JP2003501007A - 50 human secreted proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel human secreted proteins and isolated nucleic acids comprising the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies for producing human secreted proteins, as well as recombinant methods for producing such human secreted proteins. The present invention further relates to diagnostic and therapeutic methods that are useful for diagnosing and treating disorders relating to these novel human secreted proteins.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to newly identified polynucleotides and polypeptides encoded by these polynucleotides, the use of such polynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION Unlike bacteria, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotic cells are subdivided by the membrane into many functionally distinct compartments. Each membrane-separated compartment, or organelle, contains different proteins that are essential for the function of that organelle. Cells use "sorting signals," which are amino acid motifs located inside proteins, to target the protein to specific cellular organelles.

One type of selection signal, called a signal sequence, signal peptide, or leader sequence, directs a class of proteins to the organelle called the endoplasmic reticulum (ER). The ER separates membrane-bound 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. Where Golgi is
The protein is distributed in vesicles, including secretory vesicles, cell membranes, lysosomes, and other organelles.

Proteins targeted to the ER by signal sequences can be released into the extracellular space as secreted proteins. 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 an evoked signal. In the latter case, the protein is stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly,
Proteins that reside on cell membranes are also “linkers” that hold the protein to the membrane
It can be secreted into the extracellular space by proteolytic cleavage of.

Despite significant advances in recent years, only a few genes encoding human secretory proteins have been identified. These secreted proteins include commercially valuable human insulin, interferon, factor VIII, human growth hormone, tissue plasminogen activator, and erythropoietin. Thus, given the widespread role of secreted proteins in human physiology, there is a need to identify and characterize new human secreted proteins and the genes that encode them. This finding makes it possible to detect, treat and prevent medical diseases, disorders and / or conditions by using secreted proteins or the genes encoding them.

SUMMARY OF THE INVENTION The present invention relates to novel polynucleotides and their encoded polypeptides. Further, the 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 associated with the polypeptides and polynucleotides, and therapeutic methods for treating such disorders and conditions. The invention further relates to screening methods for identifying binding partners of this polypeptide.

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 a substance taken from its original environment (eg, the natural environment if it is naturally occurring), and thus It has been altered "by the hand of man" from its natural state. For example, an isolated polynucleotide can be part of a vector or composition of matter, or contained within a cell, and still be "isolated." This is because the vector, composition of matter, or particular cell is not the natural environment for the polynucleotide. The term "isolated" refers to a genomic library or cDNA
Libraries, whole cells or mRNA preparations, genomic DNA preparations (
Sheared whole cell genomic DNA preparations, including those separated by electrophoresis and transferred to a blot, or other compositions that the art does not exhibit the distinguishing characteristics of the polynucleotides / sequences of the invention. I don't say anything.

In the present invention, “secreted” proteins are proteins that can be directed as a result of a signal sequence in the ER, secretory vesicles, or extracellular space, as well as those that do not necessarily contain a signal sequence but are released into the extracellular space. Refers to protein. If a secreted protein is released into the extracellular space, it may undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In a particular embodiment, the polynucleotide of the invention is at least 15, at least 30, at least 50, at least 100, at least 125, at least 500 or at least 1000 contiguous nucleotides, but 300 kb in length, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.
It is 5 kb, 5 kb, 2.5 kb, 2.0 kb or 1 kb or less. In a further embodiment, the polynucleotide of the invention, as disclosed herein, comprises part of the coding sequence but not all or part of any intron. In another embodiment, the polynucleotide comprising the coding sequence does not include the coding sequence of a genomic flanking gene (ie, 5'or 3'to the gene of interest in the genome). In another embodiment, the polynucleotide of the invention is 10
00, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
Does not contain more than 2 or 1 genomic flanking gene coding sequences.

As used herein, “polynucleotide” refers to SEQ ID NO: X (SE
Q ID NO: X) refers to a molecule having the nucleic acid sequence contained in it or a cDNA contained in a clone deposited with the ATCC. For example, the polynucleotide includes 5'and 3'untranslated sequences, a coding region with or without a signal sequence, a nucleotide sequence of a full-length cDNA sequence containing a secretory protein coding region, and fragments, epitopes, domains of this nucleic acid sequence. , And variants. Further, as used herein, "polypeptide", as broadly defined, refers to a molecule having a translated amino acid sequence derived from a polynucleotide.

In the present invention, the full length sequence identified as SEQ ID NO: X was often generated by duplicating sequences contained in multiple clones (contig analysis).
A representative clone containing all or most of the sequence for SEQ ID NO: X has been deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA clone ID (identifier) and ATCC accession number. ATCC is 10801 University
Boulevard, Manassas, Virginia 20110-22
09, USA. The ATCC deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Prosecution Purposes.

The “polynucleotide” of the present invention is also capable of hybridizing under stringent hybridization conditions to the sequence contained in SEQ ID NO: X, its complement, or the cDNA in the clone deposited with the ATCC. Contains nucleotides. “Stringent hybridization conditions” means 50% formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate),
Overnight incubation at 42 ° C. in a solution containing 0 mM sodium phosphate, pH 7.6, 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, followed by 0.1 × SSC. It refers to washing the filter at about 65 ° C. in.

Nucleic acid molecules that hybridize to the polynucleotides of the invention at lower stringency hybridization conditions are also contemplated. Changes in hybridization stringency and signal detection are primarily achieved through manipulation of formamide concentrations (lower percentages of formamide produce reduced stringency); salt conditions, or temperature. For example, lower stringency conditions are 6 × SSPE (20 × SSPE = 3M NaCl;
2M NaH ₂ PO ₄ ; 0.02M EDTA, pH 7.4), 0.5% SDS
, 30% formamide, 100 μg / ml salmon sperm blocking DNA, incubated overnight at 37 ° C .; then 1 × SSPE, 0.1% S
Includes washing with DS at 50 ° C. Furthermore, in order to achieve even lower stringency, the washes performed after stringent hybridization can be performed at higher salt concentrations (eg 5 × SSC).

Note that the changes in the above conditions can be achieved by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Representative 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 above hybridization conditions due to compatibility issues.

Of course, a poly A + sequence (eg, any 3 ′ end poly A + region of the cDNA shown in the sequence listing) or a poly hybridizing only to a complementary stretch of T (or U) residues. Nucleotides are not included in the definition of "polynucleotide." Because such a polynucleotide hybridizes to any nucleic acid molecule containing a poly (A) stretch or its complement (eg, virtually any double-stranded cDNA clone generated using oligo dT as a primer). Because it soybeans.

The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DN.
It may be A or modified RNA or modified DNA. For example, a polynucleotide is a DNA that is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded regions.
Single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, single-stranded, or more typically double-stranded or a mixture of single-stranded and double-stranded regions. It can be composed of hybrid molecules, including possible DNA and RNA. In addition, the polynucleotide may be composed of triple-stranded regions, including RNA or DNA or both RNA and DNA. Polynucleotides may also include one or more modified bases or DNA or RNA backbones that have been modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "
A "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

The polypeptides of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. May be included. The polypeptide is either by a natural process, such as post-translational processing, or by chemical modification techniques well known in the art.
It can be modified. Such modifications are based on basic texts and more detailed research papers,
And well described in many research literatures. 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 can be branched, eg, as a result of ubiquitination, and polypeptides can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. As a modification,
Acetylation, acylation, ADP-ribosylation, amidation, covalent binding of flavins,
Covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative, phosphatidylinositol
covalent bond, cross-linking, cyclization, disulfide bond formation,
Demethylation, covalent crosslink formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Pegylatio
n), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (For example, PROTEI
NS-STRUCTURE AND MOLECULAR PROPERTIE
S. Second Edition, T.S. E. Creighton, W.C. H. Freeman and
Company, New York (1993); POSTTRANSLATI
ONAL COVALENT MODIFICATION OF PROTEI
NS, B. C. Edited by Johnson, Academic Press, New Y
ork, pp. 1-12 (1983); Seifter et al., Meth Enzymo.
l 182: 626-646 (1990); Rattan et al., Ann NY A.
cad Sci 663: 48-62 (1992)).

“SEQ ID NO: X (SEQ ID NO: X)” refers to a polynucleotide sequence, while “SEQ ID NO: Y (SEQ ID NO: Y)” refers to a polypeptide sequence, both sequences being , Identified by the integers specified in Table 1.

A “polypeptide having biological activity” refers to a polypeptide of the invention (mature form (Including), but not necessarily the same. If a dose dependence is present, it need not be identical to that of this polypeptide, but rather is substantially similar to that of a given activity when compared to a polypeptide of the invention ( That is, the candidate polypeptide is
Shows greater activity or about 1/25 compared to the polypeptides of the invention
More, and preferably about 1/10 or more of activity, and most preferably about 1/3
The above activity is shown).

Many proteins (and translated DNA sequences) contain regions whose amino acid composition is highly offset to a small subset of available residues. For example, the transmembrane domain and signal peptide, which is also transmembrane, are typically
Includes long stretches dominated by leucine (L), valine (V), alanine (A), and isoleucine (I). The polyadenosine region at the end of the cDNA (poly A
) Appears as polylysine (poly-K) in forward translation, and polyphenylalanine (poly-F) when the reverse complement is translated. These areas are often referred to as "lower complexity" areas.

Such regions may allow a database similarity search program such as BLAST to find high scoring sequence matches that do not imply true homology. Most weight matrices (used to score alignments produced by BLAST) are commonly found in groups of hydrophobic amino acids (L, V and I) found in certain low complexity regions. To a match between either,
The problem is exacerbated by the fact that it gives high scores almost as well as high scores for exact matches.

To correct this, BLASTX. 2 (Version 2.0a5MP-
WashU) "masks" lower complexity in certain sequences.
) ”Using two filters (“ seg ”and“ xnu ”). These filters analyze the sequences for such regions and create new sequences in which amino acids in the lower complexity regions are replaced by the letter "X". This is then the input sequence to the BLASTX program (sometimes referred to herein as "
"Query" and / or "Q"). This regimen helps ensure that high-scoring matches represent true homology, while negative consequences in that the BLASTX program uses a query sequence masked by a filter to draw the alignment. Exists.

[0024] Thus, the stretch of "X" in the alignment shown in the following application does not necessarily indicate that either the underlined DNA sequence or the translated protein sequence is unknown or unspecified. And also the presence of such a stretch is not meant to indicate that this sequence is or is not identical to the sequences disclosed in the alignment of the present invention. Such a stretch is defined by the BLASTX program as defined above.
Due to the detection of regions of low complexity, it may simply be shown to mask the amino acids in that region. In all cases, the specification, sequence listing (Table 1), and / or
Alternatively, the reference sequence shown in the deposited clone (sometimes referred to herein as “Subject”, “Sbjct”, and / or “S”) is critical to the practice of the invention. It should not be construed as limiting the invention to the partial sequences shown in the alignment, unless it is in the form and is shown in detail elsewhere in the specification.

Polynucleotides and Polypeptides of the Invention (Characteristics of the protein encoded by Gene No. 1) This gene was found to be expressed primarily in the following tissue / cDNA library: neutrophils control. .

Many polynucleotide sequences (eg, 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 the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 587 of SEQ ID NO: 11, and b is 15 to 601.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 11, and b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 2) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1106 of SEQ ID NO: 12, and b is 15 to 11
Is an integer of 20, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 12, and where b is a + 14 or more) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 3) It was discovered that this gene is mainly expressed in neutrophils control.

A preferred epitope is the residue in sequence number 63: Arg-17 to Asp.
An epitope including the sequence shown as -22 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 741 of SEQ ID NO: 13, and b is 15 to 755.
, Both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 13, and b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 4) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1231 of SEQ ID NO: 14, and b is 15 to 12
Is an integer of 45, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 14, and b is a + 14 or greater) and one or more polynucleotides comprising It

Characterization of the Protein Encoded by Gene No: 5 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Soares placenta Nb2HP, and to a lesser extent, Human Pituitary. , Subtraced; Syn
optical hypoxia-RSF substructured; Soares_
multiple_sclerosis_2NbHMSP; Soares_se
nescent_fibroblasts_NbHSF; Human Pitu
itary, subix IX; Hemangiopericytoma; So
ares breath 2NbHBst; Stratagene lung
(# 937210); Soares breath 3NbHBst; Prim
ary Dendritic Cells, lib 1; Soares inf
ant brain 1 NIB; Human kidney Cortex, s
humanized; Human Kidney Tumor; Rectum
normal; Soares_placenta_8to9weeks_2Nb
HP8to9W; Weizmann Olefactory Epitheliu
m; Aorta endotherial cells + TNF-a; So
ares retina N2b4HR; Stratagene hNT ne
uron (# 937233); Stratagene neuroepith
eium (# 937231); Soares_NhHMPu_S1; Re
sting T-Cell, re-excision; Raji Cells,
cyclohexamide treated; Soares_total_f
etus_Nb2HF8_9w; Messangial cell, frac
2; B Cell lymphoma; Human Hypothalamus
, Schizophrenia, re-excision; Healing g
roin wound, 6.5 hours post incision;
Stratagene NT2 neuronal precursor 93
7230; Human Adipose Tissue, re-excisio.
n; Jurkat T-Cell, S phase; Soares_feta
l_lung_NbHL19W; Soares_parathyroid_tu
mor_NbHPA; Spinal Cord, re-excision; H
human Infant Brain; Human Neutrophil; T
-Cell PHA 16 hrs; Human Brain, Striatu
m; Human Uterine Cancer; Human Hypotha
lmus, Schizophrenia; Liver, Hepatoma; Hu
man Activated Monocytes; Epithelial-T
NFa and INF induced; Bone Marrow Stro
mal Cell, unprocessed; Soares_fetal_hear
t_NbHH19W; Human Fetal Brain; Pancreas
Islet Cell Tumor; Human Placenta; Hum
an Neutrophil, Activated; Human Adult
Pulmonary, re-excision; Endothelial-in
reduced; neutrophils control; Human 8 W
eek Whole Embryo, and Stratagene colon
(# 937204).

Many polynucleotide sequences (eg, 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 the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 417 of SEQ ID NO: 15, and b is 15 to 431.
, Where both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 15, and b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 6) It was discovered that this gene is mainly expressed in neutrophils control.

As a preferred epitope, residues in SEQ ID NO: 66: Met-1 to Ser-
An epitope including the sequence shown as 6 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 997 of SEQ ID NO: 16, and b is 15 to 101.
1 is an integer of 1, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 16, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 7) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 555 of SEQ ID NO: 17, and b is 15 to 569.
, Both of which a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 17, and where b is a + 14 or more) are excluded. .

Characterization of Protein Encoded by Gene No: 8 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Human Cerebellum; H Amygdala.
Depression, subtracted; neurophils co
ntroll; and Soares infant brain 1 NIB.

The dominant tissue distribution in the brain indicates that the polynucleotides and polypeptides corresponding to this gene are useful in the detection, treatment and / or prevention of neurodegenerative disease states, behavioral disorders or inflammatory conditions. Representative applications are described below in the "Regeneration" and "Hyperproliferative Disorders" sections, Examples 11, 15 and 18, and elsewhere herein. Briefly, this application is used for Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, meningitis, encephalitis, demyelinating disease, peripheral neuropathy, neoplasia, trauma, birth defects, spinal cord injury, ischemia and Infarction, aneurysm,
Bleeding, schizophrenia, mania, dementia, delusions, obsessive-compulsive disorder, depression, panic disorder, learning disability, ALS, psychosis, autism, and behavioral changes (nutrition, sleep patterns, balance, and sensory disturbances) Detection), treatment and / or prophylaxis (including, but not limited to). Furthermore, elevated expression of this gene product in regions of the brain indicates that it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, recognition, homeostasis, or neuronal differentiation or survival. In addition, the protein, in addition to its use as a nutritional supplement, is also used to determine biological activity, to raise antibodies, to isolate cognate ligands or receptors as tissue markers. ,
It can be used to identify agents that modulate their interaction. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or targets for immunotherapy.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 18 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1015 in SEQ ID NO: 18, and b is 15 to 10
Is an integer of 29, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 18, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 9) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1209 of SEQ ID NO: 19, and b is 15 to 12
Is an integer of 23, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 19, and b is a + 14 or more) are excluded. It

(Characteristics of Protein Encoded by Gene No. 10) It was discovered that this gene is mainly expressed in neutrophils control.

A preferred epitope is represented by the residues: Thr-38 to Asn in SEQ ID NO: 70.
An epitope is included that includes the sequence shown as -45.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 20 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1333 of SEQ ID NO: 20, and b is 15 to 13
47 is an integer of 47, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 20, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 11) It was discovered that this gene is mainly expressed in neutrophils control.

As a preferred epitope, residues: Met-1 to Trp- in SEQ ID NO: 71 are included.
An epitope including the sequence shown as 6 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 21 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 727 of SEQ ID NO: 21, and b is 15 to 741.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 21, and b is a + 14 or greater) