and preferably the secreted form, 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 Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated piocesses Thus, it is well known in the art that the N-teiminal methionine encoded by the translation initiation codon geneially is removed with high efficiency from any piotein after translation in all eukaryotic cells While the N terminal methionine on most proteins also is efficiently iemoved in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the ammo acid to which the N-terminal methionine is covalently linked
In addition to encompassing host cells containing the vectoi constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, paiticularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e g , coding sequence), and/or to include genetic matenal (e g , heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides For example, techniques known in the art may be used to operably associate heterologous control regions (e g , promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e g , U S Patent No 5,641 ,670, issued June 24, 1997, U S Patent No 5,733,761, issued March 31 , 1998, International Publication No WO 96/2941 1, published September 26, 1996, International Publication No WO 94/12650, published August 4, 1994, Koller et al Proc Natl Acad Sci USA 86 8932-8935 (1989), and Zijlstra et al , Nature 342 435-438 (1989), the disclosures of each of which are incoφorated by reference in their entireties)
In addition, 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, 310 105-1 11 ( 1984)) For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer Furthermore, if desired, 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-dιamιnobutyπc acid, a-amino lsobutyπc acid, 4- aminobutyπc acid, Abu, 2-amιno butyric acid, g-Abu, e-Ahx, 6-amιno 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 (dextrorotary) or L (levorotary).
The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation. acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH,; 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.
Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent NO: 4,179,337). 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. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects 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. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incoφorated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, 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. One may specifically desire proteins chemically modified at the N-terminus.
Using 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) may be by purification of the N-terminally pegylated matenal fiom a population of pegylated piotein molecules Selective proteins chemically modified at the N-tei minus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available foi 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 (l e , dimers, tπmers, tetramers and higher multimers) Accordingly, the piesent invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them In specific embodiments, the polypeptides of the invention are monomers, dimers, tπmers or tetramers In additional embodiments, the multimers of the invention are at least dimers, at least tπmers, or at least tetramers Multimers encompassed by the invention may be homomers or heteromers
As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO Y or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein) These homomers may contain polypeptides having identical or different amino acid sequences 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 In specific embodiments, the multimer of the invention is a homodimer (e g , containing polypeptides having identical or different amino acid sequences) or a homotπmer (e g , containing polypeptides having identical and/or different amino acid sequences) In additional embodiments, the homomeπc multimer of the invention is at least a homodimer, at least a homotπmer, or at least a homotetramer As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i e , polypeptides of different proteins) in addition to the polypeptides of the invention In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, 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. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, 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 heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such 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). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide nkeis Examples include those peptide linkeis descπbed in U S Pat No 5.073,627 (hereby lncoipoiated by reference) Pioteins compiising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology Another method for piepaπng multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zippei 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 seveial 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 aie naturally occurring peptides and derivatives thereof that dimeπze or tπmeπze Examples of leucine zipper domains suitable for producing soluble multimenc proteins of the invention are those described in PCT application WO 94/10308, hereby incoφorated by reference Recombinant fusion proteins compπsing a polypeptide of the invention fused to a polypeptide sequence that dimeπzes or tπmeπzes in solution are expressed in suitable host cells, and the resulting soluble multimenc fusion protem is recovered from the culture supernatant using techniques known in the ait
Trimeπc polypeptides of the invention may offer the advantage of enhanced biological activity Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form tπmers One example is a leucine zipper derived from lung surfactant protem 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 incoφorated by reference Other peptides deπved from naturally occurring trimeπc proteins may be employed in preparing trimeπc polypeptides of the invention
In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide seuqence In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti- Flag® antibody The multimeis of the invention may be geneiated using chemical techniques known in the ait Foi example polypeptides desired to be contained in the multimeis of the invention may be chemically cioss-hnked using linker molecules and linker molecule length optimization techniques known in the ait (see, e L S Patent Numbei 5,478,925, which is herein incoφoiated by refeience in its entirety)
Additionally, multimers of the invention may be geneiated using techniques known in the ait to form one or more mter-molecule cioss-hnks between the cy steme 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 heiein incorporated by reference in its entirety) Further, polypeptides of the invention may be routinely modified by the addition of cysteine oi biotin to the C terminus oi 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 incorporated by reference in its entirety) Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e g , US Patent Number 5,478,925. which is herein incorporated by reference in its entirety)
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art In one embodiment, 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 incoφorated by reference in its entirety) In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by hgating 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) In another embodiment, 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 incorporated by membrane leconstitution techniques into liposomes (see. e g , US Patent Number 5 478,925, which is herein mcoipoiated by reference in its entirety)
Uses of the Polynucleotides
Each of the polynucleotides identified heiein can be used in numerous ways as reagents The following description should be consideied exemplary and utilizes known techniques
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 polymoφhisms), are presently available Each polynucleotide of the piesent invention can be used as a chromosome marker
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO X Pnmeis can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes Only those hybrids containing the human gene corresponding to the SEQ ID NO X will yield an amplified fragment
Similarly, 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, prescieening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries
Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread This technique uses polynucleotides as short as 500 or 600 bases, however, polynucleotides 2,000-4,000 bp are prefened For a review of this technique, see Verma et al.. "Human Chromosomes: a Manual of Basic Techniques." Pergamon Press, New York ( 1988).
For chromosome mapping, 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). Preferred 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.
Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. 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) .) Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymoφhism is identified, this polymoφhic polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Thus, the invention also provides a diagnostic method useful dining diagnosis of a disordei , involving measuπng the expression level of polynucleotides of the present invention in cells 01 body fluid fiom an individual and comparing the measured gene expiession level with a standaid level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compaied to the standard is indicative of a disordei
In still another embodiment the invention includes a kit for analyzing samples for the presence of pro feiative and/or cancerous polynucleotides derived from a test subject In a geneial embodiment 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 In a specific embodiment, 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 In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification
Where a diagnosis of a disorder, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a woise clinical outcome relative to patients expressing the gene at a level nearer the standard level
By ' 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) Preferably, 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 As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended 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. As indicated, 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 preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, 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. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including cancerous diseases and conditions. Such a method is described in US Patents 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incoφorated by reference in their entirety herein.
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. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L Chπstensen. C Behiens. S M Fieiei , D A Dπvei R H Beig, S K Kim. B Noiden. and P E Nielsen, Natuie 365, 666 ( 1993), PNAs bind specifically and tightly to complementaiy DNA strands and aie not degi aded by nucleases In fact, PNA binds more stiongly to DNA than DNA itself does This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is moie flexible Because of this. PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easiei to pertoim multiplex hybridization Smaller probes can be used than with DNA due to the strong binding In addition, it is moie likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T sub m) by 8°-20° C, vs 4°- 16° C for the DNA DNA 15- mer duplex Also, the absence of charge gioups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis The piesent invention is useful for detecting cancer in mammals In particular the invention is useful during diagnosis of pathological cell prohferative neoplasms which include, but are not limited to acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc , and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc Preferred mammals include monkeys, apes, cats dogs, cows, pigs, horses, rabbits and humans Particularly preferred are humans
Pathological cell prohferative disorders are often associated with inappropriate activation of proto-oncogenes (Gelmann, E P et al , "The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood, Vol 1 , Wiermk, P H et al eds , 161- 182 ( 1985)) Neoplas s 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 othei tissues and cell types (Gelmann et al supra) Indeed the human counterpaits of the oncogenes involved in some animal neoplasias have been amplified oi translocated in some cases of human leukemia and carcinoma (Gelmann et al , supi a) For example c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60 When HL-60 cells aie chemically induced to stop prohfeiation, the level of c-myc is found to be downregulated (International Publication Number WO 91/15580) However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5 end of c-myc oi c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells (International Publication Number WO 91/15580, Wickstrom et al , Proc Natl Acad Sci 85 1028 (1988), Anfossi et al . Proc Natl Acad Sci 86 3379 ( 1989)) However, the skilled artisan would appreciate the piesent invention s usefulness would not be limited to treatment of prohferative disorders of hematopoietic cells and tissues, in light of the numeious cells and cell types of vaiying origins which are known to exhibit prohferative phenotypes
In addition to the foregoing, 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),
"Ohgodeoxynucleotides as Antisense Inhibitois of Gene Expression, CRCPress, Boca Raton, FL (1988) Triple helix formation is discussed in, for instance Lee et al , Nucleic Acids Research 6 3073 (1979), Cooney et al , Science 241 456 (1988), and Dervan et al , Science 251 1360 ( 1991 ) Both methods rely on binding of the polynucleotide to a complementary DNA or RNA For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al , Nucl Acids Res 6 3073 (1979), Cooney et al , Science 241 456 (1988), and Dervan et al , Science 251 1360 (1991) ) or to the mRNA itself (antisense - Okano, J Neurochem 56 560 ( 1991), Ohgodeoxy-nucleotides as Antisense
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 hybudization blocks tianslation oi an mRNA molecule into polypeptide Both techniques aie effecti e in model systems, and the information disclosed herein can be used to design antisense oi tuple helix polynucleotides in an effort to tieat disease Polynucleotides of the present invention are also useful in gene therapy One goal of gene theiapy is to insert a normal gene into an organism having a defective gene, in an effort to coirect 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 tiait 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) foi identification of its personnel In this technique, 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 These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples
Forensic biology also benefits from using DNA-based identification techniques as disclosed herein 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 surf actant,uπne, fecal matter, etc , can be amplified using PCR In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, aie used in foiensic biology to identify individuals (Eihch, H , PCR Technology, Freeman and Co (1992) ) Once these specific polymoφhic loci aie amplified, they aie digested with one or more lestnction enzymes, yielding an identifying set of bands on a Southern blot piobed with DNA conesponding to the DQa class II HLA gene Similarly, polynucleotides of the present invention can be used as polymoiphic markers for forensic puφoses
There is also a need foi reagents capable of identifying the souice of a particular tissue Such need arises, for example, in forensics when piesented with tissue of unknown origin Appropriate reagents can compπse, 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 In the very least, 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 ohgomers 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
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways The following description should be considered exemplary and utilizes known techniques A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques For example, protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M , et al., J. Cell Biol 101 976-985 ( 1985). Jalkanen, M , 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 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 ( 1 12In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to assaying secreted protein levels in a biological sample, 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. For X- radiography. 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 incoφorated 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, 1 12In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or mtraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, 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).)
Thus, 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. With respect to cancer, the presence of a relatively high amount of ti nscπpt in biopsied tissue fiom an individual may indicate a predisposition foi the development of the disease 01 may piovide a means for detecting the disease prior to the appearance of actual clinical symptoms A more definitive diagnosis of this type may allow health piofessionals to employ pieventative measures or aggressive treatment earlier thereby preventing the development oi fuither progiession of the cancer
Moieover, polypeptides of the present invention can be used to treat disease For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or deci eased 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 repan 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 prohferative cells or tissues)
Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce oveφroduction of the polypeptide Similarly, administration of an antibody can activate the polypeptide. such as by binding to a polypeptide bound to a membrane (receptor)
At the very least, the 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 theiapy methods foi tieating disordei s, diseases and conditions The gene therapy methods ielate 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 promotei and any othei genetic elements necessary for the expression of the polypeptide by the target tissue Such gene therapy and dehveiy techniques are known in the art, see. foi example, WO90/11092, which is heiem incorporated by reference
Thus, foi example, cells from a patient may be engineered with a polynucleotide (DNA oi RNA) comprising a piomoter 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 Such methods are well-known in the art For example, see Belldegrun et al , J Natl Cancer Inst , 85 207-216 (1993), Ferrantini et al , Cancer Research, 53 107-1 1 12 ( 1993), Ferrantim et al , J Immunology 153 4604-4615 (1994), Kaido, T , et al , Int J Cancer 60 221-229 (1995), Ogur et al , Cancer Research 50 5102-5106 (1990), Santodonato, et al , Human Gene Therapy 7 1-10 (1996), Santodonato, et al , Gene Therapy 4 1246-1255 (1997), and Zhang, et al , Cancer Gene Therapy 3 31-38 (1996)), which are herein incorporated by reference In one embodiment, 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 As discussed in more detail below, 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
In one embodiment, the polynucleotide of the invention is delivered as a naked polynucleotide The term "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. However, 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 incorporated by reference.
The 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.
Any strong promoter known to those skilled in the art can be used for driving the expression of polynucleotide sequence of the invention. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (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 Heφes 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.
Unlike other gene therapy techniques, 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, mucopolysacchaπde matnx among the leticular fibeis of oigan tissues elastic tibeis in the walls of vessels 01 chambers collagen fibeis of fibrous tissues or that same matnx within connective tissue ensheathmg muscle cells or in the lacunae of bone It is similaily the space occupied by the plasma of the cnculation and the lymph fluid of the lymphatic channels Delivery to the interstitial space of muscle tissue is prefened for the reasons discussed below They may be convementlv delivered by injection into the tissues comprising these cells They aie preferably delivered to and expressed in persistent, non dividing cells which aie diffeientiated although delivery and expression may be achieved in non-diffeientiated or less completely differentiated cells, such as for example, stem cells of blood or skin fibroblasts In xtvo muscle cells aie particularly competent in their ability to take up and express polynucleotides
For the naked /c/e/c acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0 05 mg/kg body weight to about 50 mg/kg body weight Preferably the dosage will be from about 0 005 mg/kg to about 20 mg/kg and more preferably from about 0 05 mg/kg to about 5 mg/kg Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues However, other parenteral routes may also be used, such as. inhalation of an aerosol formulation particularly for delivery to lungs oi bronchial tissues, throat or mucous membranes of the nose In addition, 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 In certain embodiments, 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. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA , 84:7413-7416 ( 1987), which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 ( 1989), which is herein incorporated by reference); and purified transcription factors (Debs et al.. J. Biol. Chem.,
265: 10189-10192 ( 1990), which is herein incorporated by reference), in functional form.
Cationic liposomes are readily available. For example, 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. See, e.g. PCT Publication NO: WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) 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 incoφorated by reference.
Similar methods can be used to prepare liposomes from other cationic lipid materials.
Similarly, 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. These materials can also be mixed with the DOTMA and DOTAP staiting mater ials in appropπate ratios Methods tor making liposomes using these materials are well known in the art
For example commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glyceiol (DOPG). and dioleoylphosphatidyl ethanolamine (DOPE) can be used in vaπous combinations to make conventional liposomes, with or without the addition of cholesterol Thus, for example DOPG/DOPC vesicles can be prepaied by drying 50 mg each of DOPG and DOPC under a stieam of nitrogen gas into a sonication vial The sample is placed under a vacuum pump overnight and is hydiated the following day with deiomzed water The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 somcator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce umlamellar 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 umlamellar vesicles (SUVs), oi large umlamellar vesicles (LUVs), with SUVs being preferred The various hposome-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 For example, MLVs containing nucleic acid can be prepared by depositing a thm film of phosphohpid 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 umlamellar liposomes The material to be entrapped is added to a suspension of preformed MLVs and then sonicated When using 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 Tπs/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 Ca2+-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. Acad. Sci. USA . 76: 145 ( 1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255: 10431 ( 1980); Szoka et al., Proc. Natl. Acad. Sci. USA . 75:145 ( 1978); Schaefer-Ridder et al.. Science, 215: 166 ( 1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10: 1 to about 1: 10. Preferably, 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 incoφorated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Patent Nos. 4,897,355, 4,946.787, 5,049,386, 5,459, 127. 5.589,466. 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incoφorated 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 incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals. In certain embodiments, 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. Examples of 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+env Am 12, and DAN cell lines as described in Miller, Human Gene Therapy , 1 :5-14 (1990), which is incoφorated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the ait. Such means include, but aie not limited to. electroporation, the use of liposomes, and CaP04 piecipitation In one alternative, the letiovnal plasmid vector may be encapsulated into a liposome. or coupled to a lipid, and then administered to a host. The producei cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such letroviral vectoi particles then may be employed, to transduce eukaryotic cells, eithei in vitro or in vivo. The transduced eukaryotic cells will expiess polypeptides of the invention. In certain other embodiments, cells aie engineered, ex vivo oi 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 msertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Re . Respir. Dis., 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha- 1-antιtrypsιn and CFTR to the lungs of cotton rats (Rosenfeld et al.,Scιence , 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. Opm. 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 For example, 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. In addition to Ad2, other varieties of adenovnus (e g Ad3 Ad5 and Ad7) aie also useful in the piesent invention
Preferably, the adenovnuses used in the piesent invention aie lephcation deficient Replication deficient adenovnuses lequne the aid of a helper vnus and/or packaging cell line to form infectious particles The resulting vnus is capable of infecting cells and can express a polynucleotide of inteiest 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 oi a portion of the following genes Ela, Elb, E3, E4, E2a, or LI through L5 In certain other embodiments, the cells are engineered, e\ xivo or in vivo, using an adeno-associated virus (AAV) AAVs are natuially occurπng defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr 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
For example, 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 Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses 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 Anothei method of gene theiapy involves opeiably associating heteiologous contiol regions and endogenous polynucleotide sequences (e g encoding the polypeptide sequence of inteiest) v ia homologous recombination (see e g U S Patent NO 5,641 ,670 issued June 24 1997. International Publication NO WO 96/2941 1 , published Septembei 26, 1996, International Publication NO WO 94/12650, published August 4, 1994, Koller et l , Pioc Natl Acad Sci USA, 86 8932-8935 ( 1989), and Zijlstra et al , Natuie. 342 435-438 ( 1989) This method involves the activation of a gene which is piesent in the target cells but which is not normally expressed in the cells, or is expressed at a lower level than desired Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the piomoter 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 Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends Preferably, the 3' 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-facihtatmg 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 promotei taigeting sequence constiuct is taken up by cells Homologous recombination between the construct and the endogenous sequence takes place such that an endogenous sequence is placed undei the contiol of the piomotei The promoter then drives the expression of the endogenous sequence The polynucleotides encoding polypeptides of the present invention may be administered along with othei polynucleotides encoding other angiongemc prote s Angiogenic proteins include but aie not limited to, acidic and basic fibroblast growth factors, VEGF-1 , VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal giowth factor alpha and beta, platelet-derived endothelial cell giowth factor, platelet-deπved 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
Preferably, the polynucleotide encoding a polypeptide of the invention contains a secretory signal sequence that facilitates secretion of the protein Typically, 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 heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected Additionally the signal sequence may be chemically synthesized using methods known in the art Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (I e , "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e g , Alza mimpumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery For example, 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 the foreign gene in the rat livers (Kaneda et al , Science, 243 375 (1989))
A preferred method of local administration is by direct injection Preferably, 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. For example, 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.
Therapeutic 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.
Preferred 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: 11277-1 1281 (1992), which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin. 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 piesent invention can be administeied to any animal, prefeiably to mammals and buds Prefened mammals include humans, dogs, cats, mice rats, rabbits sheep cattle, horses and pigs with humans being paiticulaily
Biological Activities
The polynucleotides or polypeptides or agonists oi antagonists of the present invention can be used in assays to test foi 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
Immune Activity
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 fiom pluπpotent 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 oi toxins), or infectious Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the piesent invention can be used as a marker or detectoi 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 pluπpotent stem cells, m an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells Examples of lmmunologic deficiency syndromes include, but are not limited to blood protein disorders (e g agammaglobulinemia, dysgammaglobuhnemia), ataxia telangiectasia, common vanable immunodeficiency Digeoige Syndiome, HIV infection HTLV-BLV infection, leukocyte adhesion deficiency syndiome lymphopenia phagocyte bactericidal dysfunction, seveie combined immunodeficiency (SCIDs) Wiskott-Aldπch Disoidei anemia, thiombocytopema or hemoglobinuπa
Moreovei, a polynucleotides or polypeptides oi agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation) For example, by increasing hemostatic or thrombolytic activity a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to tieat blood coagulation disorders (e g , afibπnogenemia, factor def crencies), blood platelet disorders (e g thrombocytopenia), oi wounds resulting from trauma, surgery, or other causes Alternatively, 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 scarring
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
Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to Addison's Disease, hemolytic anemia, antiphosphohpid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyehtis, glomerulonephπtis, Goodpasture's Syndrome, Graves' Disease. Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocπnopathies, Puφura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.
