CA2370489A1 - 49 human secreted proteins - Google Patents

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 diseases, disorders, and/or conditions related to these novel human secreted proteins.

Description

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49 Human Secreted Proteins Field of the 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 of the 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 role of secreted proteins in human physiology. a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical diseases, disorders, and/or conditions by using secreted proteins or the genes that encode them.
Summary of the Invention The present invention relates to novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, andlor conditions.
The invention further relates to screening methods for 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 other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
In the present invention, a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
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 portion of the coding sequences, as =t disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
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 Number. The ATCC is located at 10801 University Boulevard Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to WO 00/61629 PCTlUS00/09071 sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within the clone deposited with the ATCC. "Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, Sx SSC
(750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 y~g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions.
Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M
NaCI;
0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ugJml salmon sperm blocking DNA; followed by washes at 50 degree C with IXSSPE, 0.1 % SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. SX
SSC).
Note that variations in the above conditions may be accomplished through 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, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

WO 00/61629 PCTlUS00109071 Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. 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 mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. In addition, the polynucleotide can be composed of IS triple-stranded regions comprising RNA or DNA or both RNA and DNA. A
polynucleotide may also contain one or more modified bases or DNA or RNA
backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. 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 or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, 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 Press, New York, pgs. I-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).) "SEQ tD 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 I.

"A polypeptide having biological activity" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.) Many proteins (and translated DNA sequences) contain regions where the amino acid composition is highly biased toward a small subset of the available residues. For example, membrane spanning domains and signal peptides (which are also membrane spanning) typically contain long stretches where Leucine (L), Valine (V), Alanine (A), and lsoleucine (I) predominate. Poly-Adenosine tracts (polyA) at the end of cDNAs appear in forward translations as poly-Lysine (poly-K) and poly-Phenylalanine (poly-F) when the reverse complement is translated. These regions are often referred to as "low complexity" regions.
Such regions can cause database similarity search programs such as BLAST to find high-scoring sequence matches that do not imply true homology. The problem is exacerbated by the fact that most weight matrices (used to score the alignments generated by BLAST) give a match between any of a group of hydrophobic amino acids (L,V and I) that are commonly found in certain tow complexity regions almost as high a score as for exact matches.
In order to compensate for this, BLASTX.2 (version 2.Oa5MP-WashU) employs two filters ("seg" and "xnu") which "mask'' the low complexity regions in a WO 00161629 PCT/US00l09071 particular sequence. These filters parse the sequence for such regions, and create a new sequence in which the amino acids in the low complexity region have been replaced with the character "X". This is then used as the input sequence (sometimes referred to herein as "Query" and/or "Q") to the BLASTX program. While this regime helps to ensure that high-scoring matches represent true homology, there is a negative consequence in that the BLASTX program uses the query sequence that has been masked by the filters to draw alignments.
Thus, a stretch of "X"s in an alignment shown in the following application does not necessarily indicate that either the underlying DNA sequence or the translated protein sequence is unknown or uncertain. Nor is the presence of such stretches meant to indicate that the sequence is identical or not identical to the sequence disclosed in the alignment of the present invention. Such stretches may simply indicate that the BLASTX program masked amino acids in that region due to the detection of a low complexity region, as defined above. In all cases, the reference sequences) (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 embodiments) of the present invention, and should not be construed as limiting the present invention to the partial sequence shown in an alignment, unless specifically noted otherwise herein.
Polvnucleotides and Polvpeptides of the Invention FEATURES OF PROTEIN ENCODED BY GENE NO: 1 It has been discovered that this gene is expressed primarily in Activated T-cell(12h)/Thiouridine-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:11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically 5 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 826 of SEQ ID
NO:11, b is an integer of 15 to 840, where both a and b correspond to the positions of 10 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 translation product of this gene shares sequence homology with, as a non-limiting example, the sequence accessible through the following database accession no.
gnIIPIDle1346972 (all information available through the recited accession number is incorporated herein by reference) which is described therein as a C. elegans protein.
A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PID~e1346972 cDNA EST yk478b4.5 comes from this gene; cDNA EST
EMBL:D74716 gene;
elegans]
comes from this gene; cDNA EST yk456b12.5 comes from this cDNA EST EMBL:T00892 comes from this gene [Caenorhabditis >sp~E1346972~E1346972 F52B11.1 PROTEIN.
Length -- 523 Plus Strand HSPs:

Score 139 (48.9 bits), Expect = l.Oe-15, Sum P(3) = = l.Oe-15 Identities =

(25%), Positives =

(470), Frame =
+2 S Q: 1706FCDVYNPQSKTYCKRLQVLCPEHSRDPKVPADEVCGCP------------LVRDVFELTG

+C+ Y+ ++ ++CKRL+ LCPEH + +VCG P V ++ E+

S: 313 YCEKYDSRTNSFCKRLKSLCPEHRKLGDEQHLKVCGYPKKWEDGMIETAKTVSELIEMED

Q: 1850DF----CRLPKRQCNRHYCWEKLRRAEVDLERVRVWYKLDELFEQERNVRTAMTNRAGLL

IO F CR K C++H+ W R ++LE+ ++ K+ EL + + L

S: 373 PFGEEGCRTKKDACHKHHKWIPSLRGTIELEQACLFQKMYELCHEMHKLNAHAEWTTNAL

Q: 2018ALMLHQTIQHDPLTTDLRSSA 2080 ++M+H+ + + LR+ A

IS S: 433 SIMMHKQPSTEKCSFFLRNFA 453 The segment of gnllPIDlel346972 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 109.
Preferred polypeptides of the invention comprise a polypeptide having the 20 amino acid sequence set out in the sequence listing as SEQ ID NO. 110 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
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 2S NCI CGAP_GCB1; Human Primary Breast Cancer Reexcision; Activated T-cell(12h)/Thiouridine-re-excision; Stratagene fetal spleen (#937205);
Soares_parathyroid tumor NbHPA; normalized infant brain cDNA; Human Thymus;
Rejected Kidney, lib 4; Human fetal brain S. Meier-Ewert; Activated T-Cell (l2hs)lThiouridine IabelledEco; Soares_pregnant uterus NbHPU; T cell helper II;
30 Human Cerebellum; Normal trachea; Corpus Callosum; Activated T-Cells, 8 hrs, subtracted; Human White Adipose; Healing Abdomen wound,70&90 min post incision; Supt Cells, cyclohexamide treated; Human adult small intestine,re-excision;
Stratagene ovary (#937217); Myoloid Progenitor Cell Line; Human Adult Small Intestine; Human Thymus; Human Umbilical Vein Endothelial Cells, uninduced;

Human Activated T-Cells; Human Pancreas Tumor; Human Adult Testes, Large Inserts, Reexcision; Human Heart; Human colorectal cancer; Soares testis_NHT;
Soares total fetus Nb2HF8 9w; Soares fetal liver spleen_1NFLS S1; Stratagene schizo brain SI1; Stratagene lung carcinoma 937218; Human Pancreas Tumor, Reexcision; Ulcerative Colitis; Human Testes Tumor, re-excision;
Hemangiopericytoma; Human Fetal Brain; Stratagene NT2 neuronal precursor 937230; Pancreas Islet Cell Tumor; Human T-Cell Lymphoma; Human Testes Tumor; Colon Tumor 11; H. Frontal cortex,epileptic,re-excision; Human Synovial Sarcoma; Anergic T-cell; NCI CGAP_SSl; NCI CLAP ColO; NCI CGAP-Thyl;
Human Endometrial Tumor; Hodgkin's Lymphoma II; Nine Week Old Early Stage Human; Soares placenta Nb2HP and Primary Dendritic Cells, lib 1.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ 1D N0:12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 I to 2373 of SEQ ID
N0:12, b is an integer of 15 to 2387, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:12, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3 The computer algorithm BLASTX has been used to determine that the translation product of this gene shares sequence homology with, as a non-limiting WO 00/61629 PCTlUS00/09071 example, the sequence accessible through the following database accession no.
gnIIPIDle1343996 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "similar to guanine nucleotide binding protein." A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PID~e1343996 similar to guanine nucleotide binding protein; cDNA EST
EMBL:T00917 comes from this gene; cDNA EST CEMSE07F comes from this 1~ gene; cDNA EST EMBL:T00918 comes from this gene; cDNA EST
EMHL:D70900 comes from this gene; cDNA EST EMBL:D7D636 comes >
Length = 376 Minus Strand HSPs:
Score = 1216 (428.1 bits), Expect = S.le-123, P = S.le-123 Identities = 222/323 (68°s), Positives = 264/323 (81%), Frame = -2 Q: 1886 PTPSSSATQSKPTPV------KPNYALKFTLAGHTKAVSSVKFSPNGEWLASSSADKLIK 1725 ZO P P +SA P NY L TL GHTK++SS KFSP G++L +SSADK +K
S: 53 PAPGASAQTPNPNAAGASASGSANYKLMCTLEGHTKSISSAKFSPCGKYLGTSSADKTVK 112 Q: 1724 IWGAYDGKFEKTISGHKLGISDVAWSSDSNLLVSASDDKTLKIWDVSSGKCLKTLKGHSN 1545 IW E+T++GHKLG++D+AWSSDS +VSASDDKTLKI+++ + + KTLKGH+N
ZS S: 113 IWNMDHMICERTLTGHKLGVNDIAWSSDSRCVVSASDDKTLKIFEIVTSRMTKTLKGHNN 172 Q: 1544 YVFCCNFNPQSNLIVSGSFDESVRIWDVKTGKCLKTLPAHSDPVSAVHFNRDGSLIVSSS 1365 YVFCCNFNPQS+L+VSGSFDESVRIWDVKTG C+KTLPAHSDPVSAV FNRDGSLI S S
S: 173 YVFCCNFNPQSSLWSGSFDESVRIWDVKTGMCIKTLPAHSDPVSAVSFNRDGSLIASGS 232 Q: 1364 YDGLCRIWDTASGQCLRTLIDDDNPPVSFVKFSPNGKYILAATLDNTLKLWDYSKGKCLK 1185 YDGL RIWDTA+GQC+KTL+DD+NPPV+FVKFSPNGKYILA+ LD+TLKLWD+SKGK LK
S: 233 YDGLVRIWDTANGQCIKTLVDDENPPVAFVKFSPNGKYILASNLDSTLKLWDFSKGKTLK 292 3J Q: 1184 TYTGHKNEKYCIFANFSVTGGKWIVSGSEDNLWIWNLQTKEIVQKLQGHTDWISTACH 1005 YTGH+N KYCIFANFSVTGGKWI+SGSED +YIWNLQT+EIVQ L+GHT V+++ CH
S: 293 QYTGHENSKYCIFANFSVTGGKWIISGSEDCKIYIWNLQTREIVQCLEGHTQPVLASDCH 352 Q: 1004 PTENIIASAALENDKTIKLWKSD 936 4O P +NIIAS ALE D I +W+SD
S: 353 PVQNIIASGALEPDNKIHIWRSD 375 The segment of gnllPIDle1343996 that is shown as "S" above is set out in the sequence listing, as SEQ 1D NO. 111.

WO 00/61629 PCTlUS00/09071 Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 112 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: CD34 positive cells (Cord Blood) and to a lesser extent in NCI CGAP_GCB 1; Soares fetal liver spleen 1 NFLS;
Soares_pregnant uterus_NbHPU; Stratagene colon (#937204); LNCAP, differential expression; Amniotic Cells - TNF induced; Human Soleus; Human Skin Tumor;
Human Pineal Gland; Soares adult brain N2b4HB55Y; Human Stomach,re-excision;
Jurkat T-Cell, S phase; Apoptotic T-cell; Human Jurkat Membrane Bound Polysomes; Human Adult Testes, Large Inserts, Reexcision; Epithelial-TNFa and INF
induced; Human Testes Tumor, re-excision; Bone Marrow Stromal Cell, untreated;
Smooth muscle, serum induced,re-exc; Pancreas Islet Cell Tumor; 12 Week Old Early Stage Human; Adipocytes; Human Testes, Reexcision; Human Adult Pulmonary,re-excision; NCI CGAP_GC3; Colon Normal III;
Soares_multiple sclerosis 2NbHMSP; Human Bone Marrow, treated; Activated T-cell(12h)/Thiouridine-re-excision; Nine Week Old Early Stage Human; Human Cerebellum; Soares fetal liver spleen_1NFLS S1; Primary Dendritic Cells, lib 1 and Soares infant brain INIB.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
62 as residues: Gly-30 to Thr-44.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically I>
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 2050 of SEQ 1D
N0:13, b is an integer of 15 to 2064, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:13, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4 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.
gi12149634 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "terminal deoxynucleotidyltransferase [Monodelphis domestica]." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~2149634 terminal deoxynucleotidyltransferase [Monodelphis domestica]
Length = 518 Minus Strand HSPs:
Score = 216 (76.0 bits), Expect = 4.7e-32, Sum P(4) = 4.7e-32 Identities = 42/83 (SOo), Positives = 62/83 (740), Frame = -3 Q: 1896 VERVRRSERYQTMKLFTQIFGVGVKTADRWYREGLRTLDDLR-EQPQKLTQQQKAGLQHH 1720 V+ V ERYQ+ KLFT +FGVG+KTAD+WYR G RTL+ +R ++ KLT+ QKAGL ++
S: 239 VQAVLNDERYQSFKLFTSVFGVGLKTADKWYRMGFRTLNKIRSDKTLKLTKMQKAGLCYY 298 3O Q: 1719 QDLSTPVLRSDVDALQQWEEAV 1651 +DL V +++ DA+ +V++AV
S: 299 EDLIDCVSKAEADAVSLLVQDAV 321 The segment of gi12149634 that is shown as "S" above is set out in the sequence listing as 5EQ ID NO. 113. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
_S Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 114 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: NCI CGAP GCB 1 and to a lesser extent in Soares_pregnant uterus NbHPU; Soares_multiple sclerosis 2NbHMSP; Human Thymus Stromal Cells; Human Leukocytes; Human OB HOS treated (1 nM E2) fraction I; Smooth muscle, ILlb induced; LNCAP prostate cell line; Brain Frontal Cortex, re-excision; Ovarian Tumor 10-3-95; Human Gall Bladder;
NCI CLAP AA 1; NCI CGAP_Br2; NCI CGAP_CoB; NCI CGAP_Pr~;
NCI CGAP_Lei2; NCI CGAP_Brn23; Colon Normal II; Human Synovial Sarcoma;
Hodgkin's Lymphoma II and T cell helper II.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
63 as residues: Pro-26 to Met-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 N0:14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1884 of SEQ ID
N0:14, b is an integer of 1S to 1898, where both a and b correspond to the positions of S nucleotide residues shown in SEQ ID N0:14, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5 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.
gi11906596 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "kinesin-73 [Drosophila melanogaster~." A partial alignment demonstrating the observed 1S homology is shown immediately below.
>giI1906596 kinesin-73 [Drosophila melanogaster] >sp~001349~D01349 KINESIN-73.
Length = 1921 Plus Strand HSPS:
Score = 1695 (596.? bits), Expect = 2.3e-184, Sum P(2) = 2.3e-184 Identities = 339/504 (67~), Positives = 396/504 (780), Frame = +1 Q: 166 ESFQRRCPGPAEVFAYDHCFWSMDESVKEKYAGQDIVFKCLGENILQNAFXGYNACIFAY 345 E +R+ P + FA+DHCF+S++ E +A Q+ VF C+G IL NAF GYNACIFAY
S: 44 EKIERKQP---KTFAFDHCFYSLNPE-DENFASQETVFDCVGRGILDNAFQGYNACIFAY 99 3O Q: 346 GQTGSGKSYTMMGTADQPGLIPRLCSGLFERTQKEGNEEQSFKVEVSYMEIYNEKVRDLL 525 GQTGSGKSYTMMGT + G+IPRLC LF + E +KVEVSYMEIYNEKV DLL
S: 100 GQTGSGKSYTMMGTQESKGIIPRLCDQLFSAIANKSTPELMYKVEVSYMEIYNEKVHDLL 159 Q: 526 DPKGSRQTLKVREHSVLGPYVDGLSKLAVTSYKDIESLMSEGNKSRTVAATNMNEESSRS 705 3S DPK ++Q+LKVREH+V+GPYVDGLS+LAVTSY+DI++LM+EGNKSRTVAATNMN ESSRS
S: 160 DPKPNKQSLKVREHNVMGPYVDGLSQLAVTSYQDIDNLMTEGNKSRTVAATNMNAESSRS 219 IR
Q: 706 HAVFKITLTHTLYDVKSGTSGEKVGKXSLVDLXGSERATKTGAAGDRLKEGSNINKSLTT 885 HAVF + LT L D +G SGEKV + SLVDL GSERA KTGA GDRLKEGSNINKSLTT
S: 220 HAVFSWLTQILTDQATGVSGEKVSRMSLVDLAGSERAVKTGAVGDRLKEGSNINKSLTT 279 S Q: 886 LGLVISALADQSAGK-SXN-KFVPYRDSVLTWLLKDSLGGNSKXAMVATVSPAADNYDET 1059 LGLVIS LADQS GK S N KFVPYRDSVLTWLLKD+LGGNS+ MVAT+SP+ADNY+ET
S: 280 LGLVISKLADQSNGKKSGNDKFVPYRDSVLTWLLKDNLGGNSRTVMVATISPSADNYEET 339 Q: 1060 LSTLRYADRAKHIVNHAVVNEDPNARIIRDLREEVEKLREQLTKAEAMKSPELKDRLEES 1239 lO LSTLRYADRAK IVNHAVVNEDPNARIIR+LR EVE LR L A +++D+L ES
S: 340 LSTLRYADRAKRIVNHAVVNEDPNARIIRELRHEVETLRSMLKHATGSPVGDVQDKLAES 399 Q: 1240 EKLIQEMTVTWEEKLRKTEEIAQERQKQLESLGISLQSSGIKVGDDKCFLVNLNADPALN 1419 E L+++++ TWEEKL KTE I ERQ+ LE +GIS+Q+.SGIKV +K +LVNLNADP+LN
IS S: 400 ENLMKQISQTWEEKLVKTERIQNERQQALEKMGISVQASGIKVEKNKYYLVNLNADPSLN 459 Q: 1420 ELLVYYLKEHTLIG----SANSQDIQLCGMGILPEHCIIDITSEGQVMLTPQKNTRTFVN 1587 ELLVYYLK+ TLIG S DIQL G+GI PEHC+I I G M P + R FVN
S: 460 ELLVYYLKDRTLIGGRTISGQOPDIQLSGLGIQPEHCVITIEDSGI,YDI-EPVQGARCFVN 518 Q: 1588 GSSVSSPIQLHHGDRILWGNNHFFRLNLP 1674 GS+ L +GDRILWGN+HFFR+N P
S: 519 GSAAVEKTPLQNGDRILWGNHHFFRVNSP 547 2S The segment of gi11906596 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 115. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 116 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
3S It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Colon Normal II; Soares_fetal liver spleen_1NFLS S1 and to a lesser extent in Pancreatic Islet; and Human Fetal Heart.

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 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 1687 of SEQ ID
NO:15, b is an integer of 15 to 1701, 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 breast 2NbHBst; and Ovarian Tumor 10-3-95.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:16 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 I to 1161 of SEQ ID
N0:16, b is an integer of 15 to 1175, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:16, 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 S example, the sequence accessible through the following database accession no.
gnIIPIDIe1321S23 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ATP(GTP)-binding protein [Homo sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PID~e1321523 (AJ010842) ATP(GTP)-binding protein [Homo sapiens]
>sp~0760041076004 ATP(GTP)-BINDING PROTEIN (FRAGMENT).
Length = 358 1S Minus Strand HSPs:
Score = 1594 (561.1 bits), Expect = 4.3e-163, P = 4.3e-163 Identities = 319/358 (890), Positives = 319/358 (890), Frame = -1 2O Q: 1761 RHPVCLLVLGMAGSGKTTFVQRLTGHLHAQGTPPYVINLDPAVHEVPFPANIDIRDTVKY

RHPVCLLVLGMAGSGKTTFVQRLTGHLHAQGTPPYVINLDPAVHEVPFPANIDIRDTVKY
S: 1 RHPVCLLVLGMAGSGKTTFVQRLTGHLHAQGTPPYVINLDPAVHEVPFPANIDIRDTVKY
Q: 1581 KEVMKQYGLGPNGGIVTSLNLFATRFDQVMKFIEKAQNMSKYVLIDTPGQIEVFTWSASG

KEVMKQYGLGPNGGIVTSLNLFATRFDQVMKFIEKAQNMSKYVLIDTPGQIEVFTWSASG
S: 61 KEVMKQYGLGPNGGIVTSLNLFATRFDQVMKFIEKAQNMSKYVLIDTPGQIEVFTWSASG

Q: 1401 TIITEALASSFPTWIYVMDTSRSTNPVTFMSNMLYACSILYKTKLPFIWMNKTDIIDH

TIITEALASSFPTWIYVMDTSRSTNPVTFMSNMLYACSILYKTKLPFIWMNKTDIIDH
3S S: 121 TIITEALASSFPTWIYVMDTSRSTNPVTFMSNMLYACSILYKTKLPFIWMNKTDIIDH

Q: 1221 SFAVEWMQDFEAFQDALNQETTYVSNLTRSMSLVLDEFYSSLRVVGVSAVLGTGLDELFV

S: 181 SFAVEWMQDFEAFQDALNQETTYVSNLTRSMSLVLDEFYSSLRVVGVSAVLGTGLDELFV

Q: 1041 QVTSAAXXXXXXXXXXXXXLKKSLANAESXXXXXXXXXXXKDMGSVALDAGTAKDSLSPV
4S s62 QVTSAA LKKSLANAES KDMGSVALDAGTAKDSLSPV
S: 241 QVTSAAEEYEREYRPEYERLKKSLANAESQQQREQLERLRKDMGSVALDAGTAKDSLSPV

S Q: 861 LHPSDLILTRGTLXXXXXXXXXXXXXXXHRVTEESHEEPAFQNFMQESMAQYWKRNNK

LHPSDLILTRGTL HRVTEESHEEPAFQNFMQESMAQYWKRNNK
S: 301 LHPSDLILTRGTLDEEDEEADSDTDDIDHRVTEESHEEPAFQNFMQESMAQYWKRNNK

The segment of gnlIPIDIe1321S23 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 117. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
1S Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 118 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Stratagene ovarian cancer (#937219); Soares infant brain 1NIB; Soares placenta Nb2HP; NC1 CGAP_GCB1; Human Adult Small Intestine; Stratagene NT2 neuronal 2S precursor 937230; NTERA2 + retinoic acid, 14 days; Stratagene lung (#937210); 12 Week Old Early Stage Human; NCI CGAP_Ew 1; Anergic T-cell;
Soares fetal liver_spleen_1 NFLS_S 1; Stratagene endothelial cell 937223;
Keratinocyte; Human 8 Week Whole Embryo; Human Infant Adrenal Gland, Subtracted; H. Atrophic Endometrium; Adipocytes,re-excision; Human Thyroid;
Human Liver; Human Adult Heart,re-excision; Apoptotic T-cell, re-excision; H

WO 00/61629 PCTlUS00/09071 Female Bladder, Adult; Healing groin wound, 7.5 hours post incision; Synovial hypoxia-RSF subtracted; Spleen metastic melanoma; Ovarian Tumor 10-3-95; Fetal Liver, subtraction II; Human Prostate; Human Thymus; Human Uterine Cancer; T-Cell PHA 24 hrs; Human Ovarian Cancer Reexcision; Human Thymus Stromal Cells;
S Epithelial-TNFa and INF induced; Human Testes Tumor, re-excision;
Soares_NhHMPu_S1; Human Substantia Nigra; NCI CGAP_AA1;
NCI CGAP_LuS; Colon Normal II; Human Placenta; Adipocytes; Pancreatic Islet;
Primary Dendritic cells,frac 2; Human Fetal Heart; human tonsils; Endothelial cells-control; Human Microvascular Endothelial Cells, fract. A; Monocyte activated;
T Cell helper I; Soares_pregnant uterus_NbHPU and Stratagene hNT neuron (#937233).
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1813 of SEQ ID
N0:17, b is an integer of 15 to 1827, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:17, and where b is greater 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.

gi12197085 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ORF2-like protein [Homo Sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
S
>gi~2197085 (AF003535) ORF2-like protein [Homo Sapiens] >gi~2197085 (AF003535) ORF2-like protein [Homo Sapiens] >sp~000549~000549 ORF2-LIKE
PROTEIN (FRAGMENT).
1() Length = 573 Plus Strand HSPs:
Score = 176 (62.0 bits), Expect = 9.3e-17, Sum P(4) = 9.3e-17 1S Identities = 57/179 (31e), Positives = 90/179 (500), Frame = +2 Q: 1721 QTNLELISSRL*QGCRIKLNT*KXIPFLYTSXEPLEFEIKIXHH*XYYQKREREXICIML 1900 Q L+LIS+ + K+N K FLYT+ E +I KR + + I L
S: 230 QNLLKLISN-FSKVSGYKINVQKSQAFLYTNNRQTESQIMGELPFTIASKRIK-YLGIQL 287 Q: 1901 TKXVXKVCEKNYKSLMK*IKGDLN*WKDKLC*WIRKLNLVMCQFIP--I*SIDSMQ-S*K 2071 T+ V + ++NYK L+K IK D N WK+ C W+ ++N+V +p I +++
S: 288 TRDVKDLFKENYKPLLKEIKEDTNKWKNIPCSWVGRINIVKMAILPKVIYRFNAIPIKLP 347 2S Q: 2072 ATLWTSAN*----F*GLHEKAKRCNSRNNIEEEQRSRTLLDFKSFYKATIIKIVWHW*KN 2239 T +T F ++A+ S + + + TLLDFK +YKAT+ K W+W +N
S: 348 MTFFTELEKTTLKFIWNQKRARIAKSILSQKNKAGGITLLDFKLYYKATVTKTAWYWYQN 407 Q: 2240 R 2242 R
S: 408 R 408 The segment of gi12197085 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 119.
3S Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 120 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in LA.28.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
67 as residues: Lys-89 to Glu-94.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:18 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 2331 of SEQ ID
N0:18, b is an integer of 15 to 2345, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:18, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 9 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Tonsils, Lib 2; and Keratinocyte.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 840 of SEQ ID
N0:19, b is an integer of 15 to 854, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:19, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10 It has been discovered that this gene is expressed primarily in Adipocytes.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:20 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 1436 of SEQ ID
N0:20, b is an integer of 15 to 1450, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:20, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Chronic Synovitis; NCI CGAP GCB1; Human Umbilical Vein Endothelial Cells, uninduced; NCI CGAP_KidS; and H. Frontal cortex,epileptic,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 N0:21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1234 of SEQ ID
N0:21, b is an integer of 15 to 1248, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:21, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12 When tested against fibroblast cell lines, supernatants removed from cells containing this gene activated the EGR1 assay. Thus, it is likely that this gene activates fibroblast 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 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Cerebellum and to a lesser extent in Stratagene pancreas (#937208); Human Substantia Nigra; Human B Cell Lymphoma; Primary Dendritic Cells, lib 1; Soares infant brain 1NIB; LNCAP + o.3nM 81881;
Soares multiple sclerosis 2NbHMSP; prostate-edited; Human OB HOS treated (10 nM E2) fraction I; Human Tonsils, Lib 2; HEL cell line; pBMC stimulated w/
poly I/C; Human Uterine Cancer; Human Thymus Stromal Cells; Human T-Cell Lymphoma; Human Eosinophils; breast lymph node CDNA library; Bone marrow;
human tonsils; Human Neutrophil, Activated; Activated T-Cell ( l2hs)/Thiouridine IabelledEco; Monocyte activated and neutrophils control.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
71 as residues: Arg 4 to Met-10, Gly-21 to Met-33, Leu-54 to Ser-62, Lys-67 to Arg-79.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:22 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, where a is any integer between 1 to 3254 of SEQ ID
N0:22, b is an integer of 15 to 3268, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:22, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13 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 Pancreatic Islet; Soares_placenta 8to9weeks_2NbHP8to9W; Normal Prostate; Human Tonsils, Lib 2; B Cell lymphoma and Human Liver, normal.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
72 as residues: Glu-121 to Leu-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 N0:23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically WO 00!61629 PCT/US00/09071 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 1659 of SEQ ID
N0:23, b is an integer of IS to 1673, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:23, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: CD34+ cell, I; Human Aortic Endothelium; Human Fetal Epithelium (Skin); Prostate BPH; Fetal Liver, subtraction II; Human Osteoblasts II;
Human Adrenal Gland Tumor; Human adult testis, large inserts; Smooth muscle, serum induced,re-exc; Macrophage-oxLDL, re-excision; Smooth muscle, serum treated; Human Synovial Sarcoma; Monocyte activated; Human B Cell Lymphoma;
and Primary Dendritic Cells, lib 1.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:24 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 932 of SEQ ID
N0:24, b is an integer of 15 to 946, where both a and b correspond to the positions of WO 00/61629 PCTlUS00/09071 nucleotide residues shown in SEQ ID N0:24, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15 S It has been discovered that this gene is expressed primarily in the following tissueslcDNA libraries: Neutrophils IL-1 and LPS induced; and Endothelial-induced.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
74 as residues: Asn-25 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 N0:25 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 607 of SEQ ID
N0:25, b is an integer of 15 to 621, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:25, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16 When tested against K562 leukemia cell lines, supernatants removed from cells containing this gene activated the ISRE assay. Thus, it is likely that this gene activates leukemia cells through the Jak-STAT signal transduction pathway. The interferon-sensitive response element is apromoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
5 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Jurkat T-Cell, S phase; and Neutrophils IL-1 and LPS
induced.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are 10 related to SEQ 1D N0:26 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 15 general formula of a-b, where a is any integer between 1 to 1722 of SEQ ID
N0:26, b is an integer of 15 to 1736, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:26, and where b is greater than or equal to a + 14.
20 FEATURES OF PROTEIN ENCODED BY GENE NO: 17 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human adult testis, large inserts and to a lesser extent in Human Testes Tumor; Activated T-cell(12h)/Thiouridine-re-excision; Soares infant brain 1NIB; Soares ovary tumor NbHOT; Palate normal; Whole 6 Week Old Embryo;
25 Human Colon, subtraction; Human Fetal Spleen; Human Liver; Human Synovium;
Human Chronic Synovitis; T-Cell PHA 16 hrs; Human Fetal Kidney; Human Thymus 3l Stromal Cells; Human Adrenal Gland Tumor; Soares_pregnant uterus NbHPU;
Human Whole Six Week Old Embryo; Human T-Cell Lymphoma; Colon Carcinoma;
Adipocytes; T Cell helper I; Human Bone Marrow, treated; Bone Marrow Cell Line (RS4,11) and Keratinocyte.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
76 as residues: Pro-20 to Pro-28, Pro-36 to Cys-41.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:27 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 1789 of SEQ ID
N0:27, b is an integer of 15 to 1803, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:27, 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 multiple sclerosis 2NbHMSP and to a lesser extent in Human Hippocampus; Spinal cord; Human Manic Depression Tissue; Early Stage Human Brain; Frontal lobe,dementia,re-excision; Alzheimers, spongy change;
Human Substantia Nigra; Soares infant brain 1 NIB; Human Right Hemisphere of Brain;
Human colon cancer, metaticized to liver, subtraction; Human Kidney Cortex, re rescue; Supt Cells, cyclohexamide treated; NTERA2 teratocarcinoma cell WO 00/61629 PCTlUS00/09071 line+retinoic acid (14 days); human corpus colosum; Human Amygdala,re-excision;
Spinal Cord, re-excision; Fetal Liver, subtraction II; Human Hypothalmus,Schizophrenia; Human Fetal Brain; Human Whole Six Week Old Embryo; Human Gall Bladder; Human Testes Tumor; Human Testes; Human Endometrial Tumor and Nine Week Old Early Stage Human.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 I to 2273 of SEQ ID
N0:28, b is an integer of 15 to 2287, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:28, 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 fetal liver spleen INFLS and to a lesser extent in Colon Tumor II; Normal colon; Stratagene lung (#937210); Human Umbilical Vein Endothelial Cells, uninduced; Soares senescent fibroblasts_NbHSF; Human endometrial stromal cells-treated with progesterone; Osteoblasts; Endothelial cells-control; Colon Normal III; Stratagene ovary (#937217); 12 Week Old Early Stage Human; CD34 depleted Buffy Coat (Cord Blood), re-excision; H. Amygdala Depression, subtracted; Activated T-cells; Human Colon, re-excision;
Macrophage (GM-CSF treated); Colon Carcinoma; breast lymph node CDNA library; Neutrophils control, re-excision; Spleen, Chronic lymphocytic leukemia;
Soares fetal lung NbHLI9W; Morton Fetal Cochlea; Soares adult brain N2b5HB55Y; Soares melanocyte 2NbHM; Anergic T-cell; Hodgkin's Lymphoma II;
T cell helper II; Primary Dendritic Cells, lib 1; Human endometrial stromal cells-treated with estradiol; Stratagene endothelial cell 937223; Stratagene endothelial cell 937223; NCI CLAP GCS; Human Fetal Dura Mater; Human Rhabdomyosarcoma;
Soares fetal heart_NbHHI9W; H Macrophage (GM-CSF treated), re-excision;
Human Placenta; Bone marrow; NCI CGAP_GC3;
Soares_parathyroid tumor NbHPA; Human Colon Cancer, subtracted; Weizmann Olfactory Epithelium; Stratagene endothelial cell 937223; Resting T-Cell, re-excision; Stomach cancer (human),re-excision; Human Hypothalamus,schizophrenia, re-excision; H Female Bladder, Adult; Human Adipose Tissue, re-excision; Human Brain, Striatum; Macrophage-oxLDL; Human Activated Monocytes; Colon Normal II; Soares breast 3NbHBst; Adipocytes; Dendritic cells, pooled; Human Microvascular Endothelial Cells, fract. A; NCI CLAP Kid6; Human Bone Marrow, treated; Neutrophils IL-1 and LPS induced; Stratagene fibroblast (#937212);
Osteoarthritis (OA-4); CD40 activated monocyte dendridic cells; Activated T-Cells, 8 hrs, subtracted; CD34+cells, II, FRACTION 2; Frontal Lobe, Dementia; Human White Adipose; Hodgkin's Lymphoma I; Healing Abdomen wound,70&90 min post incision; Frontal lobe,dementia,re-excision; Aorta endothelial cells + TNF-a;
HSA
172 Ce(Is; Apoptotic T-cell, re-excision; Human Synovium; Synovial IL-1/TNF
stimulated; H. Kidney Cortex, subtracted; Stratagene ovarian cancer (#937219);
Human Osteosarcoma; Pancreas normal PCA4 No; Human endometrial stromal cells;
Jurkat T-cell G 1 phase; Stratagene lung carcinoma 937218; Myoloid Progenitor Cell Line; Human Ovary; Spleen metastic melanoma; Human Chronic Synovitis; Human 3-~
Prostate; CD34 depleted Buffy Coat (Cord Blood); T-Cell PHA 16 hrs; Human Bone Marrow, re-excision; Apoptotic T-cell; Human Jurkat Membrane Bound Polysomes;
Human Primary Breast Cancer Reexcision; T-Cell PHA 24 hrs; Stromal cell TF274;
Human umbilical vein endothelial cells, IL-4 induced; Spinal cord; Synovial Fibroblasts (control); Ulcerative Colitis; Human Thymus Stromal Cells; Human Whole Six Week Old Embryo; Macrophage-oxLDL, re-excision; Human Gall Bladder; Human T-Cell Lymphoma: Early Stage Human Brain; Clontech human aorta polyA+ mRNA (#6572); NCI CGAPPr3; Human Fetal Lung III; Endothelial-induced; Bone Marrow Cell Line (RS4,11); Nine Week Old Early Stage Human;
Stratagene NT2 neuronal precursor 937230; Soares placenta Nb2HP; Human Pancreas: Activated T-Cells, 8 hrs, differentially expressed; Human Colon Cancer, differential; H. Leukocytes, normalized cot 500 A; Stratagene pancreas (#937208);
Stratagene ovarian cancer (#937219); H. Primary Dendritic Cells,lib 3; Human Greater Omentum, fIl remake; H. Leukocytes, normalized cot S00 B; Human Astrocyte; Human Adult Spleen, fractionII; Thyroid Thyroiditis; Palate carcinoma;
Rectum normal; Larynx Normal; Human Bone Marrow; Osteoclastoma-normalized A; Larynx Carcinoma; Larynx tumor; Colon Tumor; Liver Tumour Met 5 Tu; Colon, normal; Activated T-Cells, 8 hrs.; Brain Amygdala Depression; Human B Cell 8866;
H. Adipose Tissue; Colorectal Tumor; Hypothalamus;
Soares multiple_sclerosis 2NbHMSP; Human Adult Spleen; Soares retina N2b5HR;
stomach cancer (human); A-14 cell line; Human Aortic Endothelium; Human Cerebellum, subtracted; Human Primary Breast Cancer,re-excision; Smooth Muscle Serum Treated, Norm; human colon cancer; Human Lung; Smooth Muscle- HASTE
normalized; Human Pineal Gland; Human Normal Breast; Messangial cell, frac 2;
Human Epididymus; Human Prostate Cancer, Stage C fraction; Alzheimers, spongy change; Healing groin wound, 7.5 hours post incision; Human Frontal Cortex, 3>
Schizophrenia; Healing groin wound, 6.5 hours post incision; H. Ovarian Tumor, II, OV5232; Synovial hypoxia; Synovial Fibroblasts (II1/TNF), subt;
Soares_pregnant_uterus NbHPU; H. Meningima, M1; H. Lymph node breast Cancer;
Stratagene endothelial cell 937223; Spinal Cord, re-excision; Temporal cortex-Alzheizmer, subtracted; Human Fetal Kidney; Stratagene fetal spleen (#937205);
HMl; NCI CGAP_Br3; NCI CGAP_Co9; NCI CGAP_Pr7; NCI CLAP CNSl;
NCI CGAP_Col l; NCI CGAP_Larl; NCI CGAP_Prl2; NCI CGAP_Pr25;
NCI CGAP_Thyl; NCI CGAP_Brl.l; Human Uterine Cancer; Human Activated T-Cells; Human Pancreas Tumor; Human Chondrosarcoma; Epithelial-TNFa and INF
induced; Human Testes Tumor, re-excision; Human Thymus; Bone Marrow Stromal Cell, untreated; Human Fetal Brain; Human Liver, normal; Resting T-Cell Library,II;
Neutrophils IL-1 and LPS induced; Human Substantia Nigra; NCI CLAP Lym3;
Human Synovial Sarcoma; Primary Dendritic cells,frac 2; Human Neutrophil, Activated; Human Osteoclastoma; T Cell helper I; NCI CGAP_Kid3; neutrophils IS control; NCI CGAP_Prl; NCI CGAP_Lip2; Stratagene neuroepithelium NT2RAMI
937234; and Human 8 Week Whole Embryo.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
78 as residues: Cys-22 to Ser-27.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ 1D N0:29 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 913 of SEQ ID
N0:29, b is an integer of IS to 927, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:29, and where b is greater than or equal to a + 14.
S FEATURES OF PROTEIN ENCODED BY GENE NO: 20 When tested against sensory neuron cell lines, supernatants removed from cells containing this gene activated the EGRI assay. Thus, it is likely that this gene activates sensory neuron cells through a signal transduction pathway. Early growth response 1 (EGR1) is a promoter associated with certain genes that induces various tissues and cell types upon activation, leading the cells to undergo differentiation and proliferation.
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.
gnIIPIDId1035475 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "KIAA0774 protein [Homo sapiensJ." A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PIDId1035475 (AB018317) KIAA0774 protein [Homo Sapiens]
>spID1035475~D1035475 KIAA0774 PROTEIN (FRAGMENT).
Length = 1163 Plus Strand HSPs:
Score = 371 (130.6 bits), Expect - 1.7e-29, P = 1.7e-29 Identities = 82/201 (400), Positives = 133/201 (66~), Frame = +1 Q: 667 EIKKGHEIEKKSLEDLLSEKQESLEKQINDLKSENDALNEKLKSEEQ--KXXAREKANLK 840 3O E+ HE+EKK LE+ + + SL+ Q++ L ++ +L ++ + E+ + E+ +
S: 963 ELMSTHELEKKELEENFEKLRLSLQDQVDTLTFQSQSLRDRARRFEEALRKNTEEQLEIA 1022 Q: 841 NPQIMYLEQELESLKAVLEIKNEKLHQQDIKLMKMEKLVDNNTALVDXLKRFQQENEELX 1020 +LE++++SLK VLE+KN+++H+Q+ K++++EKL + N L + ++ QQ+NE+L

S: 1023 LAPYQHLEEDMKSLKQVLEMKNQQIHEQEKKILELEKLAEKNIILEEKIQVLQQQNEDLK 1082 Q: 1021 ARMDKHMAISRQLSTEQAVLQESLEKESKVNKRLSMENEELLWKLHNGDLCSP-KRSPTS 1197 AR+D++ ++RQLS E A LQE +EKE++ KRLS NEELLWKL GD SP K SPTS
S S: 1083 ARIDQNTWTRQLSEENANLQEYVEKETQEKKRLSRTNEELLWKLQTGDPTSPIKLSPTS 1142 Q: 1198 SAIPLQSPRNSG-SFPSP-SISPR 1263 P+ +SG S P+ S +PR
S: 1143 ---PVYRGSSSGPSSPARVSTTPR 1163 The segment of gnlIPIDId1035475 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 121.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 122 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
Preferred polypeptides comprise the following amino acid sequence:
MNKMLSFFKCFVQQRPCRCRNLFLNLFLEESFGRRQKSSSRLPV SEAXIPPV V
TLQLPTViTRVLIYLLCPRSSLGSVLQTCIKMLSESLWTLGTCHLGEMLGLGKE
PEFLGDCKGMAEDVGDLLGLHRSPLCSFHRSSSFSIESRLLTFDSFSSDSCRTA
CSVESCLEIAMCLSIRAXNSSFSCWKRFNXSTNAVLLSTSFSIFINLMSC (SEQ
ID NO: ). In this preferred sequence, "X" can represent any amino acid.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human fetal heart, Lambda ZAP Express and to a lesser extent in Soares fetal lung_NbHLl9W; Human Umbilical Vein Endothelial Cells, uninduced; Human Placenta; Soares breast 3NbHBst; Endothelial cells-control;

Stratagene endothelial cell 937223; Hepatocellular Tumor; Salivary Gland, Lib 2;
Mo7e Cell Line GM-CSF treated (lng/ml); human ovarian cancer;
Soares fetal liver spleen_1NFLS_S1; Human Hippocampus; Soares adult brain N2b~HB55Y; Stratagene lung (#937210); normalized infant brain cDNA; Soares melanocyte 2NbHM; Soares multiple_sclerosis 2NbHMSP; Human Fetal Lung III;
NCI CGAP_GCB1; Colon Normal III; Soares_pregnant uterus NbHPU;
Soares total fetus_Nb2HF8 9w; Soares placenta Nb2HP; Soares fetal liver spleen INFLS: Soares infant brain 1NIB; Brain pons; Human Fetal Liver, mixed 10 & 14 week; Human Prostate, subtracted; Weizmann Olfactory Epithelium; Soares retina N2b5HR; Human White Adipose; Human Lung; Human Quadriceps; Human adult small intestine,re-excision; Human Pineal Gland; Human Normal Breast;
Apoptotic T-cell, re-excision; Human Elypothalamus,schizophrenia, re-excision; Human Colon, re-excision; Human Infant Brain; Fetal Liver, subtraction II; T-Cell PHA 16 hrs;
Gessler Wilms tumor; Soares_fetal heart NbHHI9W;
Soares multiple_sclerosis 2NbHMSP; Stromal cell TF274; Human Hypothalmus,Schizophrenia; Soares breast 2NbHBst; Human adult lung 3' directed MboI cDNA; Soares total fetus Nb2HF8 9w; Soares_parathyroid tumor_NbHPA;
Soares_senescent fibroblasts NbHSF; Stratagene liver (#937224); Macrophage-oxLDL, re-excision; Human Gall Bladder; Human Placenta; Endothelial-induced;
Human Adult Pulmonary,re-excision; 22 week old human fetal liver cDNA library;
Human adult (K.Okubo); Human heart cDNA (YNakamura); NCI CGAP_Co3;
NCI CGAP_GC2; NCI CGAP_GC4; NCI CGAP_LuS; NCI CGAP_PNS1;
NCI CGAP_Pr22; Pancreatic Islet; Human Amygdala; Stratagene colon (#937204);
Stratagene pancreas (#937208); Human Microvascular Endothelial Cells, fract.
A;
Nine Week Old Early Stage Human and Human Cerebellum.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
79 as residues: Gly-6 to Lys-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 N0:30 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 3273 of SEQ ID
N0:30, b is an integer of 15 to 3287, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:30, 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.
gi1581505 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "XYLA of Ruminococcus flavefaciens [Ruminococcus flavefaciens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gy 581505 XYLA of Ruminococcus flavefaciens [Ruminococcus flavefaciens]
>pir~S20907~S2D907 endo-1,4-beta-xylanase (EC 3.2.1.8) precursor, bifunctional - Ruminococcus flavefaciens Length = 954 WO 00/61629 PCTlUS00/09071 Plus Strand HSPs:
Score = 168 (59.1 bits), Expect = 2.6e-1D, Sum P(2) = 2.6e-10 Identities = 57/285 (20%), Positives = 107/285 (370), Frame = +1 S
Q: 1699 KGASVQKSTGSKNDSWD--NNNRSTGGSWNFGPQDSND-NKW---GEGNKMTS--GVSQG 1854 +G S +N+ W+ NNN+ WN Q +ND N+W G+ N + G
S: 240 QGGSSDNGGQQQNNDWNQQNNNQQQNNDWNNWGQQNNDWNQWNNQGQQNNDWNNWGQQNN 299 lO Q: 1855 EWKQPTGSDELKIGEWSGPNQPNSSTGAWDNQKGHPLPENQGNAQAPCWGRSSSXTGSEV 2034 +W Q + + +W+ Q N+ W+NQ G + N WG+ ++ ++
S: 300 DWNQWNNQGQQQNNDWNNWGQQNNDWNQWNNQ-GQQQNNDWNN-----WGQQNNDW-NQW 352 Q: 2035 GGQSTGSNHXAGSSDSHNSGRRSYRPTHPDCQAVLQTLLSRTDLDPRVLSNTGWGQTQIK 2214 IS Q N+ + N+ + + + Q + + + W
S: 353 NNQGQQQNNDWNNWGQQNNDWNQWNNQNNNQQNAWNGWDNNNNWNQNNQQQNNWDWNN-- 410 Q: 2215 QDTVWDIEEVPRPEGKSDKGTEGWESAATQTKNSGGWGDAPSQSNQMKSGWGELSASTEW 2394 Q+ W+ + + W + Q + W + Q+N W + + W
2O S: 411 QNN-WNNNQQQNNDWNQWNNQNNWNNNQQQNNDWNQWNNQGQQNND----WNQWNNQNNW 465 Q: 2395 KDPKNT-GGWNDYKNNNSSN-WGGGRP--DEKTPSSWNENPSKDQGWGGGRQPNQGW 2553 N WN + NNN+ N W +++ ++W+ N W +Q N W
S: 466 NQNNNQQNAWNGWDNNNNWNQWDQNNQWNNQQQNNTWDWN--NQNNWNNNQQ-NNDW 519 The segment of giIS8ISOS that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 123.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 124 which 30 corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Whole Brain, re-excision; Human Osteoblasts II
and to a lesser extent in Smooth Muscle- HASTE normalized; Synovial Fibroblasts 3S (IlI/TNF), subt; Human Hypothalmus,Schizophrenia; Brain frontal cortex;
Early Stage Human Brain; Dendritic cells, pooled; Bone marrow; human tonsils; Human Adult Pulmonary,re-excision and Primary Dendritic Cells, lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
80 as residues: Gly-4 to Gly-10.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:31 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 5069 of SEQ ID
N0:31, b is an integer of 15 to 5083, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:31, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 22 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Hepatocellular Tumor, re-excision; Stratagene ovarian cancer (#937219); Stratagene liver (#937224) and to a lesser extent in Human Liver, normal;
NCI CGAP_Kids; Soares placenta Nb2HP; Human Gall Bladder, fraction II; Soares ovary tumor NbHOT; Soares retina N2b5HR; Temporal cortex-Alzheizmer, subtracted; Human Bone Marrow, re-excision; Human Gall Bladder; Human Osteoclastoma; Human Endometrial Tumor; Human 8 Week Whole Embryo; Soares infant brain 1NIB; Human Osteoarthritic Cartilage Fraction II1; Liver Normal MetSNo; HepG2 Cells, lambda library; Human Adult Spleen; Human Adult Pulmonary; Soares NFL T GBC_S1; Hepatocellular Tumor,re-excision; Human Osteosarcoma; Fetal Liver, subtraction II; Soares_multiple sclerosis 2NbHMSP;
H.
Kidney Medulla, re-excision; Liver, Hepatoma; NTERA2, control; Human retina cDNA Tsp5091-cleaved sublibrary; NCI CGAP_Li2; NCI CGAP_Ov2;

NCI CGAP_GCB 1; NCI CLAP Brl. l ; Stratagene HeLa cell s3 937216; Soares breast 3NbHBst; Adipocytes; Colon Normal III; Human B Cell Lymphoma 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 N0:32 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 2094 of SEQ ID
N0:32, b is an integer of 15 to 2108, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:32, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 23 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Osteoclastoma and to a lesser extent in Soares_NhHMPu S1; Osteoclastoma-normalized B; 12 Week Old Early Stage Human; H. Frontal cortex,epileptic,re-excision; NCI CGAP_GCB1; Normalized infant brain, Bento Soares and Activated T-cell( 12h)/Thiouridine-re-excision.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
82 as residues: His-35 to Gln-43.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:33 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 1234 of SEQ ID
N0:33, b is an integer of 15 to 1248, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:33, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 24 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.
gi11907327 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "angiopoietin-[Homo Sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~1907327 angiopoietin-1 [Homo Sapiens] >gnl~PID~d1003298 KIAA0003 [Homo Sapiens] {SUB 307-498}
Length = 498 Plus Strand HSPs:
Score = 429 (151.0 bits), Expect = 6.5e-47, Sum P(3) = 6.5e-47 Identities = 95/215 (44~), Positives = 125/215 (580), Frame = +2 Q: 722 RDCQELFQVGERQSGLFEIQPQGSP-PFLVNCKM-TSDGGWTVIQRRHDGSVDFNRPWEA 895 RDC +++Q G +SG++ I P P V C M + GGWTVIQ R DGS+DF R W+
3O S: 284 RDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKE 343 Q: 896 YKAGFGDPHGEFWLGLEKVHSIMGDRNSRLAVQLRDWDGNAELLQFS-VHLGGEDTAYSL 1072 YK GFG+P GE+WLG E + +I R L ++L DW+GN Q+ H+G E Y L
S: 344 YKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRL 403 WO 00!61629 PCT/US00/09071 ~~-4 Q: 1073 QLTAPVAGQLGATTVPPSGL---SVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNL 1243 L G G T S L FST D D+D K CA L+GGWWF C SNL
S: 404 YLK----GHTG-TAGKQSSLILHGADFSTKDADNDNCMCK-CALMLTGGWWFDACGPSNL 457 S Q: 1249 NGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPM 1366 NG ++ + Q KL GI W ++G Y L++TTM+I+P+
S: 458 NGMFYTA-GQNHGKLN-GIKWHYFKGPSYSLRSTTMMIRPL 496 The segment of gi11907327 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 125. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 126 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Bone Marrow Stromal Cell, untreated and to a lesser extent in Stratagene placenta (#937225); Soares_pregnant uterus NbHPU; Human osteoarthritic,fraction II; Soares senescent fibroblasts NbHSF; Human Adipose;
Epithelial-TNFa and INF induced; Soares melanocyte 2NbHM; Soares infant brain 1NIB; Soares ovary tumor NbHOT; Human osteoarthritis,fraction I; Human Synovium; Healing groin wound, 7.5 hours post incision; Synovial hypoxia;
Human Infant Brain; Human Pancreas Tumor, Reexcision;
Soares_placenta_8to9weeks 2NbHP8to9W; Human Activated T-Cells, re-excision;
Adipocytes; NCI CGAP_Kids; Human Microvascular Endothelial Cells, fract. A;
Human Hippocampus; H Kidney Cortex, subtracted III; Human Bone Marrow;

WO 00/61629 PCTlUS00/09071 ~4~
Larynx Carcinoma; HM3; Human adult (K.Okubo); NCI CGAP_Pr22; Human Pre-Differentiated Adipocytes; Human epithelioid sarcoma; Smooth muscle, control, re-excision; H. Epididiymus, caput & corpus; Human adult small intestine,re-excision;
Human Lung Cancer,re-excision; Healing groin wound - zero hr post-incision (control); Hepatocellular Tumor,re-excision; Synovial hypoxia-RSF subtracted;
Human endometrial stromal cells-treated with progesterone;
Soares fetal heart_NbHHI9W; H. Kidney Medulla, re-excision; Human Brain, Striatum; Human Adult Testes, Large Inserts, Reexcision; Liver, Hepatoma;
Soares_pineal Gland N3HPG; Human Rhabdomyosarcoma; Soares testis NHT;
Human Thymus Stromal Cells; Rejected Kidney, lib 4; Human Liver, normal; Human Placenta; Human Fetal Lung III; Bone marrow; Endothelial-induced; Endothelial cells-control; NCI CGAP_Co3; NCI CGAP_Co9; NCI CLAP Lul;
NCI CLAP Brn23; Colon Normal III; Smooth muscle,control and Nine Week Old Early Stage Human.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
83 as residues: Pro-27 to Arg-34.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1897 of 5EQ ID
N0:34, b is an integer of 15 to 1911, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:34, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 25 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Pineal Gland and Human Testes.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:35 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 982 of SEQ ID
N0:35, b is an integer of 15 to 996, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:35, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: prostate-edited; Human Bone Marrow, re-excision; and Primary Dendritic cells,frac 2.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:36 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1306 of SEQ ID
N0:36, b is an integer of 15 to 1320, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:36, 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.
gnlIPlDId1016408 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "Copper homeostasis protein CutC. [Escherichia coli]." A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PIDId1016408 Copper homeostasis protein CutC. [Escherichia coli]
>gnl~PID~d1016415 Copper homeostasis protein CutC.
[Escherichia coli]
Length = 248 Plus Strand HSPs:
Score = 494 (173.9 bits), Expect -- 1.6e-46, P = 1.6e-46 Identities = 107/241 (440), Positives = 141/241 (580), Frame = +3 Q: 180 LMEVCVDSVESAVNAERGGADRIELCSGLSEGGTTPSMGVLQWKQSVQIPVFVMIRPRG 359 3O L+E+C S+E A+ A++ GADR+ELC+ EGG TPS+GVL+ V+Q V IPV +IRPRG
S: 3 LLEICCYSMECALTAQQNGADRVELCAAPKEGGLTPSLGVLKSVRQRVTIPVHPIIRPRG 62 Q: 36D GDFLYSDREIEVMKADIRLAKLYGADGLVFGALTEDGHIDKELCMSLMAICRPLPVTFHR 539 GDF YSD E + D+R + G GLV G L DG++D +MA PL VTFHR
3S S: 63 GDFCYSDGEFAAILEDVRTVRELGFPGLVTGVLDVDGNVDMPRMEKIMAAAGPLAVTFHR 122 Q: 540 AFDMVHDPMAALETLLTLGFERVLTSGCDSSALEGLPLIKRLIEQAKGRIWMPGGGITD 719 AFDM +P+ L L LG RVLTSG S AL+GL I LI I+ M G G+
S: 123 AFDMCANPLYTLNNLAELGIARVLTSGQKSDALQGLSKIMELIAHRDAPII-MAGAGVRA 181 S Q: 720 RNLQRILEGSGATEFHCSARSTRDSGMKFRNSSVAMGASLSCSEYSLKVTDVTKVRTLNA 899 NL L+ +G E H SA + + S M++RN ++M + EYS + D V +
S: 182 ENLHHFLD-AGVLEVHSSAGAWQASPMRYRNQGLSMSSDEHADEYSRYIVDGAAVAEMKG 240 Q: 900 I 902 I
S: 241 I 241 The segment of gnlIPIDId1016408 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 127.
1S Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 128 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent in Human Prostate Cancer, Stage C fraction; Human Testes;
Soares_pregnant_uterus NbHPU; Soares fetal heart NbHHI9W; Human Eosinophils; Smooth muscle, serum treated; Stratagene muscle 937209; Hodgkin's Lymphoma II; Stratagene muscle 937209; T cell helper II; Soares placenta Nb2HP;
2S Testis, normal; Prostate; Activated T-cells; Human Soleus; Synovial IL-lITNF
stimulated; Human Ovary; Breast Cancer Cell line, angiogenic; NCI CGAP_Br2;
Human Umbilical Vein Endothelial Cells, uninduced; Human Hypothalmus,Schizophrenia; Human T-Cell Lymphoma;
Soares total fetus Nb2HF8 9w; Colon Tumor II; NCI CGAP_AA1;
NCI CGAP_GCB 1 and T Cell helper I.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1362 of SEQ ID
N0:37, b is an integer of 1~ to 1376, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:37, 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.
gi12654161 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "GPR39 [Homo sapiens~." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~2654161 (AF034633) GPR39 [Homo Sapiens] >spI043194IGP39 HUMAN PUTATIVE
G
PROTEIN-COUPLED RECEPTOR GPR39.
Length = 453 Minus Strand HSPs:
Score = 752 (264.7 bits), Expect = 7.1e-74, P -- 7.1e-74 Identities = 149/167 (89%), Positives = 149/167 (89%), Frame = -1 SO
Q: 1626 LIVVTLAVCWMPNQIRRIMAAAKPKHDWTRSYFRAYMILLPFSETFFYLSSVINPLLYTV 1447 LIWTLAVCWMPNQIRRIMAAAKPKHDWTRSYFRAYMILLPFSETFFYLSSVINPLLYTV
S: 287 LIWTLAVCWMPNQIRRIMAAAKPKHDWTRSYFRAYMILLPFSETFFYLSSVINPLLYTV 346 S Q: 1446 SSQQFRRVFVQVLCCRLSLQHANHEKRLRVHAHSTTDSARFVQRPLLFASRRQSSARRTE 1267 SSQQFRRVFVQVLCCRLSLQHANHEKRLRVHAHSTTDSARFVQRPLLFASRRQSSARRTE
S: 347 SSQQFRRVFVQVLCCRLSLQHANHEKRI,RVHAHSTTDSARFVQRPLLFASRRQSSARRTE 406 Q: 1266 KIFLSTFQSEAXXXXXXXXXXXXXXXXXXGAKPANSAAENGFQEHEV 1126 KIFLSTFQSEA GAKPANSAAENGFQEHEV
5: 407 KIFLSTFQSEAEPQSKSQSLSLESLEPNSGAKPANSAAENGFQEHEV 453 The segment of gi126S4161 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 129. Based on the structural similarity these 1S homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 130 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in Stratagene 2S ovarian cancer (#937219); Soares ovary tumor NbHOT; H. Kidney Cortex, subtracted; Stratagene neuroepithelium NT2RAMI 937234; Human Cerebellum, subtracted; NTERA2 + retinoic acid, 14 days; Human Manic Depression Tissue;
Human Infant Brain; H. Kidney Medulla, re-excision; 12 Week Old Early Stage Human, 11; Human Ovarian Cancer Reexcision; Human Adrenal Gland Tumor;
Stratagene pancreas (#937208); Stratagene endothelial cell 937223; Pancreas Islet WO 00/61629 PCTlUS00/09071 JI
Cell Tumor; PC3 Prostate cell line; Endothelial-induced and Nine Week Old Early Stage Human.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1939 of SEQ ID
N0:38, b is an integer of 15 to 1953, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:38, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29 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 Soares melanocyte 2NbHM; Human Umbilical Vein, Endo. remake;
Soares_pregnant uterus NbHPU; Soares multiple sclerosis 2NbHMSP; Human B
Cell Lymphoma; Soares fetal heart NbHHI9W; Kidney Pyramids; Stromal-Osteoclastoma; NTERA2 + retinoie acid, 14 days; LNCAP prostate cell line;
Stratagene HeLa cell s3 937216; Synovial hypoxia; Human Adult Small Intestine;
KMH2; T-Cell PHA 16 hrs: Human Brain, Striatum; human ovarian cancer; 12 Week Old Early Stage Human, II; Stratagene lung carcinoma 937218; Macrophage-oxLDL;
PC3 Prostate cell line; Human Placenta; Soares breast 3NbHBst; Adipocytes;
Human Testes Tumor; Human Neutrophil, Activated; Endothelial cells-control; Spleen, ~?
Chronic lymphocytic leukemia; Soares placenta Nb2HP; Human adult (K.Okubo);
Human fetal heart, Lambda ZAP Express; NCI CGAP_Co3 and Primary Dendritic Cells, lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
88 as residues: Arg-48 to Tyr-54.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:39 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 comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2779 of SEQ ID
N0:39, b is an integer of 15 to 2793, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:39, 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 infant brain 1 NIB and to a lesser extent in Hepatocellular Tumor; Human Placenta; Palate normal; Human Thyroid; Human Hypothalamus,schizophrenia, re-excision; Human Adult Small Intestine;
Hodgkin's Lymphoma II; Keratinocyte 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 N0:40 and may have been publicly available prior to conception of s3 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 1353 of SEQ ID
N0:40, b is an integer of 15 to 1367, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:40, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 31 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.
gnlIPIDIe1254351 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "Exportin(tRNA) [Homo sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gnllPID~e1254351 Exportin(tRNA) [Homo Sapiens] >sp1043784~043784 2O EXPORTIN.
Length = 962 Minus Strand HSPs:
Score = 1607 (565.7 bits), Expect = 1.9e-169, P = 1.9e-164 Identities = 312/312 (1000), Positives = 312/312 (1000 , Frame = -3 Q: 1724 LNHAVGFASRTSKAFSNKQTVKQCGCSEVYLDCLQTFLPALSCPLQKDILRSGVRTFLHR 1545 LNHAVGFASRTSKAFSNKQTVKQCGCSEVYLDCLQTFLPALSCPLQKDILRSGVRTFLHR
3O S: 651 LNHAVGFASRTSKAFSNKQTVKQCGCSEVYLDCLQTFLPAI~SCPLQKDILRSGVRTFLHR 710 Q: 1544 MIICLEEEVLPFIPSASEHMLKDCEAKDLQEFIPLINQITAKFKIQVSPFLQQMFMPLLH 1365 MIICLEEEVLPFIPSASEHMLKDCEAKDLQEFIPLINQITAKFKIQVSPFLQQMFMPLLH
S: 711 MIICLEEEVLPFIPSASEHMLKDCEAKDLQEFIPLINQITAKFKIQVSPFLQQMFMPLLH 770 i4 Q: 1364 AIFEVLLRPAEENDQSAALEKQMLRRSYFAFLQTVTGSGMSEVIANQGAENVERVLVTVI 1185 AIFEVLLRPAEENDQSAALEKQMLRRSYFAFLQTVTGSGMSEVIANQGAENVERVLVTVI
S: 771 AIFEVLLRPAEENDQSAALEKQMLRRSYFAFLQTVTGSGMSEVIANQGAENVERVLVTVI 830 S Q: 1189 QGAVEYPDPIAQKTCFIILSKLVELWGGKDGPVGFADFVYKHIVPACFLAPLKQTFDLAD 1005 QGAVEYPDPIAQKTCFIILSKLVELWGGKDGPVGFADFVYKHIVPACFLAPLKQTFDLAD
S: 831 QGAVEYPDPIAQKTCFIILSKLVELWGGKDGPVGFADFVYKHIVPACFLAPLKQTFDLAD 890 Q: 1004 AQTVLALSECAVTLKTIHLKRGPECVQYLQQEYLPSLQVAPEIIQEFCQALQQPDAKVFK 825 AQTVLALSECAVTLKTIHLKRGPECVQYLQQEYLPSLQVAPEIIQEFCQALQQPDAKVFK
S: 891 AQTVLALSECAVTLKTIHLRRGPECVQYLQQEYLPSLQVAPEIIQEFCQALQQPDAKVFK 950 Q: 824 NYLKVFFQRAKP 789 NYLKVFFQRAKP
S: 951 NYLKVFFQRAKP 962 The segment of gnIIPIDIe1254351 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 131. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 132 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Synovial Fibroblasts (control); Soares_fetal_heart_NbHHI9W; Soares placenta Nb2HP;
Soares infant brain 1NIB; T cell helper II; NCI CGAP_GCB1;
Soares fetal_liver spleen_1 NFLS S 1; Human Fetal Lung III; Human Placenta;
Larynx Tumour; LNCAP + 30nM R 1881; Human Prostate, subtracted; Human (Caco-2) cell line, adenocarcinoma, colon, remake; Morton Fetal Cochlea; Human Gall Bladder, fraction II; Human colon carcinoma (HCC) cell line, remake; Activated T-Ss cells; Stromal-Osteoclastoma; NTERA2 + retinoic acid, 14 days; Stratagene HeLa cell s3 937216; Synovial hypoxia; Spleen metastic melanoma; Human Osteoblasts II;
Human Jurkat Membrane Bound Polysomes; Stromal cell TF274; Macrophage-oxLDL; Bone Marrow Stromal Cell, untreated; NTERA2, control; Stratagene corneal stroma (#937222); Human Liver, normal; Human T-Cell Lymphoma; Stratagene NT2 neuronal precursor 937230; Human Placenta; Adipocytes; Colon Tumor II; Primary Dendritic cells,frac 2; Human Osteoclastoma; Smooth muscle,control; Human Bone Marrow, treated; Stratagene HeLa cell s3 937216; Stratagene hNT neuron (#937233);
Keratinocyte; Human 8 Week Whole Embryo; Soares fetal liver spleen INFLS and Primary Dendritic Cells, lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
90 as residues: Lys-24 to Tyr-40, Pro-42 to Ser-48.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1892 of SEQ ID
N0:41, b is an integer of 15 to 1906, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:41, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 32 s6 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.
gnIIPIDIe1340344 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "NS1-binding protein [Homo Sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gnl~PID~e1340344 (AJ012449) NS1-binding protein [Homo Sapiens]
>Sp~E1340344~E1340344 NS1-BINDING PRDTEIN.
Length = 619 Plus Strand HSPS:
1S Score = 2286 (804.7 bits), Expect = 1.7e-238, Sum P(2) = 1.7e-238 Identities = 424/441 (96~), Positives = 425/441 (960), Frame = +2 Q: 2 LKLPRLKLEVMLEDNVCLPSNGKLYTKVINWQRSIWENGDSLXXLMEEVQTLYYSADHK 181 LKLPRLKLEVMLEDNVCLPSNGKLYTKVINWQRSIWENGDSL LMEEVQTLYYSADHK
2O S: 171 LKLPRLKLEVMLEDNVCLPSNGKLYTKVINWVQRSIWENGDSLEELMEEVQTLYYSADHK 230 Q: 182 LLDGNLLDGQAEVFGSDDDHIQFVQKKPPRENGHKQISSSSTGCLSSPNATVQSPKHEWK 361 LLDGNLLDGQAEVFGSDDDHIQFVQKKPPRENGHKQISSSSTGCLSSPNATVQSPKHEWK
S: 231 LLDGNLLDGQAEVFGSDDDHIQFVQKKPPRENGHKQISSSSTGCLSSPNATVQSPKHEWK 290 Q: 362 IVASEKTSNNTYLCLAVLDGIFCVIFLHGRNSPQXXXXXXXXXXXXXXFEMQQDELIEKP 541 IVASEKTSNNTYLCLAVLDGIFCVIFLHGRNSPQ FEMQQDELIEKP
S: 291 IVASEKTSNNTYLCLAVLDGIFCVIFLHGRNSPQSSPTSTPKLSKSLSFEMQQDELIEKP 350 3O Q: 542 MSPMQYARSGLGTAEMNGKLIAAGGYNREECLRTVECYNPHTDHWSFLAPMRTPRARFQM 721 MSPMQYARSGLGTAEMNGKLIAAGGYNREECLRTVECYNPHTDHWSFLAPMRTPRARFQM
S: 351 MSPMQYARSGLGTAEMNGKLIAAGGYNREECLRTVECYNPHTDHWSFLAPMRTPRARFQM 410 Q: 722 AVLMGQLYWGGSNGHSDDLSCGEMYDSNIDDWIPVPELRTNRCNAGVCALNGKLYIVGG 901 S: 411 AVLMGQLYWGGSNGHSDDLSCGEMYDSNIDDWIPVPELRTNRCNAGVCALNGKLYIVGG 470 Q: 902 SDPYGQKGLKNCDVFDPVTKLWTSCAPLNIRRHQSAVCELGGYLYIIGGAESWNCLNTVE 1081 SDPYGQKGLKNCDVFDPVTKLWTSCAPLNIRRHQSAVCELGGYLYIIGGAESWNCLNTVE
4O S: 471 SDPYGQKGLKNCDVFDPVTKLWTSCAPLNIRRHQSAVCELGGYLYIIGGAESWNCLNTVE 530 Q: 1082 RYNPENNTWTLIAPMNVARRGAGVAVLNGKLFVCGGFDGSHAISCVEMYDPTRNEWKMMG 1261 RYNPENNTWTLIAPMNVARRGAGVAVLNGKLFVCGGFDGSHAISCVEMYDPTRNEWKMMG
S: 531 RYNPENNTWTLIAPMNVARRGAGVAVLNGKLFVCGGFDGSHAISCVEMYDPTRNEWKMMG 590 Q: 1262 NMTSPRSNAGIATVGNTIYAV 1324 +MTSPRSNAGIATVGNTIYAV

WO 00/61629 PCTlUS00/09071 S: 591 HMTSPRSNAGIATVGNTIYAV 611 Score = 360 (126.7 bits), Expect = 6.Oe-30, P = 6.Oe-30 Identities = 76/236 (32~), Positives = 124/236 (52%), Frame = +2 Q: 683 LAPMRTPRARFQMAVLMGQLYWGGSNGHSDDLSCGEMYDSNIDDWIPVPELRTNRCNAG 862 ++pM+ R+ A + G+L GG N + L E Y+ + D W + +RT R
S: 351 MSPMQYARSGLGTAEMNGKLIAAGGYN-REECLRTVECYNPHTDHWSFLAPMRTPRARFQ 409 lO Q: 863 VCALNGKLYIVGGSDPYGQKGLKNC-DVFDPVTKLWTSCAPLNIRRHQSAVCELGGYLYI 1039 + L G+LY+VGGS+ G +C +++D W L R + VC L G LYI
S: 410 MAVLMGQLYWGGSN--GHSDDLSCGEMYDSNIDDWIPVPELRTNRCNAGVCALNGKLYI 467 Q: 1040 IGGAESWNC--LNTVERYNPENNTWTLIAPMNVARRGAGVAVLNGKLFVCGGFDGSHAIS 1213 IS +GG++ + L + ++P WT AP+N+ R + V L G L++ GG + + ++
S: 468 VGGSDPYGQKGLKNCDVFDPVTKLWTSCAPLNIRRHQSAVCELGGYLYIIGGAESWNCLN 527 Q: 1214 CVEMYDPTRNEWKMMGNMTSPRSNAGIATVGNTIYAVGGFDGNEFLNTVEVYNLESNEW 1390 VE Y+P N W ++ M R AG+A + ++ GGFDG+ ++ VE+y+ NEW
2O S: 528 TVERYNPENNTWTLIAPMNVARRGAGVAVLNGKLFVCGGFDGSHAISCVEMYDPTRNEW 586 The segments of gnIIPIDIe1340344 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 133 and SEQ ID NO. 135. Based on the structural similarity these homologous polypeptides are expected to share at least 25 some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 134 and/or SEQ
30 ID NO. 136 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Bone Marrow, treated and to a lesser extent in Human Endometrial Tumor; Colon Carcinoma; Early Stage Human Brain; Human Placenta;
35 Human Neutrophil, Activated; Human Ovary; Stratagene hNT neuron (#937233);
Colon Normal III; Human Testes; Healing groin wound, 6.5 hours post incision;

Soares multiple sclerosis 2NbHMSP; H. Kidney Medulla, re-excision; Mo7e Cell Line GM-CSF treated (lng/ml); Stratagene Human skeletal muscle cDNA library, cat.
#936215.; normalized infant brain cDNA; Human Pancreas Tumor; Synovial Fibroblasts (control); Stratagene lung (#937210); Human fetal heart, Lambda ZAP
Express; 5oares testis NHT; Human Substantia Nigra; Human Testes, Reexcision;
Bone marrow; Hodgkin's Lymphoma II; Soares fetal liver spleen 1NFLS; Human Fetal Brain, normalized 100024F; Human Fetal Brain, normalized AC5002; Thyroid Thyroiditis; Colon Tumor; Stomach Tumour; Tongue Tumour; Saos2 Cells, Vitamin D3 Treated; Saos2, Dexamethosome Treated; Dermatofibrosarcoma Protuberance;
Human (HCC) cell line liver (mouse) metastasis, remake; Human colon carcinoma (HCC) cell line, remake; Adipocytes,re-excision; Human Tonsils, Lib 2; HEL
cell line; Hepatocellular Tumor; H. Kidney Cortex, subtracted; Salivary Gland, Lib 2;
Stratagene HeLa cell s3 937216; Human Adipose Tissue, re-excision; Jurkat T-cell G 1 phase; Soares_parathyroid tumor NbHPA; Prostate BPH; Soares NhHMPu_S 1;
TF-1 Cell Line GM-CSF Treated; T-Cell PHA 24 hrs; Human Ovarian Cancer Reexcision; Human umbilical vein endothelial cells, IL-4 induced; NCI
CGAP_Co3;
NCI CLAP Ewl; NCI CGAP_GCS; NCI CGAP_Pr22; Ulcerative Colitis;
Macrophage (GM-CSF treated); Soares testis NHT;
Soares_fetal liver spleen_1NFLS_Sl; Resting T-Cell Library,II; NCI CGAP_LuS;
Soares multiple sclerosis 2NbHMSP; Smooth muscle, serum treated; Colon Normal II; Soares_placenta_8to9weeks 2NbHP8to9W; H Macrophage (GM-CSF treated), re-excision; Normal colon; Soares melanocyte 2NbHM; 12 Week Early Stage Human II, Reexcision; Neutrophils control, re-excision; Human Fetal Heart; Endothelial-induced; Endothelial cells-control; Anergic T-cell; Human Osteoclastoma; Human B
Cell Lymphoma; NCI CLAP Br2; NCI CGAP_Ewl; NCI CGAP_Ov2;
NCI CGAP_ColO; NCI CLAP Col2; NCI CGAP_Lip2; Spleen, Chronic lymphocytic leukemia; Neutrophils IL-1 and LPS induced; Osteoblasts;
Keratinocyte;
Nine Week Old Early Stage Human; T cell helper II; Primary Dendritic Cells, lib 1 and Soares infant brain 1NIB.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1709 of SEQ ID
N0:42, b is an integer of 15 to 1723, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:42, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 33 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.
gi12197085 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "ORF2-like protein [Homo sapiensJ." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~2197085 (AF003535) ORF2-like protein [Homo sapiens]
Length = 573 Plus Strand HSPs:

Score = 185 (65.1 bits), Expect -- 2.9e-10, P = 2.9e-10 Identities = 40/72 (S5e), Positives = 44/72 (610), Frame = +2 S Q: 1844 YKENLSLQXXXXXXXXXXXXNIPCSWVGRINIVKMAILPKVIYRFSAIPIKLPC-LSSQX 2020 +KEN NIPCSWVGRINIVKMAILPKVIYRF+AIPIKLP ++
5: 295 FKENYKPLLKEIKEDTNKWKNIPCSWVGRINIVKMAILPKVIYRFNAIPIKLPMTFFTEL 354 Q: 2021 GKNYFKVXWXQK 2056 S: 355 EKTTLKFIWNQK 366 The segment of gi12197085 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 137.
15 Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 138 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following 20 tissueslcDNA libraries: Soares fetal heart_NbHHI9W and to a lesser extent in pBMC stimulated w/ poly I/C; Human Neutrophil; Human Activated Monocytes;
Neutrophils IL-1 and LPS induced; Human Placenta; Human Amygdala;
NCI CGAP_GCB1; Stratagene ovarian cancer (#937219); Monocyte activated; Nine Week Old Early Stage Human and Primary Dendritic Cells, lib 1.
25 Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:43 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 30 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 2060 of SEQ ID
N0:43, b is an integer of 15 to 2074, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:43, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 34 When tested against K562 leukemia cell lines. supernatants removed from cells containing this gene activated the ISRE assay. Thus, it is likely that this gene activates leukemia cells through the Jak-STAT signal transduction pathway. The interferon-sensitive response element is apromoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: PERM TF274 Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
93 as residues: Gln-20 to Leu-25.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 808 of SEQ ID
N0:44, b is an integer of 15 to 822, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:44, and where b is greater than or equal to a + 14.
S
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 fetal liver spleen 1 NFLS and to a lesser extent in Soares adult brain N2b4HB55Y; Hippocampus, Alzheimer Subtracted; Human 8 Week Whole Embryo; Stratagene HeLa cell s3 937216; Nine Week Old Early Stage Human; Jurkat T-Cell, S phase; H. Kidney Medulla, re-excision; Human T-Cell Lymphoma; Stratagene neuroepithelium (#937231); Soares infant brain 1NIB;
Gessler Wilms tumor; Human Prostate Cancer, Stage C fraction; Soares testis NHT;
Human Whole Brain #2 - Oligo dT > I.SKb; Human Rhabdomyosarcoma; Human Whole Six Week Old Embryo; Liver HepG2 cell line.; NCI CGAP_ColO;
NCI CGAP_Kid6; Hepatocellular Tumor, re-excision; Human Substantia Nigra;
Soares fetal heart NbHHI9W; Human Adult Pulmonary,re-excision; Human Amygdala; Human Microvascular Endothelial Cells, fract. A; Human Cerebellum;
Soares placenta Nb2HP; Human Kidney Tumor; Human Amygdala Depression, re-excision; Human Whole 6 Week Old Embryo (II), subt; Human Tonsil, Lib 3; Whole 6 Week Old Embryo; Human Gall Bladder, fraction I1; Human Fetal Spleen; Human Placenta; HSA 172 Cells; Soares_NFL T GBC_S1; Stratagene ovary (#937217);
Human Tonsils, Lib 2; H. Whole Brain #2, re-excision; Apoptotic T-cell, re-excision;
Healing groin wound - zero hr post-incision (control); Stomach cancer (human),re-excision; Alzheimers, spongy change; NTERA2 + retinoic acid, 14 days; Human Amygdala,re-excision; Human Osteosarcoma; Human Ovary; Prostate BPH; Human Adult Small Intestine; Human Infant Brain; Soares fetal liver spleen_1NFLS S1;
Human Prostate; T-Cell PHA 16 hrs; Human Jurkat Membrane Bound Polysomes;
Human Ovarian Cancer Reexcision; Human Hippocampus; Human Thymus;
NCI CLAP Br2; NCI CGAP_Co 11; NCI CGAP_GCB 1; NTERA2, control; Adult S heart, Lambda gtl l; Gessler Wilms tumor; Human fetal heart, Lambda ZAP
Express;
Human retina cDNA randomly primed sublibrary; NCI CGAP_GC2;
NCI CGAP_LuS; NCI CGAP_Kid3; NCI CGAP_Kids; NCI CGAP_PNS1;
NCI CGAP_Brn23; Soares total fetus_Nb2HF8 9w; Pancreas Islet Cell Tumor;
Human Gall Bladder; Fetal Heart; Colon Carcinoma; Smooth muscle, serum treated;
breast lymph node CDNA library; Adipocytes; Dendritic cells, pooled; H.
Frontal cortex,epileptic,re-excision; Human Fetal Heart; human tonsils; Human Neutrophil, Activated; Human B Cell Lymphoma; Spleen, Chronic lymphocytic leukemia; Bone Marrow Cell Line (RS4,11) and Activated T-cell(12h)/Thiouridine-re-excision.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
94 as residues: His-25 to Phe-30.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 2392 of SEQ ID
N0:45, b is an integer of 15 to 2406, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:45, and where 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 primarily in the following tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent in Soares placenta Nb2HP; Dendritic cells, pooled; Soares melanocyte 2NbHM; Early Stage Human Lung, subtracted; Soares retina N2b4HR; Stratagene ovarian cancer (#937219); human ovarian cancer; Stratagene fetal spleen (#937205);
Soares multiple sclerosis 2NbHMSP; NTERA2, control;
Soares_total fetus Nb2HF8 9w; Human Osteoclastoma; Human Cerebellum;
Soares fetal heart NbHHI9W; Soares senescent fibroblasts_NbHSF; Soares infant brain 1NIB; HUman Fetal Brain, normalized 100024F; Cheek Carcinoma; Human Normal Cartilage,Fraction I; Human Normal Cartilage Fraction II; Human Gall Bladder, fraction II; Human Cerebellum, subtracted; Human Skin Tumor; Human Synovium; Breast Cancer cell line, MDA 36; human corpus colosum; Human Whole I S Brain #2 - Oligo dT > l.SKb; Soares_pregnant uterus NbHPU; Human Osteoclastoma, re-excision; Synovial Fibroblasts (Ill/TNF), subt; Human Prostate;
Human Fetal Dura Mater; T-Cell PHA 24 hrs; Human Hypothalmus,Schizophrenia;
Human Activated Monocytes; Spinal cord; Ulcerative Colitis; Bone Marrow Stromal Cell, untreated; Hepatocellular Tumor, re-excision; Macrophage-oxLDL, re-excision;
Pancreas Islet Cell Tumor; 12 Week Old Early Stage Human; breast lymph node CDNA library; Early Stage Human Brain; Human Fetal Lung III; Human Amygdala;
T Cell helper I; Hodgkin's Lymphoma II; Soares_parathyroid_tumor NbHPA; and Primary Dendritic Cells, lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
95 as residues: Arg-76 to Lys-91.

6s Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:46 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. 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 2441 of SEQ ID
N0:46, b is an integer of 15 to 2455, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:46, 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 tissueslcDNA libraries: Soares infant brain 1NIB and to a lesser extent in Soares placenta Nb2HP; Early Stage Human Brain; Nine Week Old Early Stage Human;
Soares_pregnant uterus NbHPU; Morton Fetal Cochlea; Human Adult Small Intestine; NCI CGAP_GCBl; Stromal cell TF274; Soares fetal heart NbHHI9W;
Soares senescent fibroblasts_NbHSF; Human Chondrosarcoma; Stratagene lung carcinoma 937218; 12 Week Old Early Stage Human; Soares fetal liver spleen 1NFLS; Messangial cell, frac 1; Human Prostate, subtracted; Human Adult Pulmonary; Human Stomach; NTERA2 teratocarcinoma cell line+retinoic acid (14 days); HEL cell line; Stomach cancer (human),re-excision; Morton Fetal; Human Ovary; Human Infant Brain; L428; Human Osteoblasts II;
Soares_placenta_8to9weeks 2NbHP8to9W; T-Cell PHA 24 hrs; Liver, Hepatoma;
Human umbilical vein endothelial cells, IL-4 induced; Bone Marrow Stromal Cell, untreated; Human Gall Bladder; PC3 Prostate cell line; Colon Tumor; Resting T-Cell Library,ll; Colon Normal II; Human Testes Tumor; Dendritic cells, pooled;
Human Placenta; Human Fetal Heart; Human Osteoclastoma; Human Endometrial Tumor and 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 N0:47 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 described by the general formula of a-b, where a is any integer between 1 to 2560 of SEQ ID
N0:47, b is an integer of 15 to 2574, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:47, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 38 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.
gi11407655 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "SH3P7 (Mus musculus]." A partial alignment demonstrating the observed homology is shown immediately below.
>giI1407655 SH3P7 [Mus musculus] >sp~Q62418~Q62418 DREBRIN-LIKE SH3 DOMAIN-CONTAINING PROTEIN SH3P7.

Length = 433 Plus Strand HSPS:
Score = 748 (263.3 bits), Expect = 9.1e-106, Sum P(3) = 9.1e-106 Identities = 149/201 (740), Positives = 160/201 (790), Frame = +2 Q: 344 SPQRTWEQQQEVVSRNRNEQESA----VHPREIFKQKERAMSTTSISSPQPGKLRSPFLQ 511 +P RT E +QE VSR R E ESA HPREIFKQKERAMSTTS++S QPGKLRSPFLQ
lO S: 233 APSRTGEPEQEAVSRTRQEWESAGQQAPHPREIFKQKERAMSTTSVTSSQPGKLRSPFLQ 292 Q: 512 KQLTQPETHFGREPAAAISRPRADLPAEEPAPSTPPCLXXXXXXXXXXXXXXXXTFYEQP 691 KQLTQPET +GREP A +SRP A + EEPAPST T YE+P
S: 293 KQLTQPETSYGREPTAPVSRPAAGV-CEEPAPSTLSS-AQTEEEPTYEVPPEQDTLYEEP 350 Q: 692 PLVQQQGAGSEHIDHHIQGQGLSGQGLCARALYDYQAADDTEISFDPENLITGIEVIDEG 871 PLVQQQGAGSEHID+++Q QG SGQGLCARALYDYQAADDTEISFDPENLITGIEVIDEG
S: 351 PLVQQQGAGSEHIDNYMQSQGFSGQGLCARALYDYQAADDTEISFDPENLITGIEVIDEG 410 2O Q: 872 WWRGYGPDGHFGMFPANYVELIE 940 WWRGYGPDGHFGMFPANYVELIE
S: 411 WWRGYGPDGHFGMFPANYVELIE 433 The segment of gi11407655 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 139. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 140 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissueslcDNA libraries: Primary Dendritic Cells, lib 1 and to a lesser extent in Human Rhabdomyosarcoma; Human Thymus; Soares placenta Nb2HP; Human Primary Breast Cancer Reexcision; Human Epididymus; Human Pituitary, subt IX; Merkel Cells; Soares breast 2NbHBst; Stratagene lung (#937210); Human Infant Adrenal Gland, subtracted; Colon, tumour; Larynx Tumour; Human Whole 6 Week Old Embryo (II), subt; Human Aortic Endothelium; Aorta endothelial cells + TNF-a;
Activated T-cells; Breast Lymph node cDNA library; Synovial IL-1/TNF
stimulated;
Breast Cancer cell line, MDA 36; H Female Bladder, Adult; NTERA2 + retinoic acid, 14 days; Synovial hypoxia-RSF subtracted; Human Whole Brain #2 - Oligo dT >
l.SKb; Soares_NhHMPu S1; Stratagene pancreas (#937208); Human endometrial stromal cells; Human Colon, re-excision; Jurkat T-cell G 1 phase; Human Manic Depression Tissue; Human Chronic Synovitis; Fetal Liver, subtraction II; Human Brain, Striatum; Human Activated Monocytes; Rejected Kidney, lib 4; Macrophage-oxLDL, re-excision; Human Gall Bladder; Colon Carcinoma; breast lymph node CDNA library; Soares breast 3NbHBst; Soares total fetus Nb2HF8 9w; Normal colon; Stratagene corneal stroma (#937222): Human Neutrophil, Activated; Human Adult Pulmonary,re-excision; Activated T-Cell (l2hs)/Thiouridine IabeIledEco;
Human Microvascular Endothelial Cells, fract. A; Monocyte activated; Human B
Cell Lymphoma; Human Bone Marrow, treated; Hodgkin's Lymphoma II; Osteoblasts; T
cell helper II; Human Cerebellum; Soares fetal liver spleen INFLS and Soares infant brain 1 NIB.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
97 as residues: His-48 to Ser-61, Ala-66 to Val-72.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:48 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 WO 00/61629 PCT1US00/090?1 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 1746 of SEQ ID
N0:48, b is an integer of 15 to 1760, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:48, and where b is greater than or equal to a S + 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.
gi1425474 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "SMDR1 [Schistosoma mansoni]." A partial alignment demonstrating the observed homology is shown immediately below.

>gi~425474 SMDR1 [Schistosoma mansoni] >sp~Q26598~Q26598 SMDR1.
Length = 691 Plus Strand HSPs:
Score = 391 (137.6 bits), Expect = 6.2e-35, P = 6.2e-35 Identities = 89/221 (40~), Positives = 122/221 (550), Frame = +3 Q: 3 EGLLTFGYLVLLSHVGERMAVDMRRALFSSLLRQDITFFDANKTGQLVSRLTTDVQEFKS 182 2S + L TF Y+ LL VGERMA MR LF L+ QD+ +FD + +G+LV + +DVQ FKS
S: 158 QSLSTFLYIGLLGSVGERMARRMRIQLFRKLVYQDVAYFDVHSSGKLVEIIGSDVQNFKS 217 Q: 183 SFKLVISQGLRSCTQVAGCXXXXXXXXXXXXXXXXXATPALMGVGTLMGSGLRKLSRQCQ 362 SFK ISQGLR+ QV G P + +G+LMG+ LR +SR+ Q
3O S: 218 SFKQCISQGLRNGIQWGSVFALLSISPTLTAALIGCLPCVFLIGSLMGTELRHISREVQ 277 Q: 363 EQIARAMGVADEALGNVRTVRAFAMEQREEERYGXXXXXXXXXXXXXGRGIALFQGLSNI 542 Q + + DEA ++RTV++ AME + GI FQGLSN+
S: 278 SQNSLFASLIDEAFSHIRTVKSLAMEDFLINKINYNVDKAKMLSEKLSFGIGSFQGLSNL 337 Q: 543 AFNCMVLGTLFIGGSLVAGQQLTGGDLMSFLVASQTVQSFL 665 N +VLG L++GG L++ +L G LMSFL +QT+Q h S: 338 TLNGWLGVLYVGGHLMSRGELDAGHLMSFLATTQTLQRSL 378 The segment of gi1425474 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 141. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities 5 are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 142 which 10 corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Pituitary, subt IX and to a lesser extent in Stratagene pancreas (#937208); Soares adult brain N2b4HB55Y; Human Adult Testes, Large 15 Inserts, Reexcision; Spinal cord; Spleen, Chronic lymphocytic leukemia;
Human Fetal Brain, normalized CSOOH; Crohn's Disease; Sinus piniformis Tumour; Brain Amygdala Depression; Weizmann Olfactory Epithelium; LNCAP untreated; Aorta endothelial cells + TNF-a; Human Stomach,re-excision; Human endometrial stromal cells-treated with progesterone; Human Frontal Cortex, Schizophrenia; Human Infant 20 Brain; Chromosome 7 Fetal Brain cDNA Library; Human; Human Rhabdomyosarcoma; Human Placenta; Nine Week Old Early Stage Human; Soares fetal liver spleen 1NFLS and Primary Dendritic Cells, lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
98 as residues: Lys-30 to Cys-35, Glu-62 to Tyr-69.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically S 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 1009 of SEQ ID
N0:49, b is an integer of 15 to 1023, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:49, 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: Prostate BPH; T-Cell PHA 24 hrs and Human Hypothalmus,Schizophrenia.
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 603 of SEQ ID
NO:50, b is an integer of 15 to 617, 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/cDNA libraries: Soares breast 2NbHBst and to a lesser extent in Soares fetal liver spleen 1NFLS; Soares placenta Nb2HP; Soares_pregnant uterus NbHPU;
Human Adipose; Soares testis NHT; NCI CGAP_Prl ; Prostate BPH; Human Placenta; Human Adult Pulmonary,re-excision; Soares ovary tumor NbHOT; H
Female Bladder, Adult; NCI CLAP Pr2; NCI CGAP_Kids; Human Colon, re-excision; Stratagene fetal spleen (#937205); Soares breast 3NbHBst; Human Fetal Kidney, Reexcision; Human Synovial Sarcoma; Human Fetal Lung III; Smooth muscle,control; Nine Week Old Early Stage Human; Gessler Wilms tumor; Human Tongue, frac 1; Human osteoarthritic,fraction II; Human Uterus, normal; Human Colon, subtraction; prostate-edited; Human Colon; Human Fetal Spleen; Smooth muscle-ILb induced; Human Thyroid; Human Prostate Cancer, Stage C fraction;
Soares NhHMPu_Sl; Human Whole Brain #2 - Oligo dT > l.SKb; Stratagene muscle 937209; Human Adipose Tissue, re-excision; Human adult (K.Okubo);
Human heart cDNA (YNakamura); Human fetal heart, Lambda ZAP Express; Human retina cDNA Tsp5091-cleaved sublibrary; NCI CLAP Br3; NCl CGAP_GC4;
NCI CGAP_Lul; NCI CGAP_Pr3; NCI CGAP_Alvl; NCI CGAP_ColO;
NCI CGAP_Prl l; WATM1; Soares fetal lung_NbHLI9W; Human Ovary; wilm's tumor; Human Infant Brain; Human Chronic Synovitis; Human Uterine Cancer;
Human Hypothalmus,Schizophrenia; Ulcerative Colitis; Bone Marrow Stromal Cell, untreated; Fetal Heart; Smooth muscle, serum treated; HM3; Bone marrow; Human Fetal Heart; Endothelial cells-control; Human Amygdala; Osteoblasts and Soares infant brain 1NIB.
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 864 of SEQ ID
NO:51, b is an integer of 15 to 878, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:S1, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 42 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.
gi12547132 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "neuropilin-2(al7) [Homo sapiens]." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~2547132 (AF022860) neuropilin-2(a17) [Homo Sapiens] >sp~014B21~014821 NEUROPILIN-2.
Length = 926 Minus Strand HSPs:

Score = 619 (217.9 bits), Expect = 8.9e-135, Sum P(5) = 8.9e-135 Identities = 121/152 (790), Positives = 121/152 (79%), Frame = -1 Q: 3661 DQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFA 3482 S DQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFA
S: 749 DQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPISAFA 808 Q: 3481 VDIPEIHXXXXXXXXXXXXXXXXWXXXXXXXXXXXXXXXDKEKSWLYTLDPILITIIAMS 3302 VDIPEIH W DKEKSWLYTLDPILITIIAMS
lO S: 809 VDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTLDPILITIIAMS 868 Q: 3301 SLGVLLGATCAGLLLYCTCSYSGLSSRSCTTL 3206 SLGVLLGATCAGLLLYCTCSYSGLSSRSCTTL
S: 869 SLGVLLGATCAGLLLYCTCSYSGLSSRSCTTL 900 Score = 535 (188.3 bits), Expect = 8.9e-135, Sum P(5) = 8.9e-135 Identities = 96/100 (96°s), Positives = 99/10D (990), Frame = -3 Q: 3983 PSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYA 3804 S: 641 PSGFNCNFDFLEEPCGWMYDHAKWLRTTWASSSSPNDRTFPDDRNFLRLQSDSQREGQYA 700 Q: 3803 RLISPPVHLPRSPVCMEFQYQATGGRGVALQWREAARRA 3684 RLISPPVHLPRSPVCMEFQYQATGGRGVALQWREA++ +
2S S: 701 RLISPPVHLPRSPVCMEFQYQATGGRGVALQWREASQES 740 Score = 168 (59.1 bits), Expect = 8.9e-135, Sum P(5) = 8.9e-135 Identities = 36/65 (55~), Positives = 40/65 (61°s), Frame = -1 3O Q: 4177 PAGLQXRLLQHSLIFLCPYTDSKPTVETLGPTVKSXXXXXXXXXXXXXXXCGENCSFEDD 3998 PAG+ RL + C +TDSKPTV+TLGPTVKS CGENCSFEDD
S: 580 PAGIGMRLE----VLGCDWTDSKPTVKTLGPTVKSEETTTPYPTEEEATECGENCSFEDD 635 Q: 3997 KDLQL 3983 S: 636 KDLQL 640 The segments of gi12S47132 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 143,SEQ ID NO. 145 and SEQ ID NO. 147. Based 40 on the structural similarity these homologous polypeptides are expected to share at least some biological activities. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having 4S the amino acid sequence set out in the sequence listing as SEQ ID NO.
144,SEQ 1D

7~
NO. 146 andior SEQ ID NO. 148 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Soares fetal liver spleen 1NFLS; Soares infant brain 1NIB;
Soares multiple_sclerosis_2NbHMSP; Soares placenta Nb2HP;
Soares NhHMPu S 1; Soares_pregnant uterus NbHPU; Human T-Cell Lymphoma;
Stratagene corneal stroma (#937222); normalized infant brain cDNA; Human Pancreas Tumor, Reexcision; Soares fetal lung NbHLI9W; Macrophage-oxLDL, re-excision; Human Synovial Sarcoma; Smooth Muscle- HASTE normalized; Human Umbilical Vein, Reexcision; Soares fetal heart NbHHI9W;
Soares fetal_liver spleen_1NFLS S1; Stratagene endothelial cell 937223; Human Adipose; Hemangiopericytoma; Rejected Kidney, lib 4; Stratagene lung (#937210);
Primary Dendritic cells,frac 2; Endothelial-induced; Human Microvascular Endothelial Cells, fract. A; Human 8 Week Whole Embryo; NCI CGAP_GC4;
Primary Dendritic Cells, lib 1; Human Microvascular Endothelial Cells, fract.
B; H
Umbilical Vein Endothelial Cells, frac A, re-excision; Hypothalamus; Human Placenta; Human Stomach; HSA 172 Cells; NTERA2 teratocarcinoma cell line+retinoic acid ( 14 days); Raji Cells, cyclohexamide treated; Soares adult brain N2b4HB55Y; Human Umbilical Vein, Endo. remake; Spinal Cord, re-excision; H.
Kidney Medulla, re-excision; L428; Human Pancreas Tumor; Macrophage-oxLDL;
Human Hypothalmus,Schizophrenia; Normalized infant brain, Bento Soares;
Soares NFL T GBC_SI; Human umbilical vein endothelial cells, II. 4 induced;
Ulcerative Colitis; Jia bone marrow stroma; Soares NbHFB;
Soares total fetus_Nb2HF8_9w; Stratagene hNT neuron (#937233); Stratagene liver (#937224); H Macrophage (GM-CSF treated), re-excision; Human fetal heart, Lambda ZAP Express; Human Fetal Kidney, Reexcision; 12 Week Early Stage Human II, Reexcision; Human Fetal Heart; Human Adult Pulmonary,re-excision;
Endothelial cells-control; Nine Week Old Early Stage Human; T cell helper II;
NCI CLAP HN4; NCI CGAP_ColO and NCI CGAP_Kid6.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
101 as residues: Pro-26 to Gly-34.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:52 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 4547 of SEQ ID
N0:52, b is an integer of 15 to 4561, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:52, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 43 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.
gi11389766 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "unknown Homo sapiensJ." A partial alignment demonstrating the observed homology is shown immediately below.
>gi~1389766 unknown [Homo Sapiens] >sp~Q13629~Q13629 HYPOTHETICAL 13.3 KD
S PROTEIN.
Length = 118 Minus Strand HSPs:
Score = 137 (48.2 bits), Expect = 8.1e-08, P = 8.1e-08 Identities = 32164 (SOo), Positives = 38/64 (590), Frame = -2 Q: 281 HVGRPR*VDLLSPRV*DQPGQHGKMPFLIJKIQKCS*MWWRMPVVLATQEAEVGGSSRPRK 102 H GRPR D L V DQ GQ G+ P LLK K S WW +pV+ A +E E G S +P +
IS S: 56 HFGRPRRADYLRIGVPDQRGQRGESPSLLKNTKISWAWW-VPVIPAIREGEAGESLEPGR 114 Q: 101 LRLQ 90 RLQ
S: 115 QRLQ 118 The segment of gi11389766 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 149.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 1S0 which 2S corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Rejected Kidney, lib 4; human tonsils; Spleen, Chronic lymphocytic leukemia; 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:S3 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 1303 of SEQ ID
N0:53, b is an integer of 15 to 1317, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:S3, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 44 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.
gi13342794 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "calcium binding protein [Homo Sapiens]." A partial alignment demonstrating the observed homology 1S is shown immediately below.
>gi~3342794 (AF035606) calcium binding protein [Homo Sapiens]
>sp~075340~075340 CALCIUM BINDING PROTEIN.
Length = 191 Plus Strand HSPs:
Score = 349 (122.9 bits), Expect = 3.6e-31, P = 3.6e-31 2S Identities = 77/180 (42%), Positives = 1061180 (580), Frame = +1 Q: 319 PGLYGQGGAPPNVDPE-AYSW--FQSVDSDHSGYISMKELKQALVNCNWSSFNDETCLMM 489 PG G G A P+ ++ W FQ VD D SG IS EL+QAL N W+ FN T +
S: 10 PGA-GPGPAAGAALPDQSFLWNVFQRVDKDRSGVISDTELQQALSNGTWTPFNPVTVRSI 68 Q: 490 INMFDKTKSGRIDVYGFSALWKFIQQWKNLFQQYDRDRSGSISYTELQQALSQMGYNLSP 669 I+MFD+ ++ F+ +WK+I W+N+F+ yDRD SG I EL+QALS GY LS
S: 69 ISMFDRENKAGVNFSEFTGVWKYITDWQNVFRTYDRDNSGMIDKNELKQALSGFGYRLSD 128 3S Q: 670 QFTQLLVSRYCPRSANPAMQLDRFIQVCTQLQVLTEAFREKDTAVQGNIRLSFEDFVTMT 849 QF +L+ ++ R + D FIQ C LQ LT+ FR DT G I++S+E +++M
S: 129 QFHDILIRKF-DRQGRGQIAFDDFIQGCIVLQRLTDIFRRYDTDQDGWIQVSYEQYLSMV 187 Q. 850 AS 855 S
S: 188 FS 189 The segment of gi13342794 that is shown as "S" above is set out in the sequence listing as SEQ ID NO. 151. Based on the structural similarity these homologous polypeptides are expected to share at least some biological activities.
Such activities are known in the art, some of which are described elsewhere herein.
Assays for determining such activities are also known in the art, some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 152 which corresponds to the "Q" sequence in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent in Soares_pregnant uterus NbHPU; Soares placenta Nb2HP; Activated T-Cell (l2hs)lThiouridine labelledEco; Activated T-cell(12h)/Thiouridine-re-excision;
Human Jurkat Membrane Bound Polysomes; Human Adult Testes, Large Inserts, Reexcision; Soares fetal heart NbHHI9W; Keratinocyte; Human Cerebellum;
Amniotic Cells - Primary Culture; Stratagene ovarian cancer (#937219); Human endometrial stromal cells-treated with progesterone; Human Pituitary, subt IX;
Prostate BPH; H. Kidney Medulla, re-excision; Apoptotic T-cell; Human Pancreas Tumor; Human Rhabdomyosarcoma; Human Gall Bladder: Stratagene NT2 neuronal precursor 937230; Primary Dendritic cells,frac 2; Human Neutrophil, Activated;
Endothelial-induced; Osteoblasts; Human Primary Breast Cancer; LNCAP + o.3nM
81881; Human Colon, subtraction; Human OB HOS treated (10 nM E2) fraction I;

WO 00/61629 PCTlUS00/09071 Human Primary Breast Cancer,re-excision; Human Thyroid; Resting T-Cell, re-excision; Human Epididymus; Human Colon Cancer,re-excision;
Soares_placenta_8to9weeks 2NbHP8to9W; Human Fetal Epithelium (Skin); Human Stomach,re-excision; Human Amygdala,re-excision; Jurkat T-cell G 1 phase;
Human 5 Manic Depression Tissue; Human Infant Brain; Breast Cancer Cell line, angiogenic;
Human Fetal Kidney; Soares fetal lung_NbHLI9W; Human Primary Breast Cancer Reexcision; Humarr Hypothalmus,Schizophrenia; Human Thymus; Bone Marrow Stromal Cell, untreated; Soares breast 2NbHBst; Human Adrenal Gland Tumor;
Human adult testis, large inserts; NTERA2, control; Macrophage-oxLDL, re-10 excision; Adipocytes; Human Testes Tumor; Normal colon; Soares melanocyte 2NbHM; Human Testes, Reexcision; Human Placenta; Human Fetal Heart; human tonsils; Human Adult Pulmonary,re-excision; Human Amygdala; Human Microvascular Endothelial Cells, fract. A; Monocyte activated; Human B Cell Lymphoma; Spleen, Chronic lymphocytic leukemia; Human Testes; Human 15 Endometrial Tumor; Hodgkin's Lymphoma II and Primary Dendritic Cells, lib 1.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:54 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically 20 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 1667 of SEQ ID
N0:54, b is an integer of 15 to 1681, where both a and b correspond to the positions of 25 nucleotide residues shown in SEQ ID N0:54, and where b is greater than or equal to a + 14.

_ gl FEATURES OF PROTEIN ENCODED BY GENE NO: 45 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.
gi12443886 (all information available through the recited accession number is incorporated herein by reference) which is described therein as "Unknown protein [Arabidopsis thaliana~." A partial alignment demonstrating the observed homology is shown immediately below.

>gi~2443886 (AC002294) Unknown protein [Arabidopsis thaliana]
Length = 240 Plus Strand HSPs:
Score = 372 (131.0 bits), Expect = 9.4e-50, Sum P(2) = 9.4e-50 Identities = 68/129 (52%), Positives -- 99/129 (76~), Frame = +3 Q: 168 ITKDEWMEKLNNLHVQRADMNRLIMNYLVTEGFKEAAEKFRMESGIEPSVDLETLDERIK 347 ZO IT++EW +KLN + +++ DMN L+MN+LVTEG+ EAAEKF+ ESG +P +DL T+ +R+
S: 25 ITREEWEKKLNAVKLRKEDMNTLVMNFLVTEGYVEAAEKFQRESGTKPEIDLATITDRMA 84 Q: 348 IREMILKGQIQEAIALINSLHPELLDTNRYLYFHLQQQHLIELIRQRETEAALEFAQTQL 527 +++ + G +++AI +N L+pE+LDTN L+FHLQQQ LIELIRQ +TE ALEFAQ +L
ZS S: 85 VKKAVQNGNVEDAIEKVNDLNPEILDTNPELFFHLQQQRLIELIRQGKTEEALEFAQEEL 144 Q: 528 ADRARRAES 554 A R ++
S: 145 APRGEENQA 153 Score = 169 (59.5 bits), Expect = 9.4e-50, Sum P(2) = 9.4e-50 Identities = 37/92 (400), Positives = 51/92 (55~), Frame = +2 Q: 533 QGEESRECLTEMERTLALLAFDSPEESPFGDLLHTMQRQKVWSEVNQAVLDYENRESTPX 712 3S +GEE++ L E+E+T+ALL FD P +LL R K SEVN A+L ++ E P
S: 147 RGEENQAFLEELEKTVALLVFDDASTCPVKELLDLSHRLKTASEVNAAILTSQSHEKDPK 206 Q: 713 XXXXXXXXXWAQNELDQKKVKYPKMTDLSKGV 808 WAQ +LD+X V YP + DLS +
4O S: 207 LPSLLKMLIWAQTQLDEKAV-YPHINDLSTAI 237 The segments of gi12443886 that are shown as "S" above are set out in the sequence listing as SEQ ID NO. 153 and SEQ ID NO. 15s.
Preferred polypeptides of the invention comprise a polypeptide having the amino acid sequence set out in the sequence listing as SEQ ID NO. 154 and/or SEQ
ID NO. 156 which correspond to the "Q" sequences in the alignment shown above (gaps introduced in a sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: NCI CGAP_GCB1 and to a lesser extent in Soares testis_NHT; Human Pancreas Tumor, Reexcision; Soares fetal liver spleen 1NFLS; Soares fetal liver_spleen_1NFLS_S1; Human Rhabdomyosarcoma; Human Prostate Cancer, Stage C fraction; Stratagene placenta (#937225); human corpus colosum; Apoptotic T-cell; Soares_pregnant uterus NbHPU; Stratagene colon (#937204); Human 8 Week Whole Embryo; Soares placenta Nb2HP;
Soares fetal lung_NbHLI9W; Soares fetal heart NbHHI9W; Human Pituitary,_ _ re-excision; stomach cancer (human); Human Stomach; Aorta endothelial cells + TNF-a;
Human Tonsils, Lib 2; Glioblastoma; CHME Cell Line,untreated; Human Primary Breast Cancer Reexcision; Human Hippocampus; HM 1; Human adult (K.Okubo);
NCI CGAP_Co3; NCI CGAP_Co9; Human umbilical vein endothelial cells, IL-4 induced; Human Adipose; Human Adrenal Gland Tumor; Human Whole Six Week Old Embryo; Stratagene lung (#937210); Fetal Heart; Stratagene hNT neuron (#937233); Resting T-Cell Library,Il; Human T-Cell Lymphoma; Colon Tumor II;
Human Placenta; human tonsils; Human B Cell Lymphoma; Human Bone Marrow, treated; T cell helper 1I and Soares infant brain I NIB.
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 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 1581 of SEQ ID
NO:55, b is an integer of 15 to 1595, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:55, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 46 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Human Rhabdomyosarcoma.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:56 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 939 of SEQ ID
N0:56, b is an integer of 15 to 953, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:56, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 47 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Glioblastoma; NCI CGAP_Co3; Human Ovarian Cancer Reexcision; Anergic T-cell; and Human B Cell Lymphoma.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:57 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 comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1238 of SEQ ID
N0:57, b is an integer of 15 to 1252, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:57, 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 tissueslcDNA libraries: Soares infant brain 1NIB and to a lesser extent in Soares retina N2b4HR; Soares melanocyte 2NbHM; Soares placenta Nb2HP; Healing groin wound, 6.5 hours post incision; Human Prostate; Macrophage-oxLDL; Human Hypothalmus,Schizophrenia; NCI CGAP_GCB1; Anergic T-cell; Human Whole 6 Week Old Embryo (II), subt; Bone Cancer; Hypothalamus; Human (Caco-2) cell line, adenocarcinoma, colon, remake; H. cerebellum, Enzyme subtracted; Salivary Gland;
Human Frontal Cortex, Schizophrenia; Human Ovary; wilm's tumor; Human Adult Small Intestine; L428; Human Hippocampus; Synovial Fibroblasts (control);
Human Gall Bladder; Human Testes Tumor; Stratagene colon (#937204); Stratagene fetal 8i retina 937202; Stratagene endothelial cell 937223; H. Frontal cortex,epileptic,re-excision; Soares fetal lung_NbHLI9W; Human Synovial Sarcoma; Bone marrow;
human tonsils; Human Osteoclastoma; Spleen, Chronic lymphocytic leukemia; T
cell helper ll; Soares fetal liver spleen 1NFLS and Primary Dendritic Cells, lib 1.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:58 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. 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 1035 of SEQ ID
N0:58, b is an integer of 15 to 1049, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:58, and where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 49 It has been discovered that this gene is expressed primarily in the following tissues/cDNA libraries: Brain frontal cortex and Anergic T-cell.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID N0:59 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 1609 of SEQ ID
N0:59, b is an integer of 1~ to 1623, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID N0:59, and where b is greater than or equal to a + 14.

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d ~ z ~ o ~ a, o a ~ o x d x z Table I summarizes the information corresponding to each "Gene No." described above. The nucleotide sequence identified as "NT SEQ ID NO:X" was assembled from partially homologous ("overlapping") sequences obtained from the "cDNA
clone ID" identified in Table 1 and, in some cases, from additional related DNA
clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits contain multiple different clones corresponding to the same gene. "Vector"
refers to the type of vector contained in the cDNA Clone ID.
"Total NT Seq." refers to the total number of nucleotides in the contig identified by "Gene No." The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as "5' NT of Clone Seq."
and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ
ID
NO:X of the putative start codon (methionine) is identified as "5' NT of Start Codon."
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 amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep." The predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as "Predicted First AA of Secreted Portion." Finally, the amino acid position of 5EQ ID NO:Y of the last amino acid in the open reading frame is identified as "Last AA of ORF."
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID
NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. 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 errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide 2~ sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods.
The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID
NO: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 or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants andlor 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 WO 00/61629 PCTlLTS00/09071 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 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage points) for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are 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 appreciate, 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 within 5 residues (i.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 secreted 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 desribed below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polvnucleotide and Polvpeptide 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 WO 00!61629 PCT/US00/09071 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 l, the ORF (open reading frame), or any fragment specified 1_5 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=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=I, 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 IOJ
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 matchedlaligned 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 NO:Y) or to the amino acid sequence encoded by cDNA contained in a deposited clone can be determined conventionally using known computer programs. A preferred method for determing the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp.
App.
Biosci. 6:237-245(1990)). 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=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, 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 N- 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 N-and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- 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 N- and C-terminal of the subject sequence, which are not matchedlaligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal .
residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N-and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.
If the remaining 90 residues were perfectly matched the final percent identity would be 90%. 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 protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:

(1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-(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-la. They used random mutagenesis to generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino 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-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described 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 IS 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 Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, 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, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-(1967); Robbins et al., Diabetes 36: 838-845 (1987): Cleland et al., Crit.
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 amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. 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 or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, S-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
Polvnucleotide and Polvpeptide Fragments WO 00/61629 PCT/US00l09071 The present invention is also directed to polynucleotide fragments of the polynucleotides of the invention.
In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone. or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:X or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ
ID NO:Y. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment "at least 20 nt in length,"
for example, is intended to include 20 or more contiguous bases from the cDNA
sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID
NO:X. 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, 1051-1100, 11 O 1-1150, 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 IW
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 hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be "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 number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. 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 amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form.
Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
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 present invention. Moreover, polynucleotides encoding these domains are also contemplated.
Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Preferably, the polynucleotide fragments of the invention 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 protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a lli polypeptide of the invention for binding) to an antibody to the polypeptide of the invention, immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
S 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, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In another embodiment, where a ligand for a polypeptide of the invention identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize 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 blotting. See generally, Phizicky, E., et al., 1995, IVIicrobiol. 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, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID
NO:Y, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

The term "epitopes," as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitope," as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998- 4002 (1983)). 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 described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
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 I5, at least 20, at least 25, at least 30, at least 40, at least S0, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984);
Sutcliffe et al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. 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-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). 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 using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ~g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. 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 titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present 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 immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH 1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394.827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial 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

96122024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J.
Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide. 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 vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein.
Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred 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; 5,811,238;
5,830,721;
5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 ( 1999); and Lorenzo and Blasco, Biotechniques 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 DN,A 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 recombined 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 immunospecifieally 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, multispecifie, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, WO 00!61629 PCT/US00/09071 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 anticen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1, 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 immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. lmmunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portions) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the 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 combinations) 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 10-2 M. 10' M, 5 X 10-~ M, 10-3 M, 5 X 10-4 M, 10-~' M, 5 X 10-' M, 10-' M, 5 X 10-6 M, lO~GM, 5 X 10-' M, 10' M, 5 X 10-$ M, 10-$ M, 5 M, 10-9 M, 5 X 10-'° M, 10-'° M, 5 X 10-" M, 10-" M, 5 X 10-'Z
M,'°-'2 M, 5 X 10''3 M,10-'3M,SX10-'"M,10-'4M,SX10-''M,orlO-''M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. 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/ligand 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 ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serinelthreonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described 12i 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-ligand 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 96140281; U.S. Patent No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(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):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):I 153-1167 (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) (incorporated 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, WO 00/61629 PCTlUS00/09071 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.
lvlonoclonal 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" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art 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 or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. 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 CH1 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 expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and 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 Brinkman et al., J. lmmunol. Methods 182:41-50 (1995): Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. 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/GB91/01134; PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; 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., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRl 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 vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, WO 00!61629 PCT/US00/09071 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 r and 5,585,089), veneering or resurfacing (EP 592,106;
EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos.
4,444,887 and 4,716,111; and PCT publications WO 98146645, WO 98/50433, WO
98/24893, WO 98!16654, WO 96/34096, WO 96133735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced 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 non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenie 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 IS 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 98124893; WO 92/01047; WO 96134096; 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, Inc. (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., Biotechnology 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 Encodin~ 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 S' 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 WO 00/61629 PCTlUS00/09071 13i 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 andlor light S 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 recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen.
Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
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., Nature 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 appropriate biological activity can be used.
As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
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 promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.SK promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. 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., re~ion E1 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 entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 ( 1987)).
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 protein products may be important for the function of the protein.
Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing 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 particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc.
Natl.
Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
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 mycophenolic 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);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and 13; Dracopoli 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 incorporated 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 amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene.
Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse 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., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS

14=l 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other S 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 present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disull7de bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
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 14s consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 ( 1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the 1gG) may 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)). 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
232,262). 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 purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular 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, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), 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. Other peptide tags useful for purification include, but are not limited to, the "HA'' tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cel I 37:767 ( 1984)) and the "flag" tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No.
4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 1311, l l lIn 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, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e_g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
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, I3-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immurcol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99123105), 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"), WO 00/61b29 PCT/US00/09071 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 Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev. 62:119-58 (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 incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.

lmmunophenotyping The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning"
with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S.
Patent 5,985,660; and Morrison et al., Cell, 96:737-49 ( 1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual I5 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, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1%
Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1. John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-lil 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 125I) 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, WO 00/61629 PCTlUS00/09071 eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
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 125I) in the presence of increasing amounts of an unlabeled second antibody.
Therapeutic Uses The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment WO 00!61629 PCT/US00/09071 1~3 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 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.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

WO 00/61629 PCTlUS00/09071 It is preferred to use high affinity andlor potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10-Z M, 102 M, 5 X 10-~ M, 10-3 M, 5 M, 10" M, 5 X 10-' M, 10-' M, 5 X 10-6 M, 106 M, 5 X 10'' M, 10-' M, 5 X 10-$
M, 10-$ M, 5 X 10-y M, 10-9 M, 5 X 10-'° M, 10-'° M, 5 X 10-" M, 10-" M, 5 X 10-'Z M, 10-'2 M, 5 X 10-'3 M, 10-'3 M, 5 X 10'~ M, 10-'4 M, 5 X 10-'S M, and 10-'' M.
Gene Theranv 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 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
( 1990).
In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. 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 vitro, 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 1~6 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 administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT
Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, 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, Proc. 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 WO 00!61629 PCT/US00/09071 1~7 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 epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia 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); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993);
PCT Publication W094/12649; 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 for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).

WO 00!61629 PCT/US00/09071 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 into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth.
Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 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 recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for 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 or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral 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 Clinic 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 appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. 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/or tissue sample can be determined utilising techniques known to those of skill in the art 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 otherwise 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 intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration 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 dressing 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 Crit. 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 Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.
Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). 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, supra, vol. 2, pp.
115-138 (1984)).
Other controlled release systems are discussed in the review by 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 promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 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 receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., WO 00!61629 PCT/US00109071 Joliot 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 "Remin~ton'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 16>
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 mglkg 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 containers) 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 purposes to detect, WO 00/61629 PCTlUS00/09071 diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of 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 (112In), 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 background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
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).
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 S 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 recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

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 S 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 fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
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 WO 00/61629 PCTliIS00/09071 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 (CH1, 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 counterpart 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 purpose 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, 91311), 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 vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

WO 00/61629 PCTlUS00/09071 The polynucleotides may be joined to a vector containing a selectable marker 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 virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, 6418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
201178));
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA, l7>
pNHl6a, pNHl8A, pNH46.A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT~ available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS,pPICZ,pGAPZ, pGAPZaIph, pPlC9, pPIC3.5, PHIL-D2, PHIL-Sl, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent to the skilled artisan.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the l~ polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention, and preferably the secreted form, can 2~ also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical WO 00/61629 PCTlUS00/09071 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 processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
A
main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O,. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for Oz. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOXI) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXI
gene comprises up to approximately 30% of the total soluble protein in Pichia pastor-is. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.F., etal., Nucl. Acids Res. 15:3859-76 (1987).

Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOXI
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), andlor to include genetic material (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 96129411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-(1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated 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, 310:105-111 (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-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, b Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in Veneral. 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 incorporated 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.

WO 00/61629 PCTlUS00/09071 lAl 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 material from a population of pegylated protein molecules.
Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers.
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 S 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 homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (d.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 andlor 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 linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper 18-l polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 081446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric 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 FIagO fusion proteins of the invention and anti-Flag~ antibody.

WO 00/61629 PCT/US00l09071 18i The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,92, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, IS 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 incorporated by reference in its entirety).
In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic l86 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 reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Uses of the Polvnucleotides Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered 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 polymorphisms), are presently available. Each polynucleotide of the present 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. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:X will yield an amplified fragment.

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, prescreening 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 by are preferred. 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 l88 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 polymorphism is identified, this polymorphic 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 during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general 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 worse 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 methods) 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 incorporated 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.Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. I-I. Berg, S. K.
Kim, B.
Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more 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_m) by 8°-20° C, vs. 4°-16° C for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
The present invention is useful for detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

Pathological cell proliferative diseases, disorders, and/or conditions 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., Wiernik, P. H. et al. eds., 161-182 (1985)).
Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism.
(Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types.
(Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra) For example, c-myc expression is highly amplified in the non-lymphocytic leukemia 1~ cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, 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 or 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 present invention's usefulness would not be limited to treatment of proliferative diseases, disorders, and/or conditions of hematopoietic cells and tissues, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Anti sense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRCPress, Boca S 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); Oligodeoxy-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 hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treator prevent disease.
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
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 surfactant,urine,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, are used in forensic biology to identify individuals. (Erlich, H., PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an 19~
identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA
probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
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 oligomers for attachment to a "gene chip" or other support, to raise anti-DNA
antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polvpe"Ptides 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 (1121n), 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 incorporated 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, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. 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 lnc. (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 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.
Moreover, polypeptides of the present invention can be used to treat, prevent, andlor diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

WO 00/61629 PCTlUS00/09071 Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction 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 Theraw Methods Another aspect of the present invention is to gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the invention that operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, W090/11092, which is herein incorporated by reference.
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. 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, 53:107-1112 (1993); Ferrantini et al., J.
Immunology 153: 4604-4615 ( 1994); Kaido, T., et al., Int. J. Cancer 60: 221-(1995); Ogura 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-(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, pXT 1 and pSG available from Stratagene;
pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRclCMV2 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 Herpes 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, 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, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
For the nakednucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mglkg body weight to about 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 ~ 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 or 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 (Felgner 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[ 1-2,3-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, Felgner et al., Proc. Natl Acad. Sci. USA
, 84:?413-7416 (1987), which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).

WO 00/61629 PCTlUS00/09071 Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication NO: WO

(which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., Felgner et al., Proc.
Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated 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
starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying SO mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred.
The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology , 101:512-527 (1983), which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated.
SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. 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 Tris/NaCI, 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 Caz+-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 (Epoch 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.

20s 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 l: 1.
U.S. Patent NO: 5,676,954 (which is herein incorporated 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 9419469 (which are herein incorporated 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+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy , 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPOa precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding polypeptides of the invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express polypeptides of the invention.
In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotides of the invention contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses polypeptides of the invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA
into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir.
Dis., 109:233-238 ( 1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-I-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434 (1991);
Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993);
Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther., 4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al., Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 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 adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the present invention.
Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or L1 through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, Curr.
Topics in Microbiol. Immunol., 158:97 (1992)). It is also one 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.
Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding the polypeptide sequence of interest) via homologous recombination (see, e.g., U.S.
Patent NO: x,641,670, issued June 24, 1997; International Publication NO: WO
96129411, published September 26, 1996; International Publication NO: WO
94112650, 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). This method involves the activation of a gene which is present 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 promoter.
Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5' end of the desired endogenous WO 00/61629 PCTlUS00/09071 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 S' 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-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
The polynucleotides encoding polypeptides of the present invention may be administered along with other polynucleotides encoding other angiongenic proteins.
Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
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 S' 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 minipumps), 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 cornplexed 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 , 189:11277-11281 (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 present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly Biological Activities The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities.
If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
Immune Activity The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer or some autoimmune diseases, disorders,and/or conditions, acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
A polynucleotides or polypeptides, or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. A polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treator prevent those diseases, disorders, andlor conditions associated with a decrease in certain (or many) types hematopoietic cells.
Examples of immunologic deficiency syndromes include, but are not limited to: blood protein diseases, disorders, and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies), blood platelet diseases, disorders, and/or conditions (e.g.
thrombocytopenia), or 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 or prevention 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, preventing, and/or diagnosing autoimmune diseases, disorders, and/or conditions. Many autoimmune diseases, disorders, and/or conditions 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 diseases, disorders, and/or conditions.
Examples of autoimmune diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed or detected by the present invention include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmic, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, 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, prevented, and/or diagnosed by 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, prevent, and/or diagnose organ rejection or graft-s 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, oragonists 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, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, 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.) Hyperproliferative Disorders A polynucleotides or polypeptides, or agonists or antagonists of the invention can be used to treat, prevent, and/or diagnose hyperproliferative diseases, disorders, including neoplasms. A polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative diseases, disorders, and/or conditions can be treated, prevented, and/or diagnosed. 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 treating, preventing, and/or diagnosing hyperproliferative diseases, disorders, and/or conditions, such as a chemotherapeutic agent.
Examples of hyperproliferative diseases, disorders, and/or conditions that can be treated, prevented, and/or diagnosed by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the:colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative diseases, disorders. and/or conditions can also be treated, prevented, and/or diagnosed by a polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders, and/or conditions include, but are not limited to:

WO 00/61629 PCTlUS00109071 hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/or conditions, paraproteinemias, purpura, sarcoidosis, 5ezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
S 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 or preventing cell proliferative diseases, disorders, and/or conditions by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating or preventing cell-proliferative diseases, disorders, and/or conditions in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA
construct encoding the poynucleotides of the present invention is inserted 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 incorporated by reference).
In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. 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 oncogenic 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 messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
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, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 ( 1985) or other efficient DNA
delivery systems (Pates 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 S abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
By "cell proliferative 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 present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating, preventing, and/or diagnosing one or more of the described diseases, disorders, and/or conditions. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. 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, preventing, and/or diagnosing a subject having or developing cell proliferative and/or differentiation diseases, disorders, andlor conditions 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 diseases, disorders, andlor conditions 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 SX10-6M, 10-6M, SX10-'M, 10-'M, 8M, 10-8M, SX10-9M, 10-9M, SX10-'°M, 10-'°M, SX10-"M, 10-"M, SX10-'zM, 10-'ZM, SX 10-'3M, 10-'3M, SX 10-'''M, 10-'4M, SX 10-''M, and 10-'SM.
Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. 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 directly, or 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 proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 ( 1998), which is hereby incorporated by reference).

Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-(1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-(1998), IntJTissue React;20(1):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 proliferative cells or tissues.
Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
In another 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, hydrophilic, ionic and/or covalent interactions.

WO 00!61629 PCT/US00/09071 Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
Cardiovascular Disorders Polynucleotides or polypeptides, or agonists or antagonists of the invention may be used to treat, prevent, and/or diagnose cardiovascular diseases, disorders, and/or conditions, including peripheral artery disease, such as limb ischemia.
Cardiovascular diseases, disorders, and/or conditions include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septa!
defects, such as aortopulmonary septa! defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septa! defects.
Cardiovascular diseases, disorders, and/or conditions also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular WO 00!61629 PCT/US00/09071 hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septa! rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atria! fibrillation, atria! flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atria!
tachycardia, ectopic functional 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, mural valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

22s 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 diseases, disorders, and/or conditions, 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 diseases, disorders, and/or conditions 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-Angio~enesis Activitx 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 serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye diseases, disorders, and/or conditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995);
Auerbach et al., J. Microvasc. Res. 29:401-411 ( 1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 ( 1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). 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, disorders, and/or conditions associated with neovascularization by administration of the polynucleotides andlor polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating, preventing, and/or diagnosing 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 or prevent a cancer or tumor.
Cancers which may be treated, prevented, and/or diagnosed with polynucleotides, polypeptides, antagonists andlor agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas;
glioblastoma;
Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer;
advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superFicial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary 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 andlor agonists may be useful in treating, preventing, andlor diagnosing other diseases, disorders, and/or conditions, besides cancers, which involve angiogenesis. These diseases, disorders, and/or conditions include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations;
ischemic limb angiogenesis; Osler-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, preventing, and/or diagnosing 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 after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.
As noted above, the present invention also provides methods for treating, preventing, and/or S diagnosing neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
Moreover, Ocular diseases, disorders, and/or conditions associated with neovascularization which can be treated, prevented, and/or diagnosed with the polynucleotides and polypeptides of the present invention (including agonists andlor antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal.
85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for treating or preventing 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 described 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 vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity.
Visual loss may become complete if the cornea completely opacitates. A wide variety of diseases, disorders, and/or conditions can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
Within 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 administered several times daily. Alternatively, anti-angiogenic compositions prepared as described above, may also be administered directly to the cornea.
Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further 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 treatment, 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 morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to "protect" the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization.
In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply.
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 or preventing 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 or prevent 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 released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating or preventing proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
Within another aspect of the present invention, methods are provided for treating or preventing retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.
Additionally, diseases, disorders, and/or conditions which can be treated, prevented, and/or diagnosed with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, diseases, disorders, and/or conditions and/or states, which can be treated, prevented, and/or diagnosed 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 pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control 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 after" method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be 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 present 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 23s 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 further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence 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 margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical 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, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-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 (V1) 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 S 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, prevented, and/or diagnosed by the polynucleotides or polypeptides and/or antagonists or agonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p~3 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 diseases.
disorders, and/or conditions (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 herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic IO 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, prevented or diagnosed 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 sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic WO 00!61629 PCT/U500/09071 cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented, and/or diagnosed by the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative diseases, disorders, and/or conditions (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune diseases, disorders, and/or conditions (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, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

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 or 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, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from 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 anti metabolites. Polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to promote dermal reestablishment 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-epithelialization 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, cubs graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-24!
Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The polynucleotides or polypeptides, and/or agonists or antagonists of the invention, can be used to promote 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 or antagonists of the invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
The polynucleotides or polypeptides, andlor agonists or antagonists of the invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides, andlor agonists or antagonists of the invention, may have a cytoprotective effect on the small intestine mucosa. The polynucleotides or polypeptides, andlor agonists or antagonists of the invention, may also stimulate healing of mucositis (mouth ulcers) that result 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 skin 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 WO 00/61629 PCTlUS00/09071 2~t2 polynucleotides or poly peptides, and/or aaonists 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 reepithelialization 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 or antagonists of the invention, could be used to treat diseases associate with the under expression of the polynucleotides of the invention.
Moreover, the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, could be used to prevent and heal damage to the lungs due to various pathological states. A growth factor such as the polynucleotides or polypeptides, and/or agonists or antagonists of the invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated, prevented, and/or diagnosed 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 respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
The polynucleotides or polypeptides, andlor 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 tetraholoride 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 mellitus. 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, andlor 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 24=1 Nervous system diseases, disorders, and/or conditions, which can be treated, prevented, and/or diagnosed 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, disorders, and/or conditions which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated, prevented, and/or diagnosed 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 herpes 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, disorders, and/or conditions, 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 R'O 00/61629 PCT/US00/09071 24~
acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus 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, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are 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, prevent, and/or diagnose neural cell injury associated with cerebral hypoxia. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral ischemia. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose neural cell injury associated with cerebral infarction. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose 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, prevent, and/or diagnose neural cell injury associated with a heart attack.

2~6 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 survival 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 acetylcholinesterase 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.
1~ 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 diseases, disorders, and/or conditions that may be treated, prevented, and/or diagnosed according to the invention include, but are not limited to, diseases, disorders, andlor conditions 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 24?
system, as well as diseases, disorders, and/or conditions 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, prevent, and/or diagnose infectious agents.
For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may betreated, prevented, and/or diagnosed. 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, prevented, and/or diagnosed 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), WO 00/61629 PCTlUS00/09071 2~t8 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, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose:
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, prevent, and/or diagnose AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated, prevented, and/or diagnosed 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, DEMANDES OU BREVETS VOLUMINEUX
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Claims (23)

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 from the group consisting of:
(a) a polynucleotide fragment of SEQ ID NO:X or 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 by 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;

(i) 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.

2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein.

3. The isolated nucleic acid molecule of claim 1, wherein 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.

11. 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 NO:Y or the encoded sequence included in ATCC Deposit No: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 11, 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 11.

14. A recombinant host cell that expresses the isolated polypeptide of claim 11.

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 11 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 11 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 11 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 protein in the supernatant having the activity.

23. The product produced by the method of claim 20.