Similarly, 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. Moreover, 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. Similarly, 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.
Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate inflammation. For example, 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-1.)
Hvperproliferative Disorders A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat or detect hypeφroliferative disorders, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the piohfeiation ot the disoidei through dnect oi indiiect interactions Alternatively, a polynucleotides or polypeptides, 01 agonists or antagonists of the piesent invention may prohfeiate othei cells which can inhibit the hy peφrohferative disorder For example by lncieasmg an immune response, particularlv increasing antigenic qualities of the hypeφiohfeiative disoidei or by proliferating, differentiating, or mobilizing T-cells hyperpiohferative disorders can be tieated This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response Alternatively, decreasing an immune response may also be a method of ti eating hyperproliferativ e disorders, such as a chemotheiapeutic agent
Examples of hyperprohferative 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 (cential and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogemtal
Similarly, other hyperprohferative disorders can also be treated or detected by a polynucleotides or polypeptides, or agonists or antagonists of the present invention Examples of such hypeφrohferative disorders include, but are not limited to hypergammaglobulmemia, lymphopiohferative disorders, paraprotememias, puφura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia. Gaucher's Disease, histiocytosis, and any other hypeφrohferative disease, besides neoplasm, located in an organ system listed above
One preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof
Thus, the present invention provides a method for treating cell prohferative 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 tieatmg cell-piohferative disoideis in individuals compiising admimstiation of one or more active gene copies of the piesent invention to an abnormally prohfeiating cell or cells In a pieferred embodiment polynucleotides of the piesent invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides In another prefened embodiment of the present invention the DNA constiuct encoding the poynucleotides of the present invention is inseited into cells to be treated utilizing a retrovirus, or more preferrably an adenoviral vector (See G J Nabel. et al , PNAS 1999 96 324-326, which is hereby incoφorated by reference) In a most pieferred embodiment, the viral vector is defective and will not transform non-prohfeiating cells, only proliferating cells Moreovei, in a preferred embodiment, the polynucleotides of the present invention inserted mto 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 As such 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 oncogemc genes or antigens By 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 messengei 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
For local administration to abnormally proliferating cells, 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, micromjection of cells, or in vehicles such as liposomes, lipofectin, oi 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. 85:3014), vaccinia virus system (Chakrabarty 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. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus. or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack 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 prohferative 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.
By "cell prohferative disease" is meant 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. By "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 piesent invention is fuither dnected to antibody-based theiapies which involve administeπng of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, piefei ably human patient foi tieatmg one 01 more of the desciibed disorders Methods for pioducmg anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewheie herein Such antibodies may be provided in phaimaceutically acceptable compositions as known in the ait or as desciibed 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) oi by effector cells (ADCC) Some of these approaches aie described in more detail below Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation
In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell prohferative 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 5X106M, 106M, 5X10 ~M, 107M, 5X108M, 10 M, 5X109M, 109M, 5X10 10M, 10 IOM 5X 10 "M 10 "M, 5X 10 ' M 10 PM, 5X 10 ' M. 10 ' M 5X 10 14M, 10 14M. 5X 10 "M, and 10 "M
Moieovei , polypeptides of the piesent invention are useful in inhibiting the angiogenesis of piohferative cells oi tissues eithei alone, as a protein fusion, or in combination with other polypeptides dnectly or indirectly, as desciibed elsewhere herein In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic. tumor- specific cells, such as tumor-associated macrophages (See Joseph IB et al J Natl Cancer Inst, 90(21 ) 1648-53 (1998), which is hereby incorporated by reference) Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis dnectly, oi indirectly (See Witte L, et al , Cancer Metastasis Rev 17(2) 155-61 ( 1998), which is hereby incorporated by reference))
Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting prohferative cells or tissues through the induction of apoptosis Said polypeptides may act either directly, or indirectly to induce apoptosis of prohferative cells and tissues, for example in the activation of a death- domain receptor, such as tumor necrosis factor (TNF) receptoι-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 incorporated by reference) Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis thiough 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 apoptonm, galectins, thioredoxins, antnnflammatory proteins (See for example, Mutat Res 400(1-2) 447-55 (1998), Med Hypotheses 50(5) 423-33 (1998), Chem Biol Interact Apr 24,1 1 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 incorporated by reference)
Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of prohferative cells or tissues Inhibition may occur as a direct lesult of administeπng polypeptides oi antibodies directed to said polypeptides as described elseweie heiein, or lndnectly, such as activating the expiession of protems known to inhibit metastasis, foi example alpha 4 integrins, (See, e g , Curr Top Miciobiol Immunol 1998,231 125-41 which is hereby incoφorated by refeience) Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants
In anothei embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e g , compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydiophihc, ionic and/or covalent interactions Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the lmmunogenicity and/or antigemcity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention 'vaccinated' the immune lesponse to respond to prohferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e g chemokmes), to said antigens and immunogens
Cardiovascular Disorders
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 arteπo- arteπal fistula, arteπovenous fistula, cerebral arteπovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome Congenital heart defects include aortic coarctation, cor tπatπatum, 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, tπcuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonaiy septal defect, endocaidial cushion defects, Lutembachei s Syndiome trilogy of Fallot, ventricular heait septal defects
Cardiovascular disordeis also include heait disease, such as aπhythmias, carcinoid heait disease, high cardiac output low caidiac output, cardiac tamponade endocarditis (including bacterial) heait aneuiysm, caidiac arrest congestive heait failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heait hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypei trophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocaidial ischemia, peπcardial effusion, pericarditis (including constπctive and tuberculous), pneumopeπcardium, postpeπcardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitai Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis
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, Mah-tim- type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation Tachycaidias include paroxysmal tachycardia, supraventπcular tachycardia, accelerated ldioventπcular rhythm, atrioventncular 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, tπcuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tπcuspid atresia, tπcuspid valve insufficiency, and tπcuspid valve stenosis
Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial ftbroelastosis, 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, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
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 purpura, 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.
Anti-Angiogenesis Activity
The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al, Cell 56:345-355 ( 1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, 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 senous diseases aie dominated by abnormal neovascularization including solid tumoi growth and metastases, aithπtis, 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 Mia ovasc Res 29 401 -41 1 ( 1985), Folkman, Advances in Cancel 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 227 719-725 ( 1983) In a number of pathological conditions, the process of angiogenesis contributes to the disease state For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis Folkman and Klagsbrun, Science 235 442-447 (1987)
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, sohd 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)) Thus, 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 For example, polynucleotides, polypeptides, antagonists and/or agonists 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, endometπum, kidney, bladder, thyroid cancer, primary tumors and metastases, melanomas, ghoblastoma, Kaposi's sarcoma, leiomyosarcoma, non- small cell lung cancer, colorectal cancer, advanced malignancies, and blood born tumors such as leukemias For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to tieat cancers such as skin cancel, head and neck tumois bieast tumois. and Kaposi's sarcoma
Within yet othei aspects, polynucleotides, polypeptides, antagonists and/oi agonists may be utilized to treat superficial foims of bladder cancel by, foi example, intravesical administiation Polynucleotides, polypeptides, antagonists and/or agonists may be dehveied directly into the tumor, or near the tumor site via injection or a cathetei Of couise, as the artisan of ordinal y skill will appreciate 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 disoiders, besides cancers, which involve angiogenesis These disorders include, but are not limited to benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogemc granulomas, artheroscleπc 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, endometπosis, vasculogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, lschemic limb angiogenesis, Osier- Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma, fibromuscular dysplasia, wound granulation, Crohn's disease, and atherosclerosis
For example, within one aspect of the present invention methods are provided 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
Within one embodiment of the present invention 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 aftei the piohfeiative phase has had time to piogiess (appioximately 14 days after the initial injury), but befoie hypertrophic scar oi keloid development As noted above, the present invention also provides methods tor tieating neovascular diseases of the eye, including for example corneal neovascularization, neov scular glaucoma, prohferative diabetic letinopathy retiolental fibioplasia and macular degeneration
Moreovei Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/oi antagonists) include, but are not limited to neovasculai glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibioplasia, uveitis, retinopathy of prematurity macular degeneration corneal graft neovascularization. as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or ins neovascularization See, e g . reviews by Waltman et al , Am J Ophthal 85 704- 710 (1978) and Gartner et al , Surv Ophthal 22 291-312 (1978)
Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization) comprising the step of administering to a patient a therapeutically effective amount of a compound (as descπbed above) to the cornea, such that the formation of blood vessels is inhibited Briefly, the cornea is a tissue which normally lacks blood vessels In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus When the cornea becomes vasculaπzed, it also becomes clouded, resulting in a decline in the patient's visual acuity Visual loss may become complete if the cornea completely opacitates A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e g , trachoma, herpes simplex keratitis, leishmamasis and onchocercmsis), immunological processes (e g , graft rejection and Stevens-Johnson s syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses
With particularly preferred embodiments of the invention, 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 admimsteied seveial times daily Alternatively, anti-angiogenic compositions, prepaied as descπbed above, may also be admimsteied dnectly to the coinea Within preferred embodiments, the anti-angiogenic composition is prepared with a muco- adhesive polymer which binds to cornea Withm furthei embodiments, the anti- angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns) In these instances the tieatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications
Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance
The preferred site of injection may vary with the moφhology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (l e , interspersed between the blood vessels and the normal cornea) In most cases this would involve peπhmbic corneal injection to "protect" the cornea from the advancing blood vessels This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization
In this situation the material could be injected in the peπlimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas In a sustained-release form injections might only be required 2-
3 times per year A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself Within another aspect of the present invention, methods are provided for treating 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 In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle Within other embodiments, the compound may also be placed in any location such that the compound is continuously leleased into the aqueous humor Withm another aspect of the present invention, methods are provided foi treating piohferative diabetic retinopathy, comprising the step of administering to a patient a theiapeutically effective amount of a polynucleotide, polypeptide, antagonist and/oi agonist to the eyes, such that the loimation of blood vessels is inhibited
Within paiticularly preferred embodiments of the invention, prohferative 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 retma Preferably, this tieatment should be initiated prior to the acquisition of severe disease requiring photocoagulation
Withm another aspect of the present invention, methods are provided foi treating letrolental 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
Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atheroscleiotic plaques, delayed wound healing, granulations, hemophihc joints, hypertrophic scars, nonunion fractuies, Osier- Weber syndrome, pyogemc granuloma, scleroderma, trachoma, and vascular adhesions
Moreover, 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 born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogemc granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, lschemic limb angiogenesis, Oslei -Webber Syndrome plaque neovascularization, telangiectasia, hemophrhac jomts, angiofibroma fibromuscular dysplasia, wound gianulation, Crohn s disease, atheioscleiosis. bπth contiol agent by pieventing vascularization required for embryo implantation conti oiling menstruatron, drseases that have angrogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcei s (Hehcobacter pylori), Bartonellosis and bacillary angiomatosis
In one aspect of the birth contiol method, 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 aftei 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 incorporated into surgical sutures in order to prevent stitch granulomas
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures For example, within one aspect of the piesent invention a compositions (in the form of, for example, a spray or film) 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 Within other aspects of the present invention, compositions (e g , in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale Within yet other aspects of the present invention, 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 For example, within one embodiment of the invention 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 Within fuithei aspects of the present invention methods aie piovided foi treating tumor excision sites compπsing administering a polynucleotide, polypeptide, agonist and/oi agonist to the resectron margrns of a tumor subsequent to excision, such that the local lecurrence of cancer and the formation of new blood vessels at the site is inhibited Within one embodiment of the invention the anti-angiogenic compound is administered directly to the tumor excision site (e g , applied by swabbing, brushing or otherwise coating the resection maigins of the tumor with the anti-angiogenic compound) Alternatively, the anti-angiogenic compounds may be incorpoiated into known surgical pastes prior to administiation Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neuiosurgical operations
Within one aspect of the present invention, 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 For example, within one embodiment of the invention, 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
The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors Representative examples of other anti-angiogenic factors include Anti-Invasive Factor, retinoic acid and derivatives thereof, pachtaxel, Suramm, Tissue Inhibitor of Metalloproteιnase-1 , Tissue Inhibitor of Metalloproteιnase-2, Plasminogen Activator Inhibitor- 1, Plasminogen Activator Inhιbιtor-2, and various forms of the lighter "d group" transition metals 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
Representative examples of 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.
Representative examples of 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.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP- PG) (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. 267: 17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al.. Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST" Matsubara and Ziff, J. Clin. Invest. 79: 1440-1446, 1987); anticollagenase-serum alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4): 1659- 1664, 1987) Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4* chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions 36:312 316, 1992): Thalidomide; Angostatic steroid; AGM- 1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated or detected by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as. multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis. Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as heφes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that could be treated or detected by the polynucleotides or polypeptides, or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sai coma chordoma angrosarcoma endothehosai coma lymphangiosaicoma lymphangioendothehosaicoma synovioma mesothehoma, Ewing s tumoi leiomyosaicoma lhabdomyosarcoma, colon caicinoma, pancieatic cancer, breast cancer ovanan cancer piostate cancer, squamous cell carcinoma basal cell caicmoma, adenocaicinoma sweat gland carcinoma, sebaceous gland caicinoma, papillaiy carcinoma papillary adenocaicinomas cystadenocaicmoma, medullary carcinoma, bronchogenic carcinoma renal cell caicinoma, hepatoma, bile duct carcinoma, choπocarcinoma, semmoma, embryonal carcinoma Wilm s tumor, cervical cancer, testicular tumor lung carcinoma, small cell lung carcinoma, bladdei carcinoma, epithelial carcinoma, ghoma, astiocytoma medulloblastoma, craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma, ohgodendroghoma menangioma, melanoma, neuroblastoma, and retinoblastoma
Diseases associated with increased apoptosis that could be treated or detected by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS, neurodegenerative disoiders (such as Alzheimer s disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retimtis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease), autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet' s disease Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e g , hepatitis related liver injury, lschemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds Polynucleotides 01 polypeptides as well as agonists or antagonists of the invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oial cavity wounds, diabetic ulceis, dermal ulceis, cubitus ulcers, arterial ulceis venous stasis ulceis, burns resulting trom heat exposure or chemicals, and other abnormal wound healing conditions such as uremia malnutrition, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabohtes Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestabhshment subsequent to dermal loss
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithehalization from the wound bed The following are a non- exhaustive list of grafts that polynucleotides or polypeptides agonists or antagonists of the invention, could be used to increase adherence to a wound bed autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair- Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hypeφlastic graft, lamellar graft, mesh graft, mucosal graft, Olher- Thiersch graft, omenpal graft, patch giaft, pedicle graft, penetrating graft, split skm graft, thick split graft The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to piomote skin strength and to improve the appearance of aged skin It is believed that the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract The polynucleotides or polypeptides, and/or agonists 01 antagonists of the invention, may promote piohfeiation of endothelial cells, keiatinocytes. and basal keiatinocytes
The polynucleotides 01 polypeptides, and/oi agonists 01 antagonists of the invention, could also be used to reduce the side effects of gut toxicity that lesult from radiation, chemotherapy tieatments oi vnal infections The polynucleotides or polypeptides, and/or agonists oi antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that lesult from chemotherapy and viral infections
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could further be used in full regeneration of skm in full and partial thickness skin defects, including burns, (I e , repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithehalization of these lesions The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively Thus, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease Treatment with the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery The polynucleotides or polypeptides, and/or agonists oi antagonists of the invention, could be used to treat diseases associate with the under expiession of the polynucleotides of the invention
Moreovei, the polynucleotides oi polypeptides. and/or agonists or antagonists of the invention could be used to pievent and heal damage to the lungs due to various pathological states A giowth factor such as the polynucleotides or polypeptides and/or agonists or antagonists of the invention, which could stimulate prohfeiation and diffeientiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chionic lung damage For example, emphysema which results in the piogressive loss of aveoh, and inhalation injuries, I e , resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using the polynucleotides or polypeptides and/or agonists or antagonists of the invention Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respnatory distress syndrome and bronchopulmonary displasm, in premature infants
The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (I e , acetaminophen, carbon tetraholoπde and other hepatotoxins known in the art)
In addition, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used treat or prevent the onset of diabetes melhtus In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease Also, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function
Neurological Diseases Nervous system disorders, which can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to. nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: ( 1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or heφes simplex virus or with Lyme disease, tuberculosis, syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated . with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B 12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the coφus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis human immunodeficiency virus-associated myelopathy, tiansveise myelopathy 01 vaπous etiologies, piogiessive multifocal leukoencephalopathy, and central pontine myehnolysis
In a pref ei red embodiment the polypeptides polynucleotides oi agonists oi antagonists of the invention aie used to protect neural cells from the damaging effects of cerebral hypoxia According to this embodiment the compositions of the invention are used to treat or prevent neural cell mjuiy associated with ceiebral hypoxia In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists 01 antagonists of the invention are used to treat or pievent neural cell injury associated with cerebral ischemia In anothei aspect of this embodiment, the polypeptides polynucleotides or agonists or antagonists of the invention aie used to treat or prevent neuial cell injury associated with ceiebral infarction In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke In a further aspect of this embodiment the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a heart attack
The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the suivival or differentiation of neurons For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention (1) increased survival time of neurons in culture, (2) increased sprouting of neurons in culture or in vivo, (3) increased production of a neuron-associated molecule in culture or in vivo, e g , choline acetyltransferase or acetylchohnesterase with respect to motor neurons, or (4) decreased symptoms of neuron dysfunction in vivo Such effects may be measured by any method known in the art In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al (J Neurosci 10.3507-3515 ( 1990)), increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al (Exp Neurol 70 65-82 (1980)) or Brown et al (Ann Rev Neurosci 4 17-42 (1981)), increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding. Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polypeptide or polynucleotide and/or agonist or antagonist of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue. EBV, HIV. Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as. Cytomegalovirus. Herpes Simplex. Herpes Zoster). Mononegavirus (e.g., Paramyxoviridae. Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.. Influenza A, Influenza B, and parainfluenza). Papiloma virus. Papovaviridae. Parvoviridae. Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II. Lentivirus). and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g.. conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active. Delta), Japanese B encephalitis. Junin, Chikungunya. Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia. Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides. polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to. treat AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia). Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coh), Enteiobacteπaceae (Klebsiella Salmonella (e g Salmonella typhi, and Salmonella paiatyphi) Senatia, Yeisinia), Eiysrpelothπx, Hehcobacter Legionellosis, Leptospirosis Listeπa Mycoplasmatales, Mycobacteπum leprae. Vibrio cholerae, Neisseπaceae (e g Acinetobactei Gonoirhea, Menigococcal), Meisseria meningitidis, Pasteuiellacea Infections (e g , Actinobacillus, Heamophilus (e g , Heamophilus influenza type B) Pasteurella) Pseudomonas Ricketts ceae, Chlamydiaceae, Syphilis Shigella spp Staphylococcal Meningiococcal, Pneumococcal and Streptococcal (e g , Streptococcus pneumoniae and Group B Streptococcus) These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to bacteremia, endocarditis eye infections
(conjunctivitis, tuberculosis uveitis) gingivitis opportunistic infections (e g , AIDS related infections), paronych , piosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema sepsis, Lyme Disease. Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e g , mengitis types A and B),
Chlamydm, Syphilis Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e g , celluhtis, dermatocycoses), toxemia, urinary tract infections, wound infections Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases In specific embodiments, Ppolynucleotides, polypeptides agonists or antagonists of the invention are used to treat tetanus, Diptheπa, botulism, and/or meningitis type B
Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class Amebiasis. Babesiosis, Coccidiosis, Cryptospoπdiosis, Dientamoebiasis, Douπne, Ectoparasitic, Gmrdiasis, Helminthiasis, Leishmaniasis, Theileπasis Toxoplasmosis, Trypanosomiasis, and Tπchomonas and Sporozoans (e g , Plasmodium virax, Plasmodium falcipaπum, Plasmodium malaπae and Plasmodium ovale) These parasites can cause a variety of diseases or symptoms, including, but not limited to Scabies, Trombicuhasis, eye infections, intestinal disease (e g , dysentery, gmrdiasis), liver disease, lung disease, opportunistic infections (e g , AIDS lelated), malaπa, pregnancy complications, and toxoplasmosis polynucleotides 01 polypeptides, 01 agonists or antagonists of the invention can be used to tieat 01 detect any of these symptoms or diseases In specific embodiments, polynucleotides polypeptides, 01 agonists or antagonists of the invention are used to tieat malaria
Preferably, treatment using a polypeptide or polynucleotide and/or agonist oi antagonist of the piesent invention could eithei be by admmisteπng an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and leturnmg the engineered cells to the patient (ex vivo therapy) Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease
Regeneration A polynucleotide or polypeptide and/oi agonist or antagonist of the present invention can be used to differentiate, piohferate, and attract cells, leading to the regeneration of tissues (See, Science 276 59-87 (1997) ) The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e g osteoporosis, osteocarthπtis, penodontal disease, liver failuie). surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage
Tissues that could be regenerated using the present invention include organs (e g , pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue Preferably, regeneration occurs without or decreased scarring Regeneration also may include angiogenesis
Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase regeneration of tissues difficult to heal For example, increased tendon/ligament regeneration would quicken recovery time after damage A polynucleotide or polypeptide and/or agonist or antagonist of the present invention could also be used prophylactically in an effort to avoid damage Specific diseases that could be treated include of tendinitis, caφal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide and/or agonist or antagonist of the present invention to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotide or polypeptide and/or agonist or antagonist of the present invention.
Chemotaxis
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hypeφroliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
A polynucleotide or polypeptide and/or agonist or antagonist of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hypeφroliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that a polynucleotide or polypeptide and/or agonist or antagonist of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, a polynucleotide or polypeptide and/or agonist or antagonist of the present invention could be used as an inhibitor of chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g.. receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques. Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide. Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtuies The assay may also simplv compnse the steps of mixing a candidate compound with a solution containing a polypeptide measuπng poly peptide/molecule activity oi binding and comparing the polypeptide/molecule activity 01 binding to a standard Pre fei ably an ELISA assay can measuie polypeptide level or activity in a sample (e g , biological sample) using a monoclonal or polyclonal antibody The antibody can measuie polypeptide level oi activity by either binding directly or indirectly to the polypeptide or by competing with the polypeptide foi a substrate
Additionally, the receptor to which a polypeptide of the invention binds can be identified by numerous methods known to those of skill in the ait for example, ligand panning and FACS sorting (Cohgan, et al Current Piotocols in Immun , 1(2), Chapter 5 ( 1991 )) For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labelled The polypeptides can be labeled by a variety of means including lodination or inclusion of a recognition site for a site-specific protein kinase
Following fixation and incubation, the slides are subjected to auto- radiographic analysis Positive pools are identified and sub-pools are prepared and re- transfected using an iterative sub-pooling and le-screening process, eventually yielding a single clones that encodes the putative leceptor As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule Cross-linked material is resolved by PAGE analysis and exposed to X-ray film The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protem microsequencing The amino acid sequence obtained from microsequencmg would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors Moreovei the techniques of gene-shuffling, motif-shufflmg, exon-shuffling and/oi codon-shuffhng (collectively refened to as "DNA shuffling") may be employed to modulate the activities of polypeptides of the invention thereby effectively generating agonists and antagonists of polypeptides of the invention See generally, U S Patent Nos 5.605,793, 5,81 1 ,238, 5.830,721 , 5,834,252, and
5,837,458. and Patten. P A . et al , Cuπ Opinion Biotechnol 8 724-33 ( 1997),
Harayama, S Tiends Biotechnol 16(2) 76-82 ( 1998), Hansson, L O , et al , J Mol
Biol 287 265-76 ( 1999), and Lorenzo, M M and Blasco, R Biotechmques
24(2) 308-13 ( 1998) (each of these patents and publications are hereby incorporated by reference) In one embodiment, alteration of polynucleotides and conesponding polypeptides of the invention may be achieved by DNA shuffling DNA shuffling involves the assembly of two or moie DNA segments into a desired polynucleotide sequence of the invention molecule by homologous, or site-specific, recombination
In another embodiment, polynucleotides and corresponding polypeptides of the invention may be alterred by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to recombination In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc , of the polypeptides of the invention may be recombmed with one or more components, motifs, sections, parts, domains, fragments, etc of one or more heterologous molecules In preferred embodiments, the heterologous molecules are family members In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegιc(dpp), 60A, OP-2, dorsahn, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta5, and ghal- deπved neurotrophic factor (GDNF)
Other preferred fragments are biologically active fragments of the polypeptides of the invention Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide The biological activity of the fiagments may include an impioved desned activity, or a decreased undesirable activity
Additionally, this invention piovides a method of screening compounds to identify those which modulate the action of the polypeptide of the piesent invention An example of such an assay compπses combining a mammalian fibroblast cell, a the polypeptide of the present invention the compound to be screened and 3[H] thymidine undei cell cultuie conditions where the fibroblast cell would normally proliferate A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibioblast proliferation in the piesence of the compound to determine if the compound stimulates proliferation by determining the uptake of 3[H] thymidine in each case The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incoφoration of 3[H] thymidine Both agonist and antagonist compounds may be identified by this procedure In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound The ability of the compound to enhance or block this inteiaction could then be measured Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis All of these above assays can be used as diagnostic or prognostic markers
The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e g , blood vessel growth) by activating or inhibiting the polypeptide/molecule Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues Therefore, the invention includes a method of identifying compounds which bind to the polypeptides of the invention comprising the steps of (a) incubating a candidate binding compound with the polypeptide, and (b) determining if binding has occuπed Moieover, the invention includes a method of identifying agonists/antagonists compiising the steps of (a) incubating a candidate compound with the polypeptide (b) assaying a biological activity , and (b) determining if biological activity of the polypeptide has been alteied Also, one could identify molecules bind a polypeptide of the invention expeπmentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the piotein Accoidingly, specific embodiments of the invention aie directed to polynucleotides encoding polypeptides which compnse, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention
Targeted Delivery
In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention
As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell In another example, the invention provides a method for delivering a single stranded nucleic acid (e g , antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
Drug Screening Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a vaπety ot diug screening techniques The polypeptide 01 fiagment employed in such a test may be affixed to a solid suppoit, expiessed on a cell suiface, fiee in solution. 01 located intiacellulaily One method of diug scieening utilizes eukaryotic oi prokaiyotic host cells which are stably tiansformed with recombinant nucleic acids expressing the polypeptide oi fragment Diugs aie scieened against such tiansformed cells in competitive binding assays One may measuie, foi example, the formulation of complexes between the agent being tested and a polypeptide of the present invention
Thus, the present invention piovides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment theieof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art In such a competitive binding assay, the agents to screen are typically labeled Following incubation, free agent is separated from that present in bound form, and the amount of tree or uncomplexed label is a measuie of the ability of a particular agent to bind to the polypeptides of the present invention
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on September 13, 1984, which is incorporated herein by reference herein Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface The peptide test compounds are reacted with polypeptides of the present invention and washed Bound polypeptides are then detected by methods well known in the art Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support
This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
Antisense And Ribozvme (Antagonists) In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to nucleotide sequences contained a deposited clone. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241 :456 (1988); and Dervan et al., Science, 251: 1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA. For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRI site on the 5 end and a Hindlll site on the 3 end. Next, the pair of oligonucleotides is heated at 90°C for one minute and then annealed in 2X ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl/Hind III site of the retroviral vector PMV7 (WO 91/15580).
For example, the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22:787-797 ( 1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.. 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of interest. However, absolute complementarity, although preferred, is not required. A sequence "complementary to at least a portion of an RNA," referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids of the invention, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA sequence of the invention it may contarn and still foim a stable duplex (oi triplex as the case may be) One skilled in the ait can ascertain a tolerable degiee of mismatch by use of standaid procedures to deteimine the melting point of the hybπdized complex
Oligonucleotides that aie complementaiy to the 5' end of the message, e g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting tianslation Howevei, sequences complementary to the ' untranslated sequences of mRNAs have been shown to be effective at inhibiting tianslation of mRNAs as well See generally, Wagner, R., Nature, 372.333-335 ( 1994) Thus, oligonucleotides complementary to either the 5' - or 3' - non- translated, non-codmg regions of a polynucleotide sequence of the invention could be used m an antisense approach to inhibit tianslation of endogenous mRNA Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention Whether designed to hybridize to the 5' -, 3' - or coding region of mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides. at least 25 nucleotides or at least 50 nucleotides. The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), oi agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc Natl Acad Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci , 84-648-652 (1987): PCT Publication NO: WO88/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication NO: WO89/10134, published April 25, 1988), hybπdization- triggered cleavage agents. (See. e.g , Krol et al, BioTechniques, 6:958-976 (1988)) or intercalating agents (See, e.g.. Zon, Pharm. Res., 5-539-549 ( 1988)) To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybudization tπggeied cross-linking agent, tianspoit agent hybudization tπggeied cleavage agent, etc
The antisense oligonucleotide may compπse at least one modified base moiety which is selected from the gioup including, but not limited to. 5-fluoιouracιl, 5-bromouracιl, 5-chlorouracιl, 5-ιodouracιl, hypoxanthine xantine 4-acetylcytosιne, 5-(carboxyhydιoxylmethyl) uracil 5-carboxymethylamιnomethyl-2-thιouπdιne, 5-carboxymethylammomethyluracιl dihydrouracil, beta-D-galactosylqueosine, inosine, N6-ιsopentenyladenιne, 1-methylguanιne, 1-methyhnosιne 2,2-dιmethylguamne, 2-methyladenιne, 2-methylguanιne, 3-methylcytosιne, 5-methylcytosιne, N6-adenιne, 7-methylguanιne, 5-methylamιnomethyluracιl, 5-methoxyamιnomethyl 2-thιouracιl, beta-D-mannosylqueosine, 5 -methoxycarboxymethyluracil, 5-methoxyuracιl, 2-methylfhιo-N6- lsopentenyladenme, uracιl-5-oxyacetιc acid (v), wybutoxosine, pseudouracil, queosine, 2-thιocytosιne, 5-methyl-2-thιouracιl, 2-thιouracιl, 4-thιouracιl, 5-methyluracιl, uracιl-5-oxyacetιc acid methylester, uιacιl-5-oxyacetιc acid (v), 5-methyl-2-thιouracιl, 3-(3-amιno-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-dιamιnopuπne
The antisense oligonucleotide may also compπse at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabιnose, xylulose, and hexose
In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotπester, and a formacetal or analog thereof
In yet another embodiment, the antisense oligonucleotide is an a-anomeπc oligonucleotide An a-anomeπc oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al , Nucl Acids Res , 15 6625-6641 (1987)) The oligonucleotide is a 2-0-methylπbonucleotιde (Inoue et al , Nucl Acids Res , 15 6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al , FEBS Lett 215 327-330 (1987)) Polynucleotides of the invention may be synthesized by standaid methods known in the ait e g by use of an automated DNA synthesizei (such as aie commeicially available fiom Biosearch Applied Biosystems etc ) As examples phosphoiothioate oligonucleotides may be synthesized by the method of Stein et al (Nucl Acids Res 16 3209 ( 1988)). methylphosphonate oligonucleotides can be prepared by use of controlled poie glass polymer suppoits (Saπn et al , Proc Natl Acad Sci U S A . 85 7448-7451 ( 1988)), etc
While antisense nucleotides complementary to the coding region sequence of the invention could be used, those complementary to the transcribed untranslated region aie most preferred
Potential antagonists according to the invention also include catalytic RNA, oi a ribozyme (See, e g , PCT International Publication WO 90/1 1364, published October 4, 1990, Sarver et al, Science, 247 1222-1225 (1990) While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs corresponding to the polynucleotides of the invention, the use of hammerhead ribozymes is preferred Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA The sole requirement is that the target mRNA have the following sequence of two bases 5' -UG-3' The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature,
334 585-591 ( 1988) There are numerous potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the mRNA corresponding to the polynucleotides of the invention, I e , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts
As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e g for improved stability, targeting, etc ) and should be delivered to cells which express the polynucleotides of the invention in vivo DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA A preferred method of delivery involves using a DNA construct "encoding" the ribozyme undei the contiol of a strong constitutive promotei, such as, for example pol III 01 pol II promoter, so that transfected cells wrll produce sufficient quantities of the ribozyme to destioy endogenous messages and inhibit translation Since ribozymes unlike antisense molecules are catalytic, a lower intiacellular concentiation is iequned foi efficiency
Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the piesent invention on neoplastic cells and tissues, I e stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular giowth and piohferation, for example, in tumor foimation or growth
The antagonist/agonist may also be employed to prevent hyper-vasculai diseases, and prevent the proliferation of epithelial lens cells after extracapsulai cataract surgery Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty
The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing
The antagonist/agonist may also be employed to treat the diseases described herein Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention
Other Activities
The polypeptide of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment foi stimulating re- vasculaπzation of lschemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other caidiovascular conditions These polypeptide may also be employed to stimulate angiogenesis and limb regeneration, as discussed above The polypeptide may also be employed foi treating wounds due to injuiies, burns, post-operative tissue repair and ulceis since they aie mitogenic to vaπous cells of diffeient oirg s. such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged 01 diseased tissue The polypeptide of the piesent invention may also be employed stimulate neuronal growth and to treat and pievent neuronal damage which occurs in ceitain neuronal disorders or neuio-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex The polypeptide of the invention may have the ability to stimulate chondrocyte growth, therefore they may be employed to enhance bone and penodontal regeneration and aid in tissue transplants or bone grafts
The polypeptide of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth
The polypeptide of the invention may also be employed for preventing hair loss, since FGF family members activate hair- foi ming cells and promotes melanocyte growth Along the same lines, the polypeptides of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines
The polypeptide of the invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues
The polypeptide of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos
The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage
The polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e g , cosmetic surgery) Similarly, polypeptides or polynucleotides and/or agonist or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabohsm. piocessing utilization, and storage of eneigy
Polypeptide or polynucleotides and/oi agonist or antagonists of the piesent invention may be used to change a mammal s mental state or physical state by influencing biorhythms, caπcadic rhythms, depiession (including depiessive disorders), tendency for violence, tolerance for pain, reproductive capabilities
(preferably by Activm or Inhibin- ke activity), hoimonal or endocπne levels, appetite, libido, memory, stress, or other cognitive qualities
Polypeptide or polynucleotides and/or agonist or antagonists of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydiate, vitamins, mineials, cofactors or other nutritional components
Other Preferred Embodiments Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO X wherein X is any integer as defined in Table 1
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO X in Table 1
Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3 ' Nucleotide of the Clone Sequence as defined for SEQ ID NO X in Table 1
Similarly preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO X in the range of positions beginning with the nucleotide at about the position of the 5' Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X. A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:X beginning with the nucleotide at about the position of the 5' Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X.
Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which comprises a human cDNA clone identified by a cDN A Clone Identifier in Table 1 , which DNA molecule is contained in the material deposited with the American Type Culture Collection and given the ATCC Deposit Number shown in Table 1 for said cDNA Clone Identifier.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by a cDNA Clone Identifier in Table 1 , which DNA molecule is contained in the deposit given the ATCC Deposit Number shown in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone. A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1 ; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: .a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group. Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1 : and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1. Also preferred is a polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO: Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y. Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. Also prefeπed is a polypeptide wheiein said sequence of contiguous amino acids is included in the amino acid sequence of a secieted poition of the secieted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Numbei shown for said cDNA clone in Table 1
Also preferred is an isolated polypeptide comprising an ammo acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted poition of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone m Table 1
Also preferred is an isolated polypeptide comprising an ammo acid sequence at least 95% identical to a sequence of at least about 100 contiguous ammo acids in the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Numbei shown for said cDNA clone in Table 1
Also preferred is an isolated polypeptide comprising an ammo acid sequence at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1
Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table 1 , and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1
Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table 1 , and a complete ammo acid sequence of a prote encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Numbei shown foi said cDNA clone in Table 1 , which method compπses a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected fiom said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids
Also piefeπed is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table 1, and a complete amino acid sequence of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown foi said cDNA clone in Table 1
Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group
Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y wherein Y is any integer as defined in Table 1 , and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1 Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NON wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a secreted protein encoded by a human cDΝA clone identified by a cDΝA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence, of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
Also preferred is an isolated nucleic acid molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1 ; and a complete amino acid sequence of a secreted protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in T able 1 and contained in the deposit with the ATCC Deposit Numbei shown foi said cDNA clone in Table 1
Further preferred is a method of making a lecombinant vectoi compiising inseiting any of the above isolated nucleic acid molecule into a vectoi Also prefened is the recombinant vector pioduced by this method Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method
Also preferred is a method of making an isolated polypeptide compπsing culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO Y wherein Y is an integer set forth in Table 1 and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO Y is defined in Table 1 , and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1 The isolated polypeptide produced by this method is also preferred Also preferred is a method of treatment of an individual in need of an increased level of a secreted protein activity, which method comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual
The above-recited applications have uses in a wide variety of hosts Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non- human primate, and human In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat In preferred embodiments, the host is a mammal In most preferred embodiments, the host is a human In specific embodiments of the invention, for each "Contig ID" listed in the fourth column of Table 2, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, a nucleotide sequence referenced in the fifth column of Table 2 and described by the general formula of a-b, whereas a and b are uniquely determined for the corresponding SEQ ID NO:X referred to in column 3 of Table 2. Further specific embodiments are directed to polynucleotide sequences excluding one, two, three, four, or more of the specific polynucleotide sequences referred to in the fifth column of Table 2. In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.
TABLE 2
Figure imgf000222_0001
Figure imgf000223_0001
N21623, N22626, N27205, N27671 , N28862, N30633, N32921. N39948. N41879. N42065, N47498. N53404. N54305, N551 16, N99586. W 17080, W20304, N90682. N90857. AA027973. AA027972. AA029054, AA029157. AA043352, AA043353. AA100618. AA100619, AA180148. AA180149, AA258774, AA257972
H2MBA76 826161 T99337, T99336, R06650
HACAD42 48 634372 H14214, R88275, R89051. N62815, N78561 , W01327
HACBJ 1 1 50 797625 R47920. R48028, R54716. H28781, H28757, W69487, W6961 1 , AA009556, AA009555, AA010145, AA010146, AA010665, AA01 1255. AA024803. AA024919
HACBT91 52 789939 R47922, R50081, R71675. N41047, N47137, AA057770
HACBZ73 53 832528 R53948, H05265, H09484. H38840, H62900, H62934, H84150, AA012918, AAO 19501, AA502902, AA554787, AA588328, AA595550. AA741530
HCE4W88 792953 T51830, T51875, T55394. T55822, R23671 , R49788, R49831. H05472, H07023, HI 7470, HI 7576, H I 9056, H19346, H24371 , H25189, H41016, H41015, H79349, H79464, H97125, N23678, N33493, N43883, N46513, N74301 , N93377, N94458. N99133. W24134, W25396, W30736, W52553. W58695, W58694, W72377, W74087, AA009730, AA009445, AA022628, AA022776, AA054325, AA055666, AA079720, AA079719, AA098887, AA099230, AA101577, AA101576, AA1 13180, AA1 14261, AA147216, AA147198, AA148041, AA151615, AA156328, AA156207, AA158381, AA 158479, AA 159326, AA 159426, AA160121, AA160397, AA160414, AA165702, AA176358, AA176357, AA232191 , AA235563, AA235651, AA2431 12
HPTTQ91 23 751559 R59979, R61446, H 1941 1, H281 19, H41432, R87226, R87916. R90732, H51 135, N31687, N48255. AA088245,
Figure imgf000225_0001
Having geneiallv* desciibed the invention the same wrll be more readrly understood by reference to the follow g examples whrch are provided by way of illustration and are not intended as limiting
Examples
Example 1 Isolation of a Selected cDNA Clone Fiom the Deposited Sample
Each cDNA clone in a cited ATCC deposit is contained in a plasmid vectoi Table 1 identifies the vectors used to construct the cDNA library from which each clone was isolated In many cases, the vector used to constiuct the library is a phage vector from which a plasmid has been excised The table immediately below correlates the related plasmid for each phage vector used in constructing the cDNA library For example where a particular clone is identified in Table 1 as being isolated in the vectoi ' Lambda Zap, ' the corresponding deposited clone is in "pBluescπpt "
Vector Used to Construct Library Corresponding Deposited
Plasmid
Lambda Zap pBluescπpt (pBS) Urn-Zap XR pBluescπpt (pBS)
Zap Express pBK lafmid BA plafmid BA pSportl pSportl pCMVSport 2 0 pCMVSport 2 0 pCMVSport 3 0 pCMVSport 3 0 pCR®2 1 pCR®2 1
Vectors Lambda Zap (U S Patent Nos 5, 128.256 and 5,286,636), Urn-Zap XR (U S Patent Nos 5.128, 256 and 5.286,636), Zap Express (U S Patent Nos 5,128,256 and 5,286.636), pBluescπpt (pBS) (Short, J M et al , Nucleic Acids Res 16 7583-7600 ( 1988). Alting-Mees, M A and Shoit, J M , Nucleic Acids Res
17 9494 (1989)) and pBK (Alting-Mees, M A et al , Strategies 5 58-61 (1992)) are commercially available from Stratagene Cloning Systems, Ine , 1 101 1 N Torrey Pines Road La Jolla CA. 92037 pBS contains an ampicillin resistance gene and pBK contains a neomycin lesistance gene Both can be tiansformed into E coli strain XL- 1 Blue also available fiom Stiatagene pBS comes in 4 forms SK+, SK-. KS+ and KS The S and K refeis to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region ("S" is for Sad and "K" is for Kpnl which aie the fust sites on each respective end of the hnkei) ' + oi "-' refei to the orientation of the fl origin of iephcation ( on '), such that in one oπentation, single stranded rescue initiated fiom the fl on generates sense stiand DNA and in the other, antisense Vectors pSpoitl, pCMVSpoit 2 0 and pCMVSport 3 0, were obtained from
Life Technologies, Ine , P O Box 6009 Gaitheisbuig, MD 20897 All Sport vectois contain an ampicillin resistance gene and may be transformed into E coli strain DH10B, also available from Life Technologies (See, for instance, Gruber, C E , et al , Focus 15 59 (1993) ) Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E coli strain XL-1 Blue Vector pCR®2 1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be transformed into E coli strain DH10B, available from Life Technologies (See, for instance, Clark, J M , Nuc Acids Res 16 9677-9686 (1988) and Mead, D et al , Bio/Technology 9 (1991) ) Preferably, a polynucleotide of the present invention does not comprise the phage vectoi sequences identified for the paiticular clone in Table 1, as well as the corresponding plasmid vectoi sequences designated above The deposited material in the sample assigned the ATCC Deposit Number cited in Table 1 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each cDNA clone identified in Table 1 Typically, each ATCC deposit sample cited in Table 1 comprises a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone, but such a deposit sample may include plasmids for more or less than 50 cDNA clones, up to about 500 cDNA clones Two appioaches can be used to isolate a paiticulai clone fiom the deposited sample of plasmid DNAs cited foi that clone in Table 1 Fust, a plasmid is directly isolated by scieemng the clones using a polynucleotide piobe conesponding to SEQ ID NO X Particulaily, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported The oligonucleotide is labeled, for instance, with 32P-(-ATP using T4 polynucleotide kinase and purified according to routine methods (E g , Mamatis et al , Molecular Cloning A Laboratory Manual, Cold Spring Harbor Piess Cold Spring, NY (1982) ) The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the ait, such as those provided by the vector supplier oi in related publications or patents cited above The transformants are plated on 1 5% agar plates (containing the appropriate selection agent, e g , ampicillin) to a density of about 150 transformants (colonies) per plate These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e g , Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Edit , (1989), Cold Spring Harbor Laboratory Press, pages 1 93 to 1 104), or other techniques known to those of skill in the art
Alternatively, two primers of 17-20 nucleotides derived from both ends of the SEQ ID NO X (I e , within the region of SEQ ID NO X bounded by the 5 NT and the 3' NT of the clone defined in Table 1) are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0 5 ug of the above cDNA template A convenient reaction mixture is 1 5-5 mM MgC12, 0 01 % (w/v) gelatin 20 uM each of dATP, dCTP. dGTP, dTTP, 25 pmol of each primer and 0 25 Unit of Taq polymerase Thirty five cycles of PCR (denaturation at 94 degree C for 1 mm annealing at 55 degree C for 1 min, elongation at 72 degree C for 1 mm) are performed with a Perkm-Elmer Cetus automated thermal cycler The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product Several methods are available for the identification of the 5 ' or 3' non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5' and 3 ' "RACE" protocols which are well known in the art. For instance, a method similar to 5 ' RACE is available for generating the missing 5' end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21 (7): 1683-1684 ( 1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5 ' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the desired gene.
Example 2: Isolation of Genomic Clones Corresponding to a Polynucleotide A human genomic PI library (Genomic Systems, Inc.) is screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO:X., according to the method described in Example 1. (See also, Sambrook.) Example 3 Tissue Distribution of Polypeptide
Tissue distribution of mRNA expiession of polynucleotides of the piesent invention is determined using piotocols foi Noithern blot analysis, described by, among others, Sambiook et al For example, a cDNA probe pioduced by the method described in Example 1 is labeled with P32 using the redipπme™ DNA labeling system (Amei sham Life Science), accoiding to manufacturei's instructions After labeling, the probe is purified using CHROMA SPIN- 100™ column (Clontech Laboratories, Ine ). according to manufacturer's protocol number PTl 200-1 The purified labeled probe is then used to examine various human tissues for mRNA expression
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (Clontech) are examined with the labeled probe using ExpressHyb1 M hybridization solution (Clontech) according to manufacturer's protocol number PTl 190-1 Following hybridization and washing, the blots are mounted and exposed to film at -70 degree C overnight, and the films developed according to standard procedures
Example 4 Chromosomal Mapping of the Polynucleotides An oligonucleotide primer set is designed according to the sequence at the 5 end of SEQ ID NO X This primer preferably spans about 100 nucleotides This primer set is then used in a polymeiase chain reaction under the following set of conditions 30 seconds, 95 degree C, 1 minute, 56 degree C, 1 minute, 70 degree C This cycle is repeated 32 times followed by one 5 minute cycle at 70 degree C Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Ine) The reactions is analyzed on eithei 8% polyacrylamide gels or 3 5 % agarose gels Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid
Example 5 Bacterial Expression of a Polypeptide A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide pπmers conesponding to the 5 and 3 ends ot the DNA sequence, as outlined in Example 1 to synthesize insertion fiagments The pnmeis used to amplify the cDNA insert should piefeiably contain lestπction sites, such as BamHl and Xbal, at the 5 end of the pπmers in older to clone the amplified pioduct into the expression vector For example BamHl and Xbal correspond to the restriction enzyme sites on the bactenal expiession vectoi pQE-9 (Qiagen, Ine , Chatsworth, CA) This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (on), an IPTG-iegulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-hιstιdme tag (6-Hιs), and restriction enzyme cloning sites
The pQE-9 vector is digested with BamHl and Xbal and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS The ligation mixture is then used to transform the E coli strain M15/rep4 (Qiagen, Ine ) which contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kanr) Transformants are identified by their ability to grow on LB plates and ampicilhn/kanamycin resistant colonies are selected Plasmid DNA is isolated and confirmed by restriction analysis Clones containing the desired constructs are grown overnight (O/N) in liquid cultuie in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) The O/N culture is used to inoculate a laige culture at a ratio of 1 100 to 1 250 The cells are grown to an optical density 600 (O D 600) of between 0 4 and 0 6 IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM IPTG induces by inactivating the lad repressoi, clearing the P/O leading to increased gene expression
Cells are grown for an extra 3 to 4 hours Cells are then harvested by centrifugation (20 mms at 6000Xg) The cell pellet is solubihzed in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitπlo-tπ-acetic acid ("Ni-NTA") affinity resin column (available from QIAGEN, Ine , supra) Proteins with a 6 x His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist ( 1995) QIAGEN. Inc.. supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8. then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate- buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni- NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl. 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C or frozen at -80 degree C.
In addition to the above expression vector, the present invention further includes an expression vector comprising phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on February 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences. 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with Ndel and Xbal, BamHl, Xhol, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for Ndel (5' primer) and Xbal, BamHl, Xhol, or Asp718 (3' primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols. The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 6: Purification of a Polypeptide from an Inclusion Body The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4- 10 degree C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4- 10 degree C and the cells harvested by continuous centrifugation at 15.000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer. The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics. Coφ. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4. The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 xg centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000 xg) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron). equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems) The column is washed with 40 mM sodium acetate, pH 6 0 and eluted with 250 mM 500 mM 1000 mM and 1500 mM NaCl m the same buffei in a stepwise mannei The absorbance at 280 nm of the effluent is continuously monitored Fractions aie collected and furthei analyzed bv SDS-PAGE Fi actions containing the polypeptide aie then pooled and mixed with 4 volumes of water The diluted sample is then loaded onto a previousl) prepaied set of tandem columns of stiong anion (Poios HQ 50 Perseptive Biosystems) and weak anion (Poros CM-20 Perseptive Biosystems) exchange resins The columns are equilibrated with 40 mM sodium acetate pH 6 0 Both columns are washed with 40 mM sodium acetate pH 6 0, 200 mM NaCl The CM-20 column is then eluted using a 10 column volume linear gradient ranging fiom 0 2 M NaCl, 50 mM sodium acetate, pH 6 0 to 1 0 M NaCl, 50 mM sodium acetate, pH 6 5 Fractions are collected under constant A280 monitoring of the effluent Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps No major contaminant bands should be observed from Commassie blue stained 16% SDS PAGE gel when 5 ug of purified protein is loaded The purified protein can also be tested for endotoxm/LPS contamination and typically the LPS content is less than 0 1 ng/ml according to LAL assays
Example 7: Cloning and Expression of a Polypeptide in Baculovirus Expression System
In this example the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to expiess a polypeptide This expression vector contains the strong polyhedrin promoter of the Autographa cahfornica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHl, Xba I and Asp718 The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene The inseited genes aie flanked on both sides by vnal sequences foi cell-mediated homologous lecombmation with wild-type vnal DNA to generate a viable vnus that expiess the cloned poly nucleotide
Many othei baculovirus vectois can be used in place of the vectoi above, such as pAc373, pVL941, and pAcIM l as one skilled in the ait would leadily appieciate, as long as the constiuct piovides appropriately located signals foi transcription, translation, secietion and the like, including a signal peptide and an in-frame AUG as required Such vectors are described, for instance, in Luckow et al Virology 170 31- 39 (1989) Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified in Table 1 , is amplified using the PCR protocol described in Example 1 If the naturally occurring signal sequence is used to produce the secieted protem, the pA2 vector does not need a second signal peptide Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al , "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experimental Station Bulletin No 1555 (1987)
The amplified fragment is isolated trom a 1 % agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc , La Jolla, Ca ) The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art The DNA is then isolated from a 1 % agarose gel using a commercially available kit ("Geneclean' BIO 101 Inc , La Jolla, Ca )
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase E coli HB 101 or other suitable E coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the ligation mixture and spread on culture plates Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis The sequence of the cloned fragment is confirmed by DNA sequencing
Five ug of a plasmid containing the polynucleotide is co-tiansfected with 1 0 ug of a commeicially available linearized baculovnus DNA ( BaculoGold1 M baculovnus DNA' , Pharmingen, San Diego, CA), using the lipofection method described by Feigner et al , Proc Natl Acad Sci USA 84 7413-7417 ( 1987) One ug of BaculoGoldI M vnus DNA and 5 ug of the plasmid are mixed in a steπle well of a microtiter plate containing 50 ul of serum-free Grace s medium (Life Technologies Inc , Gaithersbuig, MD) Afterwaids, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature Then the transfection mixtuie is added drop-wise to Sf9 insect cells (ATCC CRL 171 1 ) seeded in a 35 mm tissue culture plate with 1 ml Grace s medium without serum The plate is then incubated for 5 hours at 27 degrees C The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added Cultivation is then continued at 27 degrees C for four days
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra An agarose gel with "Blue Gal" (Life Technologies Inc , Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques (A detailed description of a "plaque assay" of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc , Gaithersburg, page 9-10 ) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e g , Eppendorf) The agar containing the recombinant viruses is then resuspended in a microcentπfuge tube containing 200 ul of Grace s medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection ("MOI") of about 2 If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine 23s
(available from Life Technologies Inc Rockv ille, MD) Aftei 42 houis, 5 uCi of 35S methionine and 5 uCi 35S-cysteιne (available fiom Ameisham) aie added The cells are further incubated foi 16 hours and then aie harvested by centrifugation The proteins in the supernatant as well as the intracellulai pioteins are analyzed by SDS- PAGE followed by autoiadiography (if radiolabeled)
Microsequencing of the amino acid sequence of the ammo terminus of purified piotein may be used to determine the amino teiminal sequence of the produced protein
Example 8: Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protem coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e g , RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV) However, cellular elements can also be used (e g , the human actin promoter)
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala. Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2 0, and pCMVSport 3 0 Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells The transfected gene can also be amplified to expiess laige amounts of the encoded piotein The DHFR (dihv diofolate leductase) marker is useful in developing cell lines that carry sevei l hundred or even seveial thousand copies of the gene of interest (See, e g Alt, F W , et al J Biol Chem 253 1357 1370 ( 1978) Hamlm, J L and Ma, C , Biochem et Biophys Acta 1097 107-143 ( 1990), Page M J and Sydenham, M A Biotechnology 9 64-68 ( 1991) ) Another useful selection maikei is the enzyme glutamine synthase (GS) (Murphy et al Biochem J 227 277-279 (1991), Bebbington et al , Bio/Technology 10 169-175 ( 1992) Using these markers, the mammalian cells aie grown in selective medium and the cells with the highest resistance are selected These cell lines contain the amplified gene(s) integrated into a chiomosome Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No 37146), the expression vectors pC4 (ATCC Accession No 209646) and pC6 (ATCC Accession No 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al , Molecular and Cellular Biology, 438-447 (Maich, 1985)) plus a fragment of the CMV-enhancer (Boshart et al , Cell 41 521-530 (1985) ) Multiple cloning sites, e g , with the restriction enzyme cleavage sites BamHl, Xbal and Asp718, facilitate the cloning of the gene of interest The vectors also contain the 3' intron, the polyadenylation and termination signal of the rat preproinsulm gene, and the mouse DHFR gene under contiol of the SV40 early promoter
Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art The vector is then isolated from a 1 % agarose gel A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1 If the naturally occurring signal sequence is used to produce the secieted piotein, the vector does not need a second signal peptide Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (See, e g , WO 96/34891 ) The amplified fragment is isolated from a 1 % agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc , La Jolla, Ca ) The fragment then is digested with appiopriate iestπction enzymes and again puπfred on a 1 % agarose gel
The amplified fiagment is then digested with the same lestπction enzyme and purified on a 1 % agaiose gel The isolated fiagment and the dephosphoiylated vector are then ligated with T4 DNA ligase E colt HB 101 oi XL 1 Blue cells aie then transformed and bacteπa aie identified that contain the fragment inserted into plasmid pC6 using, for instance restriction enzyme analysis
Chinese hamster ovary cells lacking an active DHFR gene is used for transfection Five μg of the expression plasmid pC6 a pC4 is cotiansfected with 0 5 ug of the plasmid pSVneo using lipofectin (Felgnei et al , sup? a) The plasmid pSV2- neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418 The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418 After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418 After about 10-14 days single clones aie trypsinized and then seeded in 6-well petπ dishes oi 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM. 200 nM, 400 nM, 800 nM) Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate ( 1 uM, 2 uM, 5 uM, 10 mM, 20 mM) The same procedure is repeated until clones aie obtained which grow at a concentration of 100 - 200 uM Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reveised phase HPLC analysis
Example 9: Protein Fusions
The polypeptides of the present invention are preferably fused to other proteins These fusion proteins can be used for a vanety of applications Foi example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification (See Example 5, see also EP A 394,827, Traunecker, et al , Nature 331 84-86 (1988) ) Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellulai localization while covalent heteiodimei oi homodimeis can increase 01 deciease the activity ot a fusion piotein Fusion pioteins can also cieate chimeπc molecules having moie than one function Finally, fusion pioteins can increase solubility and/oi stability of the fused piotein compaied to the non-fused protein All of the types of fusion proteins described above can be made by modifying the following protocol which outlines the fusion of a polypeptide to an IgG molecule, or the protocol desciibed in Example 5
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5' and 3' ends of the sequence described below These primers also should have convenient lestπction enzyme sites that will facilitate cloning mto an expiession vector preferably a mammalian expression vector
For example, if pC4 (Accession No 209646) is used, the human Fc portion can be ligated into the BamHl cloning site Note that the 3' BamHl site should be destroyed Next, the vector containing the human Fc portion is re-restπcted with BamHl, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHl site Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protem will not be produced
If the naturally occurring signal sequence is used to pioduce the secreted protein, pC4 does not need a second signal peptide Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (See, e g , WO 96/34891 )
Human IgG Fc region GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGT GGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO: l)
Example 10: Production of an Antibody from a Polypeptide The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention is administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of the secreted protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology. (Kδhler et al., Nature 256:495 ( 1975); Kδhler et al, Eur. J. Immunol. 6:51 1 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C), and supplemented with about 10 g/1 of nonessential amino acids, about 1 ,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al (Gastioenteiology 80 225-232 ( 1981 ) ) The hybπdoma cells obtained through such a selection aie then assayed to rdentify clones which seciete antibodies capable of binding the polypeptide Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step proceduie using anti-idiotypic antibodies Such a method makes use of the fact that antibodies aie themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody In accoidance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells aie scieened to identify clones which pioduce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide Such antibodies comprise anti-idiotypic antibodies to the protein- specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies
It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments) Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry
For in vivo use of antibodies in humans, it may be pieferable to use "humanized" chimeric monoclonal antibodies Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above Methods for producing chimeric antibodies are known in the art (See, for review, Morrison, Science 229 1202 (1985), Oi et al , BioTechniques 4 214 ( 1986), Cabilly et al , U S Patent No 4,816,567, Taniguchi et al , EP 171496, Morrison et al , EP 173494, Neuberger et al , WO 8601533, Robinson et al , WO 8702671 , Bouhanne et al , Nature 312 643 (1984), Neuberger et al , Nature 314 268 (1985) ) Example 11 : Production Of Secreted Protein For High-Throughput Screening
Assa>s
The following protocol pioduces a supernatant containing a polypeptide to be tested This supernatant can then be used in the Scieening Assays descπbed in Examples 13-20
Fust, dilute Poly-D-Lysme (644 587 Boehπngei -Mannheim) stock solution ( 1 mg/ml in PBS) 1 20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50ug/ml Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes Be sure to distribute the solution over each well (note a 12-channel pipetter may be used with tips on eveiy other channel)
Aspirate off the Poly-D-Lysine solution and rinse with 1ml PBS (Phosphate Buffered Saline) The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance foi up to two weeks
Plate 293T cells (do not carry cells past P+20) at 2 x 10s cells/well in 5ml DMEM(Dulbecco's Modified Eagle Medιum)(wιth 4 5 G/L glucose and L-glutamme (12-604F Bιowhιttaker))/10% heat inactivated FBS(14-503F Bιowhιttaker)/lx Penstrep( 17-602E Biowhittakei) Let the cells grow overnight
The next day, mix together in a sterile solution basin 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5ml Optimem I (31985070 Gιbco/BRL)/96-well plate Wrth a small volume multi-channel pipetter, aliquot approximately 2ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8 or 9, into an appropriately labeled 96-well round bottom plate With a multi-channel pipetter, add 50ul of the Lipofectamme/Optimem I mixture to each well Pipette up and down gently to mix Incubate at RT 15-45 minutes After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections
Preferably, the transfection should be performed by tag-teaming the following tasks By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 5- lml PBS Person A then aspirates off PBS rinse, and person B, using al2-channel pipetter with tips on every other channel, adds the 200ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degrees C for 6 hours.
While cells are incubating, prepare appropriate media, either 1 %BSA in DMEM with lx penstrep. or CHO-5 media ( 1 16.6 mg/L of CaC12 (anhyd); 0.00130 mg/L CuSO4-5H2O; 0.050 mg/L of Fe(NO.,),-9H2O; 0.417 mg/L of FeSO4-7H2O; 311.80 mg/L of Kcl; 28.64 mg L of MgCl2; 48.84 mg/L of MgSO4; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO.,: 62.50 mg/L of NaH,PO4-H20; 71.02 mg/L of Na2HPO4; .4320 mg/L of ZnSO4-7H O: .002 mg/L of Arachidonic Acid ; 1.022 mg/L of Cholesterol; .070 mg/L of DL-alpha-Tocopherol- Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linoienic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D- Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H20; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL- H20; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L- Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H20; 99.65 mg/ml of L- Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol: 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L of Vitamin B12; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20uM of Ethanolamine; 0.122 mg/L of Ferric Citrate: 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal) with 2mm glutamine and lx penstrep. (BSA (81-068-3 Bayer) lOOgm dissolved in IL DMEM foi a 107c BSA stock solution) Filtei the media and collect 50 ul foi endotoxin assay in 15ml polystyiene conical
The transfection reaction is terminated, piefeiably by tag teaming at the end of the incubation period Person A aspnates off the transfectron medra, whrle person B adds 1 5ml approprrate medra to each well Incubate at 37 degrees C for 45 or 72 hours depending on the media used 1 %BSA for 45 hours oi CHO-5 for 72 hours On day foui, using a 300ul multichannel pipettei, aliquot 600ul in one 1ml deep well plate and the remaining supernatant into a 2ml deep well The supernatants from each well can then be used in the assays descπbed in Examples 13-20 It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide directly (e g , as a secreted piotein) or by the polypeptide inducing expression of other proteins, which are then secreted into the supernatant Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay
Example 12: Construction of GAS Reporter Construct
One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway Activated proteins in the Jaks-STATs pathway bind to gamma activation site "GAS" elements or mterferon- sensitive responsive element ("ISRE"), located in the promoter of many genes The binding of a protein to these elements alter the expiession of the associated gene GAS and ISRE elements are lecogmzed by a class of transcription factors called Signal Transducers and Activators of Transcription, or "STATs " There are six members of the STATs family Statl and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread) Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12 Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells It can be activated in tissue culture cells by many cytokines The STATs aie activated to translocate from the cytoplasm to the nucleus upon tyiosine phosphoiylation by a set of kinases known as the Janus Kinase ("Jaks' ) family Jaks represent a distinct family of soluble tyiosine kinases and include Tyk2 Jakl , Jak2, and Jak3 These kinases display significant sequence similaπty and aie generally catalytically inactive in resting cells
The Jaks aie activated by a wide range of receptois summarized in the Table below (Adapted from review by Schidler and Darnell, Ann Rev Biochem 64 621- 51 (1995) ) A cytokine teceptor family, capable of activating Jaks, is divided into two groups (a) Class 1 includes receptors for IL-2. IL 3, IL-4, IL-6, IL-7, IL-9. IL-1 1, IL- 12, IL-15, Epo, PRL. GH, G-CSF. GM-CSF, LIF, CNTF, and thrombopoielin, and (b) Class 2 includes IFN-a, IFN-g and IL- 10 The Class 1 receptors share a conserved cysteine motif (a set of foui conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Tip-Ser-Xxx-Tφ-Ser (SEQ ID NO 2)) Thus, on binding of a ligand to a receptoi, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements This entire process is encompassed in the Jaks-STATs signal transduction pathway
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element can be used to indicate protems involved in the proliferation and differentiation of cells For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below ) Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified
JAKs STATS GAS(elements or ISRE
Ligand tyk2 Jak l Jakl Jak3
IFN family IFN-a/B + + 1.2.3 ISRE IFN-g + + GAS (IRFl>Lys6>IFP) 11-10 1 ,3
gp!30 family
IL-6 (Pleiotrophic) + + + 7 1.3 GAS (IRFl>Lys6>IFP)
11- 1 1 (Pleiotrophic) ? + 7 1 ,3
OnM(Pleiotrophic) ? + + '> 1 ,3
LIF(Pleiotrophic) ? + + 7 1 ,3
CNTF(Pleiotrophic) -/+ + + 7 1 ,3
G-CSF(Pleiotrophic) ? + 7 1 ,3
IL- 12(Pleiotrophic) + - + + 1 ,3
g-C familv
IL-2 (lymphocytes) + - + 1 ,3,5 GAS
IL-4 (lymph/myeloid) - + - + 6 GAS (IRFl IFP »Ly6)(IgH)
IL-7 (lymphocytes) + - + 5 GAS
IL-9 (lymphocytes) + - + 5 GAS
IL- 13 (lymphocyte) + 7 7 6 GAS
IL- 15 ? + 7 + 5 GAS
gpl40 familv
IL-3 (myeloid) - + - 5 GAS (IRFl>IFP»Ly6)
IL-5 (myeloid) - + - 5 GAS
GM-CSF (myeloid) - - + - 5 GAS
Growth hormone familv
GH ? - + - 5
PRL ? +/- + - 1 ,3,5
EPO ? - + - 5 GAS (B -C AS>IRF 1 =IFP»Ly 6)
Receptor Tvrosine Kinases
EGF ? + + - 1 ,3 GAS (IRFl)
PDGF ? + + - 1,3
CSF-1 ? + + - 1 ,3 GAS (not IRFl) To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 13-14, a PCR based stiategy is employed to genei te a GAS-SV40 piomoter sequence The 5' primer contains foui tandem copies of the GAS binding site found in the IRFl piomotei and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1.457-468 ( 1994) ). although other GAS or ISRE elements can be used instead The 5' primer also contains 18bp of sequence complementary to the SV40 early promoter sequence and is flanked with an Xhol site The sequence of the 5* primer is: 5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID NO:3)
The downstream primer is complementary to the SV40 promotei and is flanked with a Hind III site- 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:4) PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with Xhol/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: 5 ' : CTCGAG ATTTCCCCG AA ATCT AG ATTTCCCCGAA ATG ATTTCCCCG AA A TGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCG CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT AGGCTTTTGCAAAAAGCTT:3' (SEQ ID NO:5)
With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP). or any protein detectable by an antibody. The above sequence continued synthetic GAS-SV40 piomoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech usmg HindlH and Xhol. effectively replacing the SV40 piomotei with the amplified GAS SV40 promoter element, to create the GAS-SEAP vectoi Howevei , this vector does not contain a neomycin resistance gene, and theiefoie is not prefened for mammalian expression systems
Thus, in order to geneiate mammalian stable cell lines expressing the GAS- SEAP reporter, the GAS-SEAP cassette is lemoved trom the GAS-SEAP vector using Sail and Notl, and inseited into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these lestnction sites in the multiple cloning site, to create the GAS-SEAP/Neo vectoi Once this vector is tiansfected mto mammalian cells, this vector can then be used as a repoiter molecule foi GAS binding as described in Examples 13-14
Other constructs can be made using the above description and replacing GAS with a different promoter sequence For example, constiuction of reporter molecules containing NFK-B and EGR promoter sequences are described in Examples 15 and 16 However, many other promoters can be substituted using the protocols described in these Examples For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e g , GAS/NF-KB/EGR, GAS/NF-KB, II- 2/NFAT, or NF-KB/GAS) Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial). Reh (B-cell), Saos-2 (osteoblast). HUVAC (aortic), or Cardiomyocyte
Example 13: High-Throughput Screening Assay for T-cell Activity. The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernate containing a polypeptide of the invention proliferates and/or differentiates T-cells T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12 Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway The T-cell used in this assay is Jurkat T-cells (ATCC Accession No TIB- 152), although Molt-3 cells (ATCC Accession No CRL- 1552) and Molt-4 cells (ATCC Accession No CRL- 1582) cells can also be used Juikat T-cells aie lymphoblastic CD4+ Thl helper cells In older to generate stable cell lines, appioximatelv 2 million Juikat cells are transfected wrth the GAS SEAP/neo vector usmg DMRIE-C (Lrfe Technologιes)(transfectιon piocedure described below) The tiansfected cells are seeded to a density of appioxπuately 20,000 cells per well and tiansfectants resistant to 1 mg/ml gentian selected Resistant colonies aie expanded and then tested for then response to increasing concentrations of inteifeion gamma The dose response of a selected clone is demonstrated
Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells Thus, it is either scaled up or performed in multiple to generate sufficient cells for multiple 96 well plates Jurkat cells are maintained in RPMI + 10% serum with l %Pen-Strep Combine 2 5 mis of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask Add 2 5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins During the incubation period, count cell concentration, spin down the required number of cells ( 107 per transfection), and resuspend in OPTI-MEM to a final concentration of 107 cells/ml Then add 1ml of 1 x 107 cells in OPTI-MEM to T25 flask and incubate at 37 degrees C for 6 hrs After the incubation, add 10 ml of RPMI + 15% serum The Jurkat GAS-SEAP stable reporter lines are maintained in RPMI + 10% serum, 1 mg/ml Gentian, and 1 % Pen-Strep These cells are treated with supernatants containing polypeptides of the invention and/or induced polypeptides of the invention as produced by the protocol described in Example 1 1
On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells per ml The exact number of cells required will depend on the number of supernatants being screened For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required
Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100, 000 cells per well) Aftei all the plates have been seeded, 50 ul of the supernatants are tiansreπed directly fiom the 96 well plate containing the supernatants into each well using a 12 channel pipette In addition a dose of exogenous interferon gamma (0 1 , 1 0 10 ng) is added to wells H9 H 10, and H I 1 to serve as additional positive contiols foi the assay
The 96 well dishes containing Juikat cells tieated with supernatants are placed in an incubator foi 48 his (note this time is vaπable between 48-72 his) 35 ul samples from each well are then transfeπed to an opaque 96 well plate using a 12 channel pipette The opaque plates should be covered (using sellophene covers) and stored at -20 degrees C until SEAP assays are performed according to Example 17 The plates containing the remaining treated cells are placed at 4 degrees C and seive as a souice of material for repeating the assay on a specific well if desired
As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells Over 30 fold induction is typically observed in the positive control wells
The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art
Example 14: High-Throughput Screening Assay Identifying Myeloid Activity The following protocol is used to assess myeloid activity by determining whether polypeptides of the invention piohferates and/or differentiates myeloid cells Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 12 Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 12, a DEAE-Dextran method (Kharbanda et al , 1994, Cell
Giowth & Differentiation, 5 259-265) is used Fust, harvest 2x1 Oe7 U937 cells and wash with PBS The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin Next, suspend the cells in 1 ml of 20 mM Tns-HCl (pH 7 4) buffer containing 0 5 mg/ml DEAE-Dextran. 8 ug G * S-SEAP2 plasmrd DNA, 140 mM NaCl, 5 mM KCI, 375 uM Na2HP04 7H20 1 mM MgCh, and 675 uM CaCh Incubate at 37 degrees C for 45 mm Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degrees C foi 36 hi
The GAS-SEAP/U937 stable cells are obtained by gi owing the cells in 400 ug/ml G418 The G418-free medium is used for routine growth but every one to two months, the cells should be re-grow n in 400 ug/ml G418 for couple of passages s These cells are tested by harvesting 1x10 cells (this is enough for ten 96-well plates assay) and wash with PBS Suspend the cells in 200 ml above described growth medium, with a final density of 5x10^ cells/ml Plate 200 ul cells per well in the 96-well plate (or 1x10s cells/well)
Add 50 ul of the supernatant prepared by the protocol described in Example 1 1 Incubate at 37 degrees C for 48 to 72 hr As a positive contiol, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells Over 30 fold induction is typically observed in the positive control wells SEAP assay the supernatant according to the protocol described in Example 17
Example 15: High-Throughput Screening Assay Identifying Neuronal Activity.
When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways One of these genes, EGRl (early growth response gene 1), is induced in various tissues and cell types upon activation The promoter of EGRl is responsible for such induction Using the EGRl promoter linked to reporter molecules, activation of cells can be assessed
Particularly, the following protocol is used to assess neuronal activity in PC 12 cell lines PC 12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate). NGF (nerve growth factor), and EGF (epidermal growth factor) The EGRl gene expression is activated during this treatment Thus, by stably transfecting PC 12 cells with a construct containing an EGR piomotei linked to SEAP reportei, activation of PC 12 cells can be assessed
The EGR/SEAP iepoiter constiuct can be assembled by the following protocol The EGR-1 piomoter sequence (-633 to +l)(Sakamoto K et al , Oncogene 6 867-871 ( 1991 )) can be PCR amplified fiom human genomic DNA using the following primers
5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID NO 6) 5' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3 (SEQ ID NO 7) Using the GAS SEAP/Neo vector produced in Example 12, EGRl amplified product can then be inserted into this vector Linearize the GAS SEAP/Neo vector using restriction enzymes Xhol/Hindlll, removing the GAS/SV40 stuffer Restπct the EGRl amplified product with these same enzymes Ligate the vector and the EGRl promoter
To prepare 96 well-plates for cell culture, two mis of a coating solution ( 1 30 dilution of collagen type I (Upstate Biotech Inc Cat#08- 1 15) in 30% ethanol (filter sterilized)) is added pei one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr
PC 12 cells are routinely grown in RPMI- 1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat # 12449-78P), 5% heat- inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish One to four split is done every three to four days Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times
Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine protocol described in Example 1 1 EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418 The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium Wash the cells once with PBS
(Phosphate buffered saline) Then starve the cells in low serum medium (RPMI- 1640 containing 1% horse serum and 0 5% FBS with antibiotics) overnight The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5x 10*5 cells/ml. Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to
1x10-5 cells/well). Add 50 ul supernatant produced by Example 1 1, 37°C for 48 to 72 hr. As a positive control, a growth factor known to activate PC 12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 17.
Example 16: High-Throughput Screening Assay for T-cell Activity
NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL- 1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF- KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses. In non-stimulated conditions, NF- KB is retained in the cytoplasm with I-KB
(Inhibitor KB). However, upon stimulation, I- KB is phosphorylated and degraded, causing NF- KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF- KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC. Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 1 1. Activators or inhibitors of NF-KB would be useful in treating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis. To constiuct a vector containing the NF-KB piomoter element, a PCR based strategy is employed The upstieam primer contains foui tandem copies of the NF- KB binding site (GGGGACTTTCCC) (SEQ ID NO 8), 18 bp ot sequence complementaiy to the 5 end ot the SV40 early promotei sequence and is flanked
Figure imgf000255_0001
5' GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC TTTCCATCCTGCCATCTCAATTAG 3' (SEQ ID NO 9)
The downstieam primer is complementaiy to the 3' end of the SV40 promoter and is flanked with a Hind III site 5' GCGGCAAGCTTTTTGCAAAGCCTAGGC 3' (SEQ ID NO 4)
PCR amplification is performed using the SV40 promoter template present in the pB-gal promoter plasmid obtained fiom Clontech The resulting PCR fiagment is digested with Xhol and Hmd III and subcloned into BLSK2- (Stratagene) Sequencing with the T7 and T3 pnmeis confirms the insert contains the following sequence
5 ' CTCG AGGGG ACTTTCCCGGGG ACTTTCCGGGG ACTTTCCGGG ACTTTCC ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTA TTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTT 3' (SEQ ID NO 10)
Next, replace the SV40 minimal promoter element present in the pSEAP2- promoter plasmid (Clontech) with this NF-KB/SV40 fragment using Xhol and Hindlll However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems
In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes Sail and Notl, and inserted into a vector containing neomycin resistance Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with Sail and Notl Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 13. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 13. As a positive control, exogenous TNF alpha (0.1 ,1 , 10 ng) is added to wells H9, H 10, and H 1 1. with a 5- 10 fold activation typically observed.
Example 17: Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.5x Dilution Buffer and dispense 15 ul of 2.5x dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C for 30 min. Separate the Optiplates to avoid uneven heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on luminometer, one should treat 5 plates at each time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:
# ol plates R\n butter diluent (ml) CSPD (ml
10 60 3
11 65 3.25
12 70 3.5
13 75 375
14 80 4
15 85 4.25
16 90 4.5
17 95 475
18 100 5
19 105 5.25
20 110 55
21 115 5.75
22 120 6
23 125 6.25
24 130 6.5
25 135 6.75
26 140 7
27 145 7.25
28 150 7.5
29 155 7.75
30 160 8
31 165 8.25
32 170 8.5
33 175 8.75
34 180 9
35 185 9.25
36 190 9.5
37 195 9.75
38 200 10
39 205 10.25
40 210 10.5
41 215 10.75
42 220 11
43 225 11.25
44 230 11.5
45 235 11.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
Example 18: High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium sodium pH membrane potentral or any other small molecule which is detectable by a fluoiescent probe
The following assay uses Fluoiometnc Imaging Plate Readei ("FLIPR") to measuie changes in fluoiescent molecules (Molecular Probes) that bind small molecules Cleaily any fluoiescent molecule detecting a small molecule can be used instead of the calcium fluoiescent molecule, fluo-4 (Molecular Piobes, Inc , catalog no F-14202) used hαe
For adherent cells seed the cells at 10,000 -20,000 cells/well in a Co-star black 96-well plate with clear bottom The plate is incubated in a CO, incubator for 20 hours The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash
A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO To load the cells with fluo-4 , 50 ul of 12 ug/ml fluo-4 is added to each well The plate is incubated at 37 degrees C in a CO, incubator for 60 min The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer
For non-adherent cells, the cells are spun down from culture media Cells are re-suspended to 2-5x106 cells/ml with HBSS in a 50-ml conical tube 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension The tube is then placed in a 37 degrees C water bath for 30-60 min The cells are washed twice with HBSS resuspended to lxlO6 cells/ml, and dispensed into a microplate, 100 ul/well The plate is centrifuged at 1000 φm for 5 min The plate is then washed once in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final volume
For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4 The supernatant is added to the well, and a change in fluorescence is detected
To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters (1) System gain is 300-800 mW, (2) Exposure time is 0 4 second, (3) Camera F/stop is F/2, (4) Excitation is 488 nm, (5) Emission is 530 nm, and (6) Sample addition is 50 ul Increased emission at 530 nm indicates an extracellular signaling event which has resulted in an increase in the intracellular
Ca++ concentration Example 19: High-Throughput Screening As av Identifying Tyrosine Kinase Activity
The Piote Tyrosine Kinases (PTK) repiesent a divαse group of transmembrane and cytoplasmic kinases Within the Receptor Protem Tyrosine Kinase RPTK) gioup are receptors foi a range of mitogemc and metabolic growth factors including the PDGF. FGF. EGF. NGF. HGF and Insulin receptoi subfamilies In addition there aie a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix protems
Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosohc protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine supeifamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin)
Because of the wide range of known factois capable of stimulating tyrosine kinase activity, the identification of novel human secreted proteins capable of activating tyrosine kinase signal transduction pathways are of interest Therefore, the following protocol is designed to identify those novel human secreted proteins capable of activating the tyrosine kinase signal transduction pathways
Seed target cells (e g., primary keratmocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO) or 10% Matπgel purchased from Becton Dickinson (Bedford,MA), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc (Saciamento, CA) aftei 48 hr Falcon plate covers #3071 fiom Becton Dickinson (Bedfoid MA) aie used to covei the Lopiodyne Silent Screen Plates Falcon Miαotest III cell culture plates can also be used in some piohferation experiments To prepare extiacts. A431 cells aie seeded onto the nylon membranes of
Loprodyne plates (20 000/200ml/w ell) and cultured overnight in complete medium Cells are quiesced by incubation in serum-free basal medium foi 24 hr After 5-20 minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in Example 11 , the medium was removed and 100 ml of exti ction buffei ((20 mM HEPES pH 7 5, 0 15 M NaCl, 1% Triton X-100, 0 1 % SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease mhibitois (# 1836170) obtained from Boeheπnger Mannheim (Indianapolis, IN) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4 degrees C The plate is then placed in a vacuum transfer manifold and the extract filteied thiough the 0 45 mm membrane bottoms of each well using house vacuum Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice To obtain extracts clarified by centrifugation, the content of each well, after detergent solubihzation for 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C at 16,000 x g Test the filtered extracts for levels of tyrosine kinase activity Although many methods of detecting tyrosine kinase activity are known, one method is described here
Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide) Biotinylated peptides that can be used for this puφose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastπn) Both peptides are substrates for a range of tyrosine kinases and are available from Boehπnger Mannheim
The tyrosine kinase reaction is set up by adding the following components in order First, add lOul of 5uM Biotinylated Peptide, then lOul ATP/Mg2+ (5mM
ATP/50mM MgC^), then lOul of 5x Assay Buffer (40mM imidazole hydrochloπde, pH7 3 40 mM beta-glyceiophosphate ImM EGTA lOOmM MgCh 5 mM MnCb 0 5 mg/ml BSA) then 5ul of Sodium Vanadate( ImM), and then 5ul of watei Mix the components gently and pieincubate the leaction mix at 30 degrees C for 2 min Initial the ieaction by adding lOul of the control enzyme oi the filtered supernatant The tyrosine kinase assay reaction is then teiminated by adding 10 ul of
120mm EDTA and place the reactions on ice
Tyrosine kinase activity is detei mined by tiansferπng 50 ul alrquot of reaction mixtuie to a microtitei plate (MTP) module and incubating at 37 degiees C for 20 min This allows the streptavadm coated 96 well plate to associate with the biotinylated peptide Wash the MTP module with 300ul/well of PBS four times Next add 75 ul of anti phospotyrosine antibody conjugated to horse radish peroxιdase(antι-P-Tyr POD(0 5u/ml)) to each well and incubate at 37 degrees C for one hour Wash the well as above
Next add lOOul of peroxidase substrate solution (Boehπnger Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min) Measure the absorbance of the sample at 405 nm by using ELISA reader The level of bound pei oxidase activity is quantitated using an ELISA reader and leflects the level of tyrosine kinase activity
Example 20: High-Throughput Screening Assay Identifying Phosphorylation Activity
As a potential alternative and/or compliment to the assay of protem tyrosine kinase activity described in Example 19, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk- 1 and Erk-2 kinases However, phosphorylation of other molecules, such as Raf. JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoseπne, phosphotyrosine, or phosphothreonme molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay
Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0 1ml of protein G (lug/ml) for 2 hr at room temp, (RT) The plates are then nnsed with PBS and blocked with 3% BSA/PBS foi 1 hi at RT The piotein G plates aie then treated with 2 commeiαal monoclonal antibodies ( lOOng/well) against Eik-land Eιk-2 ( 1 hr at RT) (Santa Ciuz Biotechnology) (To detect other molecules this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules ) After 3-5 πnses with PBS, the plates are stored at 4 degrees C until use
A431 cells aie seeded at 20.000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium The cells are then starved foi 48 hr in basal medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the supernatants obtained in Example 1 1 for 5-20 minutes The cells are then solubilized and extracts filtered directly into the assay plate
After incubation with the extract for 1 hr at RT, the wells are again rinsed As a positive control, a commercial preparation of MAP kinase ( lOng/well) is used in place of A431 extract Plates are then treated with a commercial polyclonal (rabbit) antibody (lug/ml) which specifically lecognizes the phosphorylated epitope of the Erk- 1 and Erk-2 kinases (1 hr at RT) This antibody is biotinylated by standard procedures The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence) An incieased fluorescent signal over background indicates a phosphorylation
Example 21: Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is be isolated cDNA is then generated from these RNA samples using protocols known in the art (See, Sambrook ) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO X Suggested PCR conditions consist of 35 cycles at 95 degrees C for 30 seconds, 60-120 seconds at 52-58 degrees C, and 60-120 seconds at 70 degrees C, using buffer solutions described in Sidransky et al , Science 252 706 (1991)
PCR products are then sequenced using primers labeled at their 5' end with T4 polynucleotide kinase, employing SequiTheim Polymerase (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations is then cloned and sequenced to validate the results of the direct sequencing. PCR products is cloned into T-tailed vectors as described in Holton et al.,
Nucleic Acids Research, 19: 1 156 ( 1991 ) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5'- triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot- 1 DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, VT) in combination with a cooled charge-coupled device camera (Photometries, Tucson, AZ) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991).) Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Coφoration, Durham, NC.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.
Example 22: Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
The coated wells are then incubated for > 2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbounded polypeptide.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbounded conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Inteφolate the concentration of the polypeptide in the sample using the standard curve.
Example 23: Formulation
The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant a polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier). The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Theiapeutic alone), the site of dehveiy, the method of administration, the scheduling of administration, and othei factois known to practitioners The "effective amount' for purposes herein is thus determined by such considerations
As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about lug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion More preferably, this dose is at least 0 01 mg/kg/day, and most preferably for humans between about 0 01 and 1 mg/kg/day foi the hormone If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump An intravenous bag solution may also be employed The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect
Therapeutics can be are administered orally, rectally, parenterally, intracistemally, intravaginally, mtraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray
"Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisohd or liquid filler, diluent, encapsulating material or formulation auxiliary of any The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion
Therapeutics of the invention are also suitably administered by sustained- release systems Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, mtraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semisohd or liquid filler, diluent, encapsulating material or formulation auxiliary of any type The term "parenteral" as used herein refers to modes of administiation which include intravenous, lntramusculai, intrapeπtoneal, intrasternal. subcutaneous and intraarticulai injection and infusion
Therapeutics of the invention are also suitably administered by sustained- release systems Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e g , films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble deπvatives (such as, for example, a sparingly soluble salt)
Sustained-release matrices include polylactides (U.S Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) (Langer et al , J. Biomed. Mater. Res 15 167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id ) or poly-D- (-)-3-hydroxybutyπc aαd (EP 133,988) Sustained-release Therapeutics also include hposomally entrapped
Therapeutics of the invention (see generally, Langer, Scιe??ce 249:1527-1533 (1990); Treat et al, in Lιposo?nes in the Therapy of Infectious Disease a?ιd Cancer. Lopez- Berestein and Fidler (eds.), Liss, New York, pp 317 -327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sα (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad Sα (USA) 77 4030-4034 ( 1980); EP 52,322, EP 36,676; EP 88,046; EP 143,949, EP 142,641 ; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544.545, and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) umlamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Cπt. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989))
Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)). For parenteial administiation, in one embodiment, the Therapeutic is formulated geneially by mixing it at the desned degiee of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, 1 e , one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic
Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both Then, if necessary, the product is shaped into the desired formulation Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides, e g , polyarginme or tπpeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, such as glycine, glutamic acid, aspartic acid, or arginine, monosaccharides, disacchaπdes, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextπns, chelating agents such as EDTA, sugar alcohols such as manmtol or sorbitol, counterions such as sodium, and or nonionic surfactants such as polysorbates, poloxamers, or PEG
The Therapeutic is typically formulated in such vehicles at a concentration of about 0 1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8 It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts
Any pharmaceutical used for therapeutic administration can be sterile Sterility is readily accomplished by filtration through sterile filtration membranes (e g , 0 2 micron membranes) Therapeutics generally aie placed into a containei having a sterile access poit, for example, an intiavenous solution bag oi vial having a stopper pierceable by a hypodermic injection needle
Therapeutics ordinarily will be stored in unit or multi-dose containers, foi example, sealed ampoules or vials, as an aqueous solution or as a lyophihzed formulation for reconstitution As an example of a lyophihzed formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophihzed The infusion solution is prepared by reconstituting the lyophihzed Therapeutic using bacteπostatic Water-for-Injection The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention Associated with such contaιner(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 In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds
The Therapeutics of the invention may be administered alone or in combination with adjuvants Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Coφ ), QS21 (Genentech, Inc ), BCG, and MPL In a specific embodiment, Therapeutics of the invention are administered in combination with alum In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21 Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl hpid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheπa, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus. cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis Combinations may be administered either concomitantly, e g , as an admixture, separately but simultaneously oi concurrently, or sequentially This includes presentations in which the combined agents aie admimsteied together as a theiapeutic mixture, and also proceduies in which the combined agents are administered separately but simultaneously, e g as through separate intravenous lines into the same individual Administration ' in combination" further includes the separate administration of one of the compounds or agents given fust, followed by the second
The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents Therapeutic agents that may be admimsteied in combination with the Therapeutics of the invention, include but not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-mflammatoπes, conventional lmmunotherapeutic agents, cytokines and/or growth factors Combinations may be administered either concomitantly, e g , as an admixture, separately but simultaneously or concurrently, or sequentially This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e g , as through separate intravenous lines into the same individual Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family TNF, TNF-related or TNF-hke molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4- 1BBL, DcR3, OX40L, TNF-gamma
(International Publication No WO 96/14328), AIM-I (International Publication No WO 97/33899), endokine-alpha (International Publication No WO 98/07880), TR6 (International Publication No WO 98/30694), OPG, and neutrokine-alpha (International Publication No WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International
Publication No WO 96/34095), DR3 (International Publication No WO 97/33904), DR4 (International Publication No WO 98/32856), TR5 (International Publication No. WO 98/30693). TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddl), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection. ■
In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIF AMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™,
KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETH AMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™. RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacteriwn avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacteriian tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™. and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplas?na go?ιdii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.
In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine. In a fuither embodiment, the Therapeutics of the invention aie administered in combination with an antibiotic agent Antibiotic agents that may be administered with the Therapeutics of the invention include, but aie not limited to amoxiαllm, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Chndamyαn, chloramphenicol, cephalospoπns, αpiofloxacin, αprofloxaαn, eiythromyαn, fluoroqumolones, macrohdes metronidazole, penicillins, quinolones, πfampin, streptomycin, sulfonamide tetracycllnes, tπmethopπm, tπmethopπm- sulfamthoxazole, and vancomyαn
Conventional nonspecific lmmunosuppressive agents, that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclospoπne. cyclospoπne analogs, cyclophosphamide methylpredmsone, prednisone, azathiopπne, FK-506, 15-deoxysperguahn, and other lmmunosuppressive agents that act by suppressing the function of responding T cells In specific embodiments, Therapeutics of the invention are administered in combination with immunosuppressants Immunosuppressants preparations that may be administered with the Therapeutics of the invention include, but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclospoπn), PROGRAF™ (tacrohmus), CELLCEPT™ (mycophenolate), Azathiopπne, glucorticosteroids, and RAPAMUNE™ (sirohmus) In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation
In an additional embodiment. Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™,
IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, and GAMIMUNE™ In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e g , bone marrow transplant) In an additional embodiment, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent Anti- lnflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to. glucocorticoids and the nonsteroidal anti- inflammatories. aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, fhiazinecarboxamides, e- acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine. bucolome. difenpiramide, ditazol, emorfazone, guaiazulene. nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime. proquazone, proxazole, and tenidap.
In another embodiment, compostions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5- FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea. procarbazine, mitomycin. busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide). In a specific embodiment, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or any combination of the components of CHOP. In another embodiment, Therapeutics of the invention are administered in combination with Rituximab. In a further embodiment, Therapeutics of the invention are administered with Rituxmab and CHOP, or Rituxmab and any combination of the components of CHOP. In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10. IL12, IL13. IL15. anti-CD40, CD40L, IFN-gamma and TNF- alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin. including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10. IL-1 1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19. IL-20, and IL-21.
In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF). as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268 ( 1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth
Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D). as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DEI 9639601. The above mentioned references are incoφorated herein by reference herein. In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, LEUKINE™ (SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).
In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4. FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-1 1, FGF-12, FGF-13, FGF-14, and FGF- 15. In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Example 24: Method of Treating Decreased Levels of the Polypeptide
The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 23.
Example 25: Method of Treating Increased Levels of the Polypeptide The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition compiising a theiapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention)
In one example, antisense technology is used to inhibit pioduction of a polypeptide of the present invention This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancel For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0 5, 1.0, 1.5, 2 0 and 3 0 mg/kg day for 21 days This tieatment is repeated aftei a 7-day rest period if the treatment was well tolerated The formulation of the antisense polynucleotide is provided in Example 23
Example 26: Method of Treatment Using Gene Therapy-Ex Vivo
One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge The monolayer is trypsinized and scaled into larger flasks pMV-7 (Kirschmeier, P.T. et al., DNA, 7.219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase The linear vector is fractionated on agarose gel and purified, using glass beads
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5' and 3' end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5' primer contains an EcoRI site and the 3' primer includes a Hindlll site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB 101 , which are then plated onto agar containing kanamycin for the puφose of confirming that the vector has the gene of interest properly inserted. The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 27: Gene Therapy Using Endogenous Genes Corresponding To Polynucleotides of the Invention Another method of gene therapv accoiding to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous iecombmation as described, foi example, in U S Patent NO 5,641 670, issued June 24. 1997, International Publication NO WO 96/2941 1 , published Septembei 26 1996 International Publication NO WO 94/12650, published August 4 1994, Koller et al Pi oc Natl Acad Sci USA, 86 8932-8935 ( 1989), and Zijlstra et al , Natiti e, 342 435-438 ( 1989) This method involves the activation of a gene which is present in the taiget cells, but which is not expressed in the cells, or is expressed at a lower level than desired Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5 non-codmg sequence of endogenous polynucleotide sequence, flanking the promotei The targeting sequence will be sufficiently near the 5' end of the 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 Preferably, the amplified promoter contains distinct restnction enzyme sites on the 5' and 3' ends Preferably, the 3' 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 the amplified targeting sequences are digested with the appropriate lestnction enzymes and subsequently treated with calf intestinal phosphatase The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase The resulting mixture is maintained under conditions appropriate for ligation of the two fragments The construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation
In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation However, the polynucleotide constructs may also be administered with transfection-facihtatmg agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc Such methods of delivery are known in the art Once the cells are transfected. homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM + 10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCI, 0.7 mM Na2 HP04, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3X106 cells/ml. Electroporation should be performed immediately following resuspension.
Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC 18 (MBI Fermentas, Amherst, NY) is digested with Hindlll. The CMV promoter is amplified by PCR with an Xbal site on the 5' end and a BamHl site on the 3'end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a Hindlll site at the 5' end and an Xba site at the 3'end; the other non-coding sequence (fragment 2) is amplified with a BamHl site at the 5'end and a Hindlll site at the 3'end. The CMV promoter and the fragments ( 1 and 2) are digested with the appropriate enzymes (CMV promoter - Xbal and BamHl; fragment 1 - Xbal; fragment 2 - BamHl) and ligated together. The resulting ligation product is digested with Hindlll, and ligated with the Hindlll- digested pUC18 plasmid.
Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.X106 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incoφorate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSe'c should be observed.
Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer- pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.
The engineered fibroblasts are then injected into the host, either alone or after- having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.
Example 28: Method of Treatment Using Gene Therapy - In Vivo
Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/1 1092, WO98/1 1779; U.S. Patent NO. 5693622,
5705151, 5580859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-41 1 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incoφorated herein by reference). The polynucleotide constructs may 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, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous earner.
The term "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. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Feigner P.L. et al. (1995) Ann. NY Acad. Sci. 772: 126- 139 and Abdallah B. et al. (1995) Biol. Cell 85( 1): 1-7) which can be prepared by methods well known to those skilled in the art.
The polynucleotide vector constructs 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. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, 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 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. It is similarly 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. lot example stem cells of blood or skin ftbioblasts /// xivo muscle cells are paiticularly competent in then ability to take up and expiess polynucleotides
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0 05 g/kg body weight to about 50 mg/kg body weight Preferably the dosage will be fiom about 0 005 mg/kg to about 20 mg/kg and moie preferably fiom about 0 05 mg/kg to about 5 mg/kg Of course, as the artisan of oidinary skill will appreciate this dosage will vary according to the tissue site of injection The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues However, other paienteral 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 In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure
The dose response effects of injected polynucleotide in muscle ι?ι vivo is determined as follows Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes The quadriceps muscles of mice are then injected with various amounts of the template DNA Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0 3 ml of 2 5% Avertm A 1 5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized The template
DNA is injected in 0 1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0 5 cm from the distal insertion site of the muscle into the knee and about 0 2 cm deep A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips After an appropriate incubation time (e g , 7 days) muscle extracts are prepared by excising the entire quadriceps Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression A time couise foi piotein expression may be done in a similai fashion except that quadriceps fiom ditfeient mice aie haivested at difteient times Peisistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellulai DNA and HIRT supernatants from injected and contiol mice The results of the above experimentation in mice can be use to extrapolate proper dosages and other tieatment parameters in humans and other animals using naked DNA
Example 29: Transgenic Animals. The polypeptides of the invention can also be expressed in transgenic animals
Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e g , baboons, monkeys, and chimpanzees may be used to generate transgenic animals In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol
Any technique known in the art may be used to introduce the transgene (l e , polynucleotides of the invention) mto animals to produce the founder lines of transgenic animals Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al , Appl Microbiol Biotechnol 40 691-698 (1994),
Carver et al , Biotechnology (NY) 1 1 1263-1270 (1993), Wright et al , Biotechnology (NY) 9 830-834 (1991), and Hoppe et al , U S Pat No 4,873, 191 ( 1989)), retrovirus mediated gene transfer into germ lines (Van der Putten et al , Proc Natl Acad Sα , USA 82 6148-6152 (1985)), blastocysts or embryos, gene targeting in embryonic stem cells (Thompson et al , Cell 56 313-321 (1989)), electroporation of cells or embryos (Lo, 1983, Mol Cell Biol 3 1803-1814 (1983)), introduction of the polynucleotides of the invention using a gene gun (see, e g , Ulmer et al , Science 259 1745 (1993), introducing nucleic acid constructs into embryonic pleuπpotent stem cells and transferring the stem cells back into the blastocyst, and sperm- mediated gene transfer (Lavitrano et al , Cell 57 717-723 (1989), etc For a review of such techniques, see Gordon, "Transgenic Animals," Intl Rev Cytol 1 15 171-229 (1989), which is incoφorated by reference herein in its entirety Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 ( 1996); Wilmut et al.. Nature 385:810- 813 (1997)).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the puφose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science . 265: 103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcπptase-PCR (it-PCR) Samples of tiansgenic gene expressing tissue may also be evaluated lmmunocytochemically oi immunohistochemically using antibodies specific for the tiansgene pioduct
Once the founder animals are pioduced, they may be bied, inbred, outbred, oi crossbred to produce colonies of the particular animal Examples of such breeding strategies include, but are not limited to outbreeding of founder animals with more than one integration site in older to establish separate lines, inbreeding of separate lines in order to produce compound transgemcs that express the transgene at higher levels because of the effects of additive expression of each transgene, crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis, crossing of separate homozygous lines to produce compound heterozygous or homozygous lines, and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders
Example 30: Knock-Out Animals.
Endogenous gene expression can also be reduced by inactivating or "knocking out" the gene and/or its promoter using targeted homologous recombination (E g , see Smithies et al , Nature 317 230-234 (1985), Thomas & Capecchi, Cell 51 503-
512 (1987), Thompson et al , Cell 5 313-321 (1989), each of which is incoφorated by reference herein in its entirety) For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art. In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or mtraperitoneally.
Alternatively, the cells can be incoφorated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349; and Mulligan & Wilson, U.S. Patent No. 5,460.959 each of which is incoφorated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 31: Production of an Antibody a) Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing polypeptide(s) of the invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of polypeptide(s) of the invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
Monoclonal antibodies specific for polypeptide(s) of the invention are prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:51 1 (1976); Kohler et al., Eur. J. Immunol. 6:292 ( 1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with polypeptide(s) of the invention or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/1 of nonessential amino acids, about 1 ,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 ( 1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide(s) of the invention.
Alternatively, additional antibodies capable of binding to polypeptide(s) of the invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by polypeptide(s) of the invention. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and are used to immunize an animal to induce formation of further protein-specific antibodies. For in vivo use of antibodies in humans, an antibody is "humanized". Such antibodies can be produced using genetic constructs derived from hybridoma cells . producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229: 1202 (1985); Oi et al, BioTechniques 4:214 ( 1986); Cabilly et al., U.S. Patent No. 4.816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671 ; Bouhanne et al., Nature 312:643 ( 1984); Neuberger et al.. Nature 314:268 (1985).)
b) Isolation Of Antibody Fragments Directed Against
Polypeptide(s) of the Present Invention From A Library Of scFvs Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against polypeptide(s) of the invention to which the donor may or may not have been exposed (see e.g., U.S. Patent 5,885,793 incorporated herein by reference in its entirety). Rescue of the Library.
A library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1 % glucose and 100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of 2xTY- AMP-GLU, 2 x 108 TU of delta gene 3 helper (M l 3 delta gene III. see PCT publication WO 92/01047) are added and the culture incubated at 37°C for 45 minutes without shaking and then at 37°C for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2xTY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in PCT publication WO 92/01047.
Ml 3 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage moφhogenesis. The culture is incubated for 1 hour at 37° C without shaking and then for a further hour at 37°C with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37°C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 1013 transducing units/ml (ampicillin-resistant clones). Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or
10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel- PBS for 2 hours at 37°C and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated foi 30 minutes at loom temperature tumbling on an ovei and undei turntable and then left to stand foi anothei 1 5 hour s Tubes aie washed 10 times with PBS 0 1 % Tween-20 and 10 times with PBS Phage are eluted by adding 1 ml of 100 mM tπethylamme and rotating 15 minutes on an under and ovei turntable after which the solution is immediately neutiahzed with 0 5 ml of 1 0M Tris- HCl, pH 7 4 Phage aie then used to infect 10 ml of mid-log E coli TGI by incubating eluted phage with bactena for 30 minutes at 37°C The E coli are then plated on TYE plates containing 1 % glucose and 100 μg/ml ampicillin The resulting bacterial hbiaiy is then rescued with delta gene 3 helper phage as described above to piepare phage for a subsequent round of selection This process is then repeated for a total of 4 lounds of affinity puπfication with tube-washing incieased to 20 times with PBS, 0 1% Tween-20 and 20 times with PBS for rounds 3 and 4 Characterization of Binders Eluted phage from the 3rd and 4th rounds of selection are used to infect E coli HB 2151 and soluble scFv is produced (Marks, et al , 1991) from single colonies for assay ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9 6 Clones positive in ELISA are fuither chaiacteπzed by PCR fingerprinting (see, e g , PCT publication WO 92/01047) and then by sequencing These ELISA positive clones may also be further characterized by techniques known in the art, such as, foi example, epitope mapping, binding affinity, leceptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity
Example 32: Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation
Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lmeage cells and their microenvironment Signals may impart a positive stimulus that allows a B-hneage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5 IL-6, IL-7, IL10, IL- 13, IL-14 and IL-15 Interestingly, these signals are by themselves weak effectors but can. in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.
One of the best studied classes of B-cell co-stimulatory proteins is the TNF- superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD 154. CD70, and CD 153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
In Vitro Assay- Purified polypeptides of the invention, or truncated forms thereof, is assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the polypeptides of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin- fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incoφoration. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220). Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 10' B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5 X lO-'M 2ME, lOOU/ml penicillin, lOug/ml streptomycin, and 10"5 dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20h pulse (luCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72h post factor addition. The positive and negative controls are IL2 and medium respectively.
In Vivo Assay- BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of a polypeptide of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days at which time they are sacπticed and various tissues and serum collected foi analyses Comparison of H&E sections fiom noπual spleens and spleens treated with polypeptides of the invention identify the results of the activity of the polypeptides on spleen cells such as the diffusion of peπ-aiteπal lymphatic sheaths and/or significant increases in the nucleated cellularity of the red pulp legions, which may indicate the activation of the differentiation and proliferation of B-cell populations Immunohistochemical studies using a B cell maiker, antι-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, aie due to incieased B-cell repiesentation within loosely defined B-cell zones that infiltrate established T-cell regions
Flow cytometπc analyses of the spleens fiom mice treated with polypeptide is used to indicate whether the polypeptide specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice
Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers Accordingly, serum IgM and IgA levels are compared between buffer and polypeptide-treated mice
The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e g , gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention
Example 33: T Cell Proliferation Assay
A CD3-mduced proliferation assay is performed on PBMCs and is measured by the uptake of Η-thymidine The assay is performed as follows Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33 1) overnight at 4 degrees C (1 μg/ml in 05M bicarbonate buffer, pH 9 5), then washed three times with PBS PBMC are isolated by F/H gradient centrifugation from human peπpheral blood and added to quadruplicate wells (5 x 104/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of polypeptides of the invention (total volume 200 ul) Relevant prote buffer and medium alone are controls Aftei 48 hr culture at 37 degrees C, plates are spun for 2 min at 1000 φm and 100 μl of supematant is removed and stored -20 degrees C for measurement of IL-2 (or other cytokines) if effect on piohfeiation is obseived Wells aie supplemented with 100 ul of medium containing 0 5 uCi of H-thymidine and cultuied at 37 degrees C for 18 24 hi Wells aie harvested and incoipoiation of Η-thymidine used as a measure of proliferation Anti CD3 alone is the positive contiol foi piohferation IL-2 ( 100 U/ml) is also used as a contiol which enhances prohfeiation Contiol antibody which does not induce proliferation of T cells is used as the negative contiols for the effects of polypeptides of the invention
The studies described in this example tested activity of polypeptides of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e g , gene therapy), agonists, and/oi antagonists of polynucleotides or polypeptides of the invention
Example 34: Effect of Polypeptides of the Invention on the Expression of MHC Class II. Costimulatorv and Adhesion Molecules and Cell Differentiation of Monocytes and Monocvte-Derived Human Dendritic Cells
Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood adherent PBMC or elutriated monocytic fractions aie cultured for 7- 10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) These dendritic cells have the characteristic phenotype of immature cells (expression of CDl. CD80. CD86, CD40 and MHC class II antigens) Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83) These changes correlate with increased antigen-presentmg capacity and with functional maturation of the dendritic cells
FACS analysis of surface antigens is performed as follows Cells are treated 1 -3 days with increasing concentrations of polypeptides of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0 02 mM sodium azide, and then incubated with 1 20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson) Effect on the pioduction of cytokines Cytokines geneiated bv dendritic cells in paiticulai IL 12, aie impoitant in the initiation of T-cell dependent immune lesponses IL- 12 stiongly influences the development of Thl helper T-cell immune response, and induces cytotoxic T and NK cell function An ELISA is used to measure the IL 12 lelease as follows Dendiitic cells ( 106/ml) aie treated with increasing concentiations of polv peptides of the invention for 24 houis LPS (100 ng/ml) is added to the cell cultuie as positive control Supematants fiom the cell cultuies are then collected and analyzed for IL-12 content using commercial ELISA kit (e g, R & D Systems (Minneapolis MN)) The standard piotocols pi ovided with the kits are used
Effect on the expiession of MHC Class II, costimulatory and adhesion molecules Three major families of cell surface antigens can be identified on monocytes adhesion molecules molecules involved in antigen presentation, and Fc receptor Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation Increase expression of Fc receptors may correlate with impioved monocyte cytotoxic activity, cytokine release and phagocytosis
FACS analysis is used to examine the surface antigens as follows Monocytes are treated 1-5 days with lnαeasmg concentrations of polypeptides of the invention or LPS (positive contiol), washed with PBS containing 1% BSA and 0 02 mM sodium azide, and then incubated with 1 20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degreesC After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson) Monocyte activation and/or increased survival Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes Polypeptides, agonists, or antagonists of the invention can be screened using the three assays descπbed below For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, MD) by centrifugation through a Histopaque gradient (Sigma) Monocytes aie isolated fiom PBMC by counterflow centπfugal elutπation
Monocyte Suivival Assay Human peπphαal blood monocv tes piogiessively lose viability when cultured in absence of seium 01 other stimuli Their death lesults from internally regulated process (apoptosis) Addition to the cultuie of activating factors, such as TNF-alpha dramatically impioves cell survival and prevents DNA fragmentation Propidium iodide (PI) staining is used to measuie apoptosis as follows Monocytes are cultured for 48 houis in polypropylene tubes in serum- free medium (positive contiol), in the piesence of 100 ng/ml TNF-alpha (negative contiol). and in the presence of varying concentrations of the compound to be tested Cells are suspended at a concentration of 2 x lOVml in PBS containing PI at a final concentration of 5 μg/ml, and then incubaed at room temperature for 5 minutes befoie FACScan analysis PI uptake has been demonstiated to correlate with DNA fragmentation in this experimental paradigm Effect on cytokine release An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation An ELISA to measure cytokine release is performed as follows Human monocytes are incubated at a density of 5x10^ cells/ml with increasing concentrations of the a polypeptide of the invention and under the same conditions, but in the absence of the polypeptide For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in piesence of a polypeptide of the invention LPS (10 ng/ml) is then added Conditioned media are collected after 24h and kept frozen until use Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e g, R & D Systems (Minneapolis, MN)) and applying the standard protocols provided with the kit Oxidative burst Purified monocytes are plated in 96-w plate at 2-l lO1 cell/well Increasing concentrations of polypeptides of the invention are added to the wells in a total volume of 0 2 ml culture medium (RPMI 1640 + 10% FCS, glutamine and antibiotics) After 3 days incubation, the plates are centrifuged and the medium is removed from the wells To the maciophage monolayers, 0 2 ml per well of phenol red solution ( 140 mM NaCl, 10 mM potassium phosphate buffer pH 7 0, 5 5 mM dextrose, 0 56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37°C for 2 hours and the reaction is stopped by adding 20 μl IN NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H20: produced by the macrophages. a standard curve of a H20: solution of known molarity is performed for each experiment. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polypeptides, polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 35: Biological Effects of Polypeptides of the Invention
Astrocyte and Neuronal Assays-
Recombinant polypeptides of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate a polypeptide of the invention's activity on these cells. Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension." Proc. Natl. Acad. Sci. USA 85:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC- 12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of a polypeptide of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incoφoration assay. Fibroblast and endothelial cell assays
Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and maintained in giowth media from Clonetics Deimal miαovasculai endothelial cells are obtamed from Cell Applications (San Diego. CA) Foi piohferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in giowth medium The cells are then incubated for one day m 0 1 % BSA basal medium Aftei replacing the medium with fresh 0 1 % BSA medium, the cells aie incubated ith the test proteins foi 3 days Alamar Blue (Alamar Biosαences, Sacramento, CA) is added to each well to a final concentration of 10% The cells are incubated for 4 hi Cell viability is measuied by reading in a CytoFluor fluorescence readei Foi the PGE, assays, the human lung fibroblasts aie cultured at 5,000 cells/well in a 96-well plate for one day After a medium change to 0 1 % BSA basal medium, the cells aie incubated with FGF-2 or polypeptides of the invention with or without IL-lα foi 24 hours The supernatants are collected and assayed foi PGE, by EIA kit (Cayman, Ann Arboi. MI) For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day After a medium change to 0 1% BSA basal medium, the cells are incubated with FGF-2 or with or without polypeptides of the invention IL-l for 24 hours The supernatants aie collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, MA) Human lung fibroblasts are cultured with FGF-2 or polypeptides of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts FGF-2 should show a stimulation at 10 - 2500 ng/ml which can be used to compare stimulation with polypeptides of the invention Parkinson Models The loss of motor function in Parkinson's disease is attributed to a deficiency of stπatal dopamine resulting from the degeneration of the nigrostπatal dopaminergic projection neurons An animal model for Parkinson's that has been extensively characterized involves the systemic administration of l-methyl-4 phenyl 1,2,3,6- tetrahydropyπdme (MPTP) In the CNS, MPTP is taken-up by astrocytes and catabohzed by monoamine oxidase B to l-methyl-4-phenyl pyridine (MPP+) and released Subsequently MPP+ is actively accumulated in dopamineigic neuions by the high-affinity reuptake tianspoitei foi dopamine MPP+ is then concentiated in mitochondna by the electrochemical giadient and selectiv ely inhibits mcotidamide adenine disphosphate ubiquinone oxidoreductionase (complex I) theieby lnteifeπng with electron transport and eventually geneiating oxygen radicals
It has been demonstiated in tissue cultuie paradigms that FGF-2 (basic FGF) has trophic activity towaids mgial dopamineigic neurons (Ferrari et al Dev Biol 1989) Recently, Di Unsickei s gioup has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J Neuroscience 1990)
Based on the data with FGF-2 polypeptides of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment The potential effect of a polypeptide of the invention is fust examined in vitro in a dopaminergic neuronal cell culture paradigm The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm" on polyorthinine-laminin coated glass covershps The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N 1 ) The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopminergic neurons, immunohistochemical staining Dissociated cell cultures are prepared from embryonic rats The culture medium is changed every third day and the factors are also added at that time
Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase liurnunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro Therefore, if a polypeptide of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the polypeptide may be involved in Parkinson's Disease The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 36: The Effect of Polypeptides of the Invention on the Growth of Vascular Endothelial Cells
On day 1 , human umbilical vein endothelial cells (HUVEC) are seeded at 2-5x104 cells/35 mm dish density in Ml 99 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with Ml 99 containing 10% FBS, 8 units/ml heparin. A polypeptide having the amino acid sequence of SEQ ID NON, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
An increase in the number of HUVEC cells indicates that the polypeptide of the invention may proliferate vascular endothelial cells.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 37: Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells For evaluation of mitogenic activity of growth factors, the colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H- tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture ( 1 :0.05) are added per well and allowed to incubate foi 1 hour at 37°C before measuπng the absoibance at 490 nm in an ELISA plate leadei Backgiound absorbance from control wells (some media no cells) rs subtracted and seven wells are performed in paiallel for each condition See, Leak et al In Vitio Cell
Figure imgf000300_0001
Biol 30A 512 518 ( 1994) The studies desciibed in this example tested activ lty of a polypeptide of the invention Howevei one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy) agonists, and/or antagonists of the invention
Example 38: Inhibition of PDGF-induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect
HAoSMC piohferation can be measured, foi example, by BrdUrd incoφoration Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories) In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degiees C for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine After counterstaining with hematoxyhn the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted The BidUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number In addition the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination See, Hayashida et al , J Biol Chem 6 271(36) 21985-21992 (1996)
The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy), agonists, and/or antagonists of the invention Example 39: Stimulation of Endothelial Migration
This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.
Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John. MD; Falk, W., et al., J. Immunological Methods 1980;33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, MA) are coated with 0.1 % gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M l 99 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent. early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5 x 10"1 cells suspended in 50 ul Ml 99 containing \ % FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37°C in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantified by counting cells of three random high-power fields (40x) in each well, and all groups are performed in quadruplicate.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 40: Stimulation of Nitric Oxide Production by Endothelial Cells
Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.
Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive contiol (such as VEGF- 1 ) and the polypeptide of the invention Nitnc oxide in the medium is detei mined by use of the Guess leagent to measure total nitiite aftei reduction of nitnc oxide-deπved nitrate by nitiate leductase The effect of the polypeptide of the invention on nitnc oxide lelease is examined on HUVEC Briefly, NO lelease fiom cultuied HUVEC monolayei is measured with a NO- speαfic polarographic electiode connected to a NO meter (Iso-NO Woild Precision Instruments Inc ) ( 1049) Calibration of the NO elements is performed according to the following equation
2 KNO, + 2 KI + 2 H,S04 6 2 NO + I, + 2 H,O + 2 K,S04 The standaid calibration curve is obtained by adding graded concentrations of
KNO, (0, 5, 10, 25. 50, 100, 250 and 500 nmol/L) into the calibration solution containing KI and H,SO4 The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050) The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates and the cell plates are kept on a slide warmer (Lab Line Instruments Inc ) To maintain the temperature at 37°C The NO sensor probe is inseited vertically mto the wells, keeping the tip of the electrode 2 mm under the surface of the solution, befoie addition of the different conditions S-mtroso acetyl peniαllamin (SNAP) is used as a positive control The amount of released NO is expressed as picomoles per lxlO6 endothelial cells All values reported are means of four to six measurements in each group (number of cell culture wells) See, Leak et / Biochem and Biophxs Res Comm 277 96-105 ( 1995)
The studies described in this example tested activity of polypeptides of the invention Howevei, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy), agonists, and/or antagonists of the invention
Example 41: Effect of Polypepides of the Invention on Cord Formation in Angiogenesis Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in xιt?o CADMEC (miciovascular endothelial cells) aie puichased from Cell Applications Inc as piohfeiating (passage 2) cells and aie cultuied in Cell Applications CADMEC Growth Medium and used at passage 5 Foi the in \ ιt?o angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications Attachment Factor Medium (200 ml/well) foi 30 min at 37°C CADMEC aie seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium The Growth Medium is then replaced with 300 mg Cell Applications Choid Formation Medium containing contiol buffer or a polypeptide of the invention (0 1 to 100 ng/ml) and the cells aie cultured for an additional 48 hr The numbers and lengths of the capillary-like choids are quantitated through use of the Boeckeler VIA- 170 video image analyzer All assays are done in triplicate
Commeiαal (R&D) VEGF (50 ng/ml) is used as a positive control b-esteradiol ( 1 ng/ml) is used as a negative control The appropriate buffer (without piotein) is also utilized as a control The studies described in this example tested activity of a polv peptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy), agonists, and/or antagonists of the invention
Example 42: Angiogenic Effect on Chick Chorioallantoic Membrane
Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis Blood vessel formation on CAM is easily visible and quantifiable The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined Fertilized eggs of the White Leghorn chick (Callus gallus) and the Japanese qual
(Coturnix cotumix) are incubated at 37 8°C and 80% humidity Differentiated CAM of 16-day-old chick and 13-day-old qual embryos is studied with the following methods
On Day 4 of development, a window is made into the egg shell of chick eggs The embryos are checked for normal development and the eggs sealed w ith cellotape They are further incubated until Day 13 Thermanox covershps (Nunc, Naperville, IL) are cut into disks of about 5 mm in diameter Sterile and salt-free growth factors are dissolved in distilled water and about 3 3 mg/ 5 ml are pipetted on the disks After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 43: Angiogenesis Assay Using a Matrigel Implant in Mouse
In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (Matrigel). The protein is mixed with the liquid Matrigel at 4 degree C and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid "plug" of Matrigel is removed and examined for the presence of new blood vessels. Matrigel is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.
When thawed at 4 degree C the Matrigel material is a liquid. The Matrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of Matrigel and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the Matrigel plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). Matrigel alone is used to determine basal levels of angiogenesis. The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g gene therapy), agonists, and/oi antagonists of the invention
Example 44: Rescue of Ischemia in Rabbit Lower Limb Model To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindhmb ischemia model is created by surgical removal of one femoial arteries as desciibed previously (Takeshita et al , Am J Pathol 147 1649- 1660 (1995)) The excision of the femoral artery results in retrograde propagation of thiombus and occlusion of the external iliac artαy Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshitaet al Am J Pathol 147 1649-1660 ( 1995)) An interval of 10 days is allowed for postoperative recovαy of rabbits and development of endogenous collateral vessels At 10 day post-operatively (day 0), after performing a baseline angiogram. the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al Hu??ι Ge?ιe The? 4 749-758 (1993), Leclerc er -;/ J Clin Invest 90 936-944 (1992)) When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 mm through an infusion catheter On day 30. various parameters are measured in these rabbits (a) BP ratio - The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb, (b) Blood Flow and Flow Reserve - Resting FL the blood flow during undilated condition and Max FL the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL resting FL, (c) Angiographic Score - This is measured by the angiogram of collateral vessels A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh, (d) Capillary density - The number of collateral capillaries determined in light microscopic sections taken from hindhmbs The studies described in this example tested activity of polynucleotides and polypeptides of the invention However, one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention Example 45: Effect of Poh peptides of the Invention on Vasodilation
Since dilation of vasculat endothelium is impoitant in leduαng blood piessuie, the ability of polypeptides of the invention to affect the blood piessure in spontaneously hypertensive rats (SHR) is examined Increasing doses (0. 10, 30, 100 300, and 900 mg/kg) of the polypeptides of the invention aie administered to 13-14 week old spontaneously hypeitensive iats (SHR) Data aie expiessed as the mean +/- SEM Statistical analysis are perfoimed with a paired t test and statistical significance is defined as p<0 05 vs the response to buffei alone The studies described in this example tested activity of a polypeptide of the invention However one skilled in the ait could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy), agonists, and/or antagonists of the invention
Example 46: Rat Ischemic Skin Flap Model
The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization The study in this model is divided mto three parts as follows a) Ischemic skin b) Ischemic skin wounds c) Normal wounds
The experimental protocol includes a) Raising a 3x4 cm, single pedicle full-thickness random skin flap (myocutaneous flap over the lower back of the animal) b) An exαsional wounding (4-6 mm in diameter) in the ischemic skin (skin-flap) c) Topical treatment with a polypeptide of the invention of the exαsional wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosage ranges lmg to 100 mg d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-wounding for histological, immunohistochemical. and in situ studies The studies desciibed in this example tested activity ol a polypeptide ot the invention Howevei one skilled in the ait could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene theiapy) agonists, and/oi antagonists of the invention
Example 47: Peripheral Arterial Disease Model
Angiogenic theiapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases The experimental protocol includes a) One side of the femoral arteiy is ligated to create ischemic muscle of the hindhmb, the other side of hindhmb seives as a control b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks c) The ischemic muscle tissue is collected after ligation of the femoral artery at 1 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology Biopsy is also performed on the other side of noimal muscle of the contralateral hindhmb
The studies described in this example tested activity of a polypeptide of the invention However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e g , gene therapy), agonists, and/or antagonists of the invention
Example 48: Ischemic Myocardial Disease Model A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion Alteration of expression of the polypeptide is investigated in situ The experimental protocol includes a) The heart is exposed through a left-side thoracotomy in the rat Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed b) a polypeptide of the invention, in a dosage range of 20 mg - 500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks. c) Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 49: Rat Corneal Wound Healing Model
This animal model shows the effect of a polypeptide of the invention on neovascularization. The experimental protocol includes: a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer. b) Inserting a spatula below the lip of the incision facing the outer corner of the eye. c) Making a pocket (its base is 1-1.5 mm form the edge of the eye). d) Positioning a pellet, containing 50ng- 5ug of a polypeptide of the invention, within the pocket. e) Treatment with a polypeptide of the invention can also be applied topically to the corneal wounds in a dosage range of 20mg - 500mg (daily treatment for five days).
The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 50: Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models A. Diabetic db+/db+ Mouse Model.
To demonstrate that a polypeptide of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characteπzed clinically lelevant and lepioduαble model of rmpaned wound healing Healrng of the drabetic wound is dependent on foimation of granulation tissue and le epithehahzation lather than contιactιon (Gaιtneι, M H et al J &u? t> Res 52 389 ( 1992), Greenhalgh, D G et l Am J Pathol 136 1235 ( 1990))
The diabetic animals have many of the chaiacteπstic features obseived in Type II diabetes melhtus Homozygous (db+/db+) mice aie obese in compaπson to their normal heterozygous (db+/+m) httermates Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al Pioc Natl Acad Sci USA 77 283-293 (1982)) Animals show polyphagia, polydipsia and polyuπa
Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al , J Immunol 120 1375 (1978), Debray-Sachs, M et al , Clin Exp Immunol 51(1) 1-7 (1983), Leiter et al , Am J of Pathol 114 46-55 (1985)) Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Noπdo, F et al , Exp Neurol 83(2) 221-232 (1984), Robertson et al , Diabetes 29(1) 60-67 (1980), Giacomelh et al , Lab Invest 40(4) 460-473 (1979), Coleman D L , Diabetes 31 (Suppl) 1-6 (1982)) These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al , J Immunol 120 1375-1377 (1978))
The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al , A?n J of Pathol 736 1235-1246 (1990))
Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous httermates are used in this study (Jackson Laboratories) The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study Animals are individually housed and received food and water ad libitum All manipulations are performed using aseptic techniques The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin. D.B.. J. Exp. Med. 172:245-251 ( 1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full- thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
A polypeptide of the invention is administered using at a range different doses, from 4mg to 500mg per wound per day for 8 days in vehicle. Vehicle control groups received 50mL of vehicle solution. Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral. buffered formalin in tissue cassettes between biopsy sponges for further processing.
Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control. 2) untreated group, and 3) treated group.
Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64mm2, the corresponding size of the dermal punch. Calculations are made using the following formula:
[Open area on day 8] - [Open area on day 1] / [Open area on day 1] Specimens aie fixed in 10% buffeied foimahn and paraffin embedded blocks aie sectioned peipendiculai to the wound suiface (5mm) and cut usmg a Reicheit-Jung miαotome Routine hematoxyhn-eosin (H&E) staining is peiformed on cioss-sections ot bisected wounds Histologic examination of the wounds aie used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with a polypeptide of the invention This assessment included verification of the presence of cell accumulation lnflammatoiy cells, capillaries fibioblasts, re- epithehahzation and epidermal maturity (Greenhalgh, D G et al Am J Pathol 136 1235 ( 1990)) A calibrated lens micrometer is used by a blinded observer
Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system Human skin is used as a positive tissue contiol while non-immune IgG is used as a negative control Keratinocyte growth is determined by evaluating the extent of reepitheliahzation of the wound using a calibrated lens micrometer
Proliferating cell nuclear antigen/cychn (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1 50) with an ABC Elite detection system Human colon cancer can serve as a positive tissue control and human brain tissue can be used as a negative tissue control Each specimen includes a section with omission of the primary antibody and substitution with non-immune mouse IgG Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation
Experimental data are analyzed using an unpaired t test A p value of < 0 05 is considered significant
B. Steroid Impaired Rat Model
The inhibition of wound healing by steroids has been well documented in various m vitro and in vivo systems (Wahl, GlucocorUcoids and Wound healing In Anti- Inflammatory Steroid Action Basic and Clinical Aspects 280-302 (1989), Wahlet al , J Immunol 115 476-481 ( 1975), Werb et al , J Exp Med 147 1684-1694 (1978))
GlucocorUcoids retard wound healing by inhibiting angiogenesis decreasing vascular permeability (Ebert et al , An Intern Med 37701-705 (1952)). fibroblast proliferation, and collagen synthesis (Beck et al Gi ow th Factoi s 5 295-304 ( 1991 ) Haynes et al J Clin Iin est 61 703-797 ( 1978)) and pioduαng a tiansient reduction of circulating monocytes (Haynes et al . J Clin Iin est 61 703-797 ( 1978) Wahl "Glucocorticoids and wound healing . In Antimflammatoiy Steioid Action Basic and Clinical Aspects Academic Piess New York, pp 280-302 ( 1989)) The systemic administration of steroids to impaiied wound healing is a well establish phenomenon in rats (Beck et al , G?
Figure imgf000312_0001
th Factors 5 295-304 ( 1991 ), Haynes et al , J Clin Invest 61 703-797 ( 1978), Wahl, "Glucocorticoids and wound healing", In Antnnflammatory Sterord Actron Basrc and Chnrcal Aspects, Academic Press. New Yoik, pp 280 302 ( 1989) Pierce et al , Proc Natl Acad Sci USA 86 2229-2233 ( 1989))
To demonstrate that a polypeptide of the invention can accelerate the healing process, the effects of multiple topical applications of the polypeptide on full thickness exαsional skin wounds in rats in which healing has been impaired by the systemic administration of mefhylprednisolone is assessed Young adult male Sprague Dawley rats weighing 250-300 g (Charles River
Laboratories) are used in this example The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study The healing response of rats is impaired by the systemic administration of methylpredmsolone ( 17mg/kg/rat intramuscularly) at the time of wounding Animals are individually housed and received food and water ad lιbιtu??ι All manipulations are performed using aseptic techniques This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc Institutional Animal Care and Use Committee and the Guidelines foi the Care and Use of Laboratory Animals
The wounding protocol is followed according to section A, above On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions The surgical area is dried with sterile gauze pπor to wounding An 8 mm full-thickness wound is created using a Keyes tissue punch The wounds are left open for the duiation of the experiment Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylpredmsolone administration Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges Wounds aie visually examined and photographed at a fixed distance at the day of wounding and at the end of tieatment Wound closure is determined by daily measuiement on days 1-5 and on day 8 Wounds aie measured horrzontally and veitically using a calibrated Jameson ca per Wounds are consideied healed if gianulation tissue is no longer visible and the wound is covered by a continuous epithelium
The polypeptide of the invention is admimsteied using at a range diffeient doses, from 4mg to 500mg pei wound pei day for 8 days in vehicle Vehicle control groups received 50mL of vehicle solution
Animals aie euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300mg/kg) The wounds and suirounding skin are then haivested for histology Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for furthei processing
Four groups of 10 animals each (5 with methylpredmsolone and 5 without glucocorticoid) are evaluated 1) Untreated group 2) Vehicle placebo control 3) treated groups
Wound closuie is analyzed by measuring the aiea in the vertical and horizontal axis and obtaining the total area of the wound Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8) The wound area on day 1 is 64mm1, the corresponding size of the dermal punch Calculations are made using the following formula
[Open area on day 8] - [Open area on day 1] / [Open area on day 1]
Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned peφendicular to the wound surface (5mm) and cut using an Olympus microtome Routine hematoxyhn-eosm (H&E) staining is performed on cross-sections of bisected wounds Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with a polypeptide of the invention A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap
Experimental data are analyzed using an unpaired t test A p value of < 0 05 is considered significant The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 51 : Lymphadema Animal Model
The puφose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of a polypeptide of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.
Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ~350g are dosed with Pentobarbital.
Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1 % Evan's
Blue. Circumference and volumetric measurements are then made following injection of dye into paws.
Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated suture ligated.
Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then and ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues. Caie is taken to contiol any mild bleeding lesulting fiom this pioceduie Aftei lymphatics are occluded the skin flaps aie sealed by using liquid skin (Vetbond) (AJ Buck) The separated sk edges are sealed to the undei lying muscle tissue while leaving a gap of ~0 5 cm aiound the leg Skm also may be anchoied by suturing to undeilymg muscle when necessaiy
To avoid infection, animals are housed individually with mesh (no bedding) Recovering animals are checked daily thiough the optimal edematous peak, which typically occurred by day 5-7 The plateau edematous peak are then observed To evaluate the intensity of the lymphedema the circumfeience and volumes of 2 designated places on each paw before operation and daily foi 7 days are measured The effect plasma protems on lymphedema is determined and whether piotein analysis is a useful testing perimeter is also investigated The weights of both control and edematous limbs aie evaluated at 2 places Analysis is performed in a blind manner
Circumference Measurements Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb cncumference Measurements are done at the ankle bone and dorsal paw by 2 different people then those 2 readings aie averaged Readings are taken from both control and edematous limbs
Volumetric Measurements On the day of surgeiy, animals are anesthetized with Pentobarbital and aie tested prior to surgery For daily volumetπcs animals are under brief halothane anesthetic (rapid immobilization and quick recovery), both legs are shaved and equally marked using waterproof marker on legs Legs are fust dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software(Chen/Vιctor) Data is recorded by one person, while the other is dipping the limb to marked area Blood-plasma protein measurements Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2+ comparison
Limb Weight Comparison After drawing blood, the animal is prepared for tissue collection The limbs are amputated using a quilhtine, then both experimental and control legs are cut at the ligature and weighed A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed
Histological Preparations The transverse muscle located behind the knee (popliteal) aiea is dissected and arranged in a metal mold, filled with freezeGel, dipped mto cold methylbutane placed into labeled sample bags at - 80EC until sectioning Upon sectioning the muscle is obseived undei fluoiescent microscopy for lymphatics
The studies described in this example tested activity of a polypeptide of the invention Howevei. one skilled in the ait could easily modify the exemplified studies to test the activity of polynucleotides (e g gene therapy), agonists, and/oi antagonists of the invention
Example 52: Suppression of TNF alpha-induced adhesion molecule expression by a Polypeptide of the Invention The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-hgand mtei actions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule- 1 (ICAM-1), vasculai cell adhesion molecule- 1 (VCAM-1), and endothelial leukocyte adhesion molecule- 1 (E-selectin) expression on endothelial cells (EC) The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an lnflammatoiy response The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs
Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokme, rs a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome
The potential of a polypeptide of the invention to mediate a suppression of TNF-a induced CAM expression can be examined A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a membα of the FGF family of proteins
To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM- 2, Clonetics, San Diego, CA) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C humidified incubator containing 5% CO2
HUVECs are seeded in 96-well plates at concentrations of 1 x 104 cells/well in EGM 3 1 S
medium at 37 degiee C foi 18 24 hrs oi until confluent The monolayeis aie subsequently washed 3 times with a seium-fiee solution of RPMI- 1640 supplemented with 100 U/ml penrαllin and 100 mg/ml stieptomyαn and tieated with a given cytokine and/oi giowth factoι(s) foi 24 h at 37 degree C Following incubation, the cells are then evaluated foi CAM expiession
Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence Growth medium is lemoved from the cells and replaced with 90 ul of 199 Medium (10% FBS) Samples foi testing and positive or negative controls aie added to the plate in tπphcate (in 10 ul volumes) Plates are incubated at 37 degree C for either 5 h (selectin and integrm expression) or 24 h (integrm expression only) Plates are aspirated to remove medium and 100 μl of 0 1 % paιafoιmaldehyde-PBS(wιth Ca++ and Mg++) is added to each well Plates are held at 4°C for 30 min
Fixative is then removed from the wells and wells aie washed IX with PBS(+Ca,Mg)+0 5% BSA and drained Do not allow the wells to dry Add 10 μl of diluted primary antibody to the test and control wells Antι-ICAM-1 -Biotin, Anti-VCAM- 1 -Biotin and Anti-E-selectm-Biotin are used at a concentration of 10 μg/ml (1 10 dilution of 0 1 mg/ml stock antibody) Cells are incubated at 37°C for 30 min in a humidified environment Wells are washed X3 with PBS(+Ca,Mg)+0 5% BSA
Then add 20 μl of diluted ExtrAvidin- Alkaline Phosphotase (1 5,000 dilution) to each well and incubated at 37°C for 30 min Wells are washed X3 with
PBS(+Ca,Mg)+0 5% BSA 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 104) 100 μl of pNPP substiate in glycine buffer is added to each test well Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin- Alkaline Phosphotase in glycine buffer 1 5,000 (10°) > 10°' > 10 ' > 10 ' ^ 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5 50 ng, 1 74 ng, 0 55 ng, 0 18 ng 100 μl of pNNP reagent must then be added to each of the standard wells The plate must be incubated at 37lC for 4h A volume of 50 μl of 3M NaOH is added to all wells The results are quantified on a plate reader at 405 nm The background subtraction option is used on blank wells filled with glycme buffer only The template is set up to indicate the concentration of AP-conjugate in each standard well [ 5 50 ng, 1 74 ng, 0 55 ng, 0 18 ng] Results are indicated as amount of bound AP- conjugate in each sample The studies desciibed in this example tested activity of a polypeptide of the invention Howevei. one skilled in the ait could easily modify the exemplified studies to test the activity of polynucleotides (e g gene therapy), agonists, and/oi antagonists of the invention It will be clear that the invention may be piacticed otherwise than as particularly desciibed in the foregoing description and examples Numeious modifications and variations of the piesent invention are possible in light of the above teachings and, therefoie, are within the scope of the appended claims
The entne disclosure of each document cited (including patents patent applications, journal articles, abstracts laboiatory manuals, books, or othei disclosures) in the Background of the Invention. Detailed Description and Examples is hereby incoφorated herein by reference Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form aie both incorporated herein by reference in their entireties
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
( PCT Rule \ 3bιs)
A. The indications made below relate to the microorganism referred to in the descπption on page 67 , hne N/A
B. IDENTIFICATION OFDEPOSIT Further deposits are identified on an additional sheet
Name of depositary institution American Type Culture Collection
Address of depositary institution (including postal code and coimtn) 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America
Date of deposit AccessionNumbei
08 April 1999 203917
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not foi all designated States)
Europe
In respect to those designations in which a European Patent is sought a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28 (4) EPC).
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications iisted below will be submitted to the International Bureau later (specify the general nature of the mdicanons eg "Accession Number of Deposit")
For receiving Office use only For International Bureau use only
I I This sheet was received with the international application [ j This sheet was received by the International Bureau on*
Authorized officer Authorized officer
Form PCT/RO/134 (July 1992) ATCC Deposit No.: 203917
CANADA
The applicant requests that, until either a Canadian patent has been issued on the basis of an application or the application has been refused, or is abandoned and no longer subject to reinstatement, or is withdrawn, the Commissioner of Patents only authorizes the furnishing of a sample ofthe deposited biological material referred to in the application to an independent expert nominated by the Commissioner, the applicant must, by a written statement, inform the International Bureau accordingly before completion of technical preparations for publication ofthe international application.
NORWAY
The applicant hereby requests that the application has been laid open to public inspection (by the Norwegian Patent Office), or has been finally decided upon by the Norwegian Patent Office without having been laid open inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Norwegian Patent Office not later than at the time when the application is made available to the public under Sections 22 and 33(3) ofthe Norwegian Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on the list of recognized experts drawn up by the Norwegian Patent Office or any person approved by the applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or prior to the lapsing, refusal or withdrawal of the application, to a person who is a skilled addressee without an interest in the invention (Regulation 3.25(3) ofthe Australian Patents Regulations).
FINLAND
The applicant hereby requests that, until the application has been laid open to public inspection (by the National Board of Patents and Regulations), or has been finally decided upon by the National Board of Patents and Registration without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art.
UNITED KINGDOM
The applicant hereby requests that the furnishing of a sample of a microorganism shall only be made available to an expert. The request to this effect must be filed by the applicant with the Internationa] Bureau before the completion ofthe technical preparations for the international publication ofthe application. ATCC Deposit No.: 203917
DENMARK
The applicant hereby requests that, until the application has been laid open to public inspection (by the Danish Patent Office), or has been finally decided upon by the Danish Patent office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the Danish Patent Office not later that at the time when the application is made available to the public under Sections 22 and 33(3) of the Danish Patents Act. If such a request has been filed by the applicant, any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Danish Patent Office or any person by the applicant in the individual case.
SWEDEN
The applicant hereby requests that, until the application has been laid open to public inspection (by the Swedish Patent Office), or has been finally decided upon by the Swedish Patent Office without having been laid open to public inspection, the furnishing of a sample shall only be effected to an expert in the art. The request to this effect shall be filed by the applicant with the International Bureau before the expiration of 16 months from the priority date (preferably on the Form PCT/RO/134 reproduced in annex Z of Volume I ofthe PCT Applicant's Guide). If such a request has been filed by the applicant any request made by a third party for the furnishing of a sample shall indicate the expert to be used. That expert may be any person entered on a list of recognized experts drawn up by the Swedish Patent Office or any person approved by a applicant in the individual case.
NETHERLANDS
The applicant hereby requests that until the date of a grant of a Netherlands patent or until the date on which the application is refused or withdrawn or lapsed, the microorganism shall be made available as provided in the 31F(1) ofthe Patent Rules only by the issue of a sample to an expert. The request to this effect must be furnished by the applicant with the Netherlands Industrial Property Office before the date on which the application is made available to the public under Section 22C or Section 25 ofthe Patents Act ofthe Kingdom ofthe Netherlands, whichever ofthe two dates occurs earlier.

Claims

What Is Claimed Is:
1 An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected fiom the group consisting of
(a) a polynucleotide fiagment of SEQ ID NO X oi a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No Z, which is hybridizable to SEQ ID NO X,
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO Y or a polypeptide fragment encoded b the cDNA sequence included in ATCC Deposit
No Z, which is hybridizable to SEQ ID NO X
(c) a polynucleotide encoding a polypeptide domain of SEQ ID NO Y or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No Z, which is hybridizable to SEQ ID NO X, (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO Y or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No Z, which is hybridizable to SEQ ID NO X,
(e) a polynucleotide encoding a polypeptide of SEQ ID NO Y or the cDNA sequence included in ATCC Deposit No Z, which is hybridizable to SEQ ID NO X, having biological activity,
(f) a polynucleotide which is a variant of SEQ ID NO X,
(g) a polynucleotide which is an allelic variant of SEQ ID NO X,
(h) a polynucleotide which encodes a species homologue of the SEQ ID NO Y, (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues 121
2 The isolated nucleic acid molecule of claim 1 wherein the polynucleotide fiagment compnses a nucleotide sequence encoding a secieted piotein
3 The isolated nucleic acid molecule of claim 1 , wheiein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO Y or the polypeptide encoded by the cDNA sequence included in ATCC Deposit No Z. which is hybridizable to SEQ ID NO X
4 The isolated nucleic acid molecule of claim 1 , wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO X or the cDNA sequence included in ATCC Deposit No Z, which is hybridizable to SEQ ID NO X
5 The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus
6 The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the
N-terminus
7 A recombinant vector comprising the isolated nucleic acid molecule of claim 1
8 A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 1
9 A recombinant host cell produced by the method of claim 8
10 The recombinant host cell of claim 9 comprising vector sequences
1 1. An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NON or the encoded sequence included in ATCC Deposit Νo:Z; (b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z, having biological activity;
(c) a polypeptide domain of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No:Z;
(d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence included in ATCC Deposit No:Z;
(e) a secreted form of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No:Z;
(f) a full length protein of SEQ ID NO: Y or the encoded sequence included in ATCC Deposit No:Z; (g) a variant of SEQ ID NO: Y;
(h) an allelic variant of SEQ ID NO:Y; or (i) a species homologue of the SEQ ID NO: Y.
12. The isolated polypeptide of claim 1 1, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C- terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated polypeptide of claim 1 1.
14. A recombinant host cell that expresses the isolated polypeptide of claim 1 1.
15. A method of making an isolated polypeptide comprising:
(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and
(b) recovering said polypeptide.
16. The polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 1 1 or the polynucleotide of claim 1.
18. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the polynucleotide of claim 1 ; and
(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the polypeptide of claim 1 1 in a biological sample; and
(b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
20. A method for identifying a binding partner to the polypeptide of claim 11 comprising:
(a) contacting the polypeptide of claim 1 1 with a binding partner; and
(b) determining whether the binding partner effects an activity of the polypeptide.
21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.
22. A method of identifying an activity in a biological assay, wherein the method comprises:
(a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant; (c) detecting an activity in a biological assay: and
(d) identifying the protem in the supernatant having the activity
23 The product produced by the method of claim 20
PCT/US2000/006043 1999-03-12 2000-03-09 50 human secreted proteins WO2000055171A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
EP00913810A EP1165587A4 (en) 1999-03-12 2000-03-09 50 human secreted proteins
JP2000605599A JP2002538807A (en) 1999-03-12 2000-03-09 50 human secreted proteins
CA002364549A CA2364549A1 (en) 1999-03-12 2000-03-09 50 human secreted proteins
AU35181/00A AU3518100A (en) 1999-03-12 2000-03-09 50 human secreted proteins
US10/105,299 US7368527B2 (en) 1999-03-12 2002-03-26 HADDE71 polypeptides
US10/472,965 US20070026454A1 (en) 1999-03-12 2002-03-26 Human secreted proteins
US10/472,964 US20070032414A1 (en) 1999-03-12 2002-03-26 Human secreted proteins
US10/670,185 US20070015162A1 (en) 1999-03-12 2003-09-25 99 human secreted proteins
US10/670,186 US20070031842A1 (en) 1999-03-12 2003-09-25 379 human secreted proteins
US10/868,184 US20070048818A1 (en) 1999-03-12 2004-06-16 Human secreted proteins
US10/994,608 US20070048297A1 (en) 1999-03-12 2004-11-23 Human secreted proteins
US11/781,665 US20070298491A1 (en) 1999-03-12 2007-07-23 Human Secreted Proteins
US12/753,401 US8410248B2 (en) 1999-03-12 2010-04-02 HWBAO62 polypeptides
US13/848,789 US20130203164A1 (en) 1999-03-12 2013-03-22 Human Secreted Proteins

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US12414699P 1999-03-12 1999-03-12
US60/124,146 1999-03-12
US16706199P 1999-11-23 1999-11-23
US60/167,061 1999-11-23

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/006012 Continuation-In-Part WO2000055198A1 (en) 1999-03-12 2000-03-09 50 human secreted proteins

Related Child Applications (7)

Application Number Title Priority Date Filing Date
PCT/US2000/006012 Continuation-In-Part WO2000055198A1 (en) 1999-03-12 2000-03-09 50 human secreted proteins
US95008201A Continuation-In-Part 1999-03-12 2001-09-12
US95008301A Continuation-In-Part 1999-03-12 2001-09-12
US10/105,299 Continuation-In-Part US7368527B2 (en) 1999-03-12 2002-03-26 HADDE71 polypeptides
PCT/US2002/009188 Continuation-In-Part WO2002077186A2 (en) 1999-03-12 2002-03-26 Human secreted proteins
PCT/US2002/009239 Continuation-In-Part WO2002077188A2 (en) 1999-03-12 2002-03-26 Human serine/threonine kinase
US10/868,184 Continuation-In-Part US20070048818A1 (en) 1999-03-12 2004-06-16 Human secreted proteins

Publications (1)

Publication Number Publication Date
WO2000055171A1 true WO2000055171A1 (en) 2000-09-21

Family

ID=26822252

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/006043 WO2000055171A1 (en) 1999-03-12 2000-03-09 50 human secreted proteins

Country Status (5)

Country Link
EP (1) EP1165587A4 (en)
JP (1) JP2002538807A (en)
AU (1) AU3518100A (en)
CA (1) CA2364549A1 (en)
WO (1) WO2000055171A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002077188A3 (en) * 2001-03-27 2003-04-17 Human Genome Sciences Inc Human serine/threonine kinase
US7368527B2 (en) 1999-03-12 2008-05-06 Human Genome Sciences, Inc. HADDE71 polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 27 September 1995 (1995-09-27), ZHAO, S.: "Human DNA sequence from cosmid L219B8, Huntington's Disease chromosome 4p16.3", XP002908798, accession no. EMBL Database accession no. Z54246 *
See also references of EP1165587A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7368527B2 (en) 1999-03-12 2008-05-06 Human Genome Sciences, Inc. HADDE71 polypeptides
WO2002077188A3 (en) * 2001-03-27 2003-04-17 Human Genome Sciences Inc Human serine/threonine kinase
WO2002077186A3 (en) * 2001-03-27 2003-12-24 Human Genome Sciences Inc Human secreted proteins
WO2003004623A3 (en) * 2001-03-27 2004-07-22 Human Genome Sciences Inc Human secreted proteins
EP1460900A4 (en) * 2001-03-27 2004-11-03 Human Genome Sciences Inc Human secreted proteins
EP1385380A4 (en) * 2001-03-27 2005-02-02 Human Genome Sciences Inc Human secreted proteins
EP1385381A4 (en) * 2001-03-27 2005-02-09 Human Genome Sciences Inc Human secreted proteins
EP1383888A4 (en) * 2001-03-27 2005-03-30 Human Genome Sciences Inc Human secreted proteins
EP1408759A4 (en) * 2001-03-27 2005-04-06 Human Genome Sciences Inc Human secreted proteins
EP1506398A4 (en) * 2001-03-27 2005-06-29 Human Genome Sciences Inc Human secreted proteins

Also Published As

Publication number Publication date
EP1165587A1 (en) 2002-01-02
JP2002538807A (en) 2002-11-19
EP1165587A4 (en) 2003-01-15
CA2364549A1 (en) 2000-09-21
AU3518100A (en) 2000-10-04

Similar Documents

Publication Publication Date Title
WO2000061629A1 (en) 49 human secreted proteins
WO2000058495A1 (en) 45 human secreted proteins
EP1161446A1 (en) 50 human secreted proteins
EP1220947A2 (en) 50 human secreted proteins
WO2000058334A1 (en) 50 human secreted proteins
EP1171591A1 (en) 48 human secreted proteins
WO2000055177A2 (en) 49 human secreted proteins
WO2000058350A1 (en) 49 human secreted proteins
WO2000061596A1 (en) 50 human secreted proteins
WO2000061625A1 (en) 48 human secreted proteins
EP1169440A1 (en) 48 human secreted proteins
EP1165587A1 (en) 50 human secreted proteins
WO2000056765A1 (en) 48 human secreted proteins
WO2000061627A1 (en) 49 human secreted proteins
EP1171456A1 (en) 50 human secreted proteins
EP1163332A1 (en) 48 human secreted proteins
EP1163331A1 (en) 50 human secreted proteins
WO2000058356A1 (en) 50 human secreted proteins
WO2000058467A1 (en) 50 human secreted proteins
WO2000058340A2 (en) 50 human secreted proteins
WO2000058468A2 (en) 47 human secreted proteins
EP1173478A1 (en) 49 human secreted proteins
WO2000058336A1 (en) 50 human secreted proteins
EP1169331A1 (en) 49 human secreted proteins
WO2000058469A1 (en) 48 human secreted proteins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 605599

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref document number: 2364549

Country of ref document: CA

Ref country code: CA

Ref document number: 2364549

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000913810

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000913810

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2000913810

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10868184

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 10868184

Country of ref document: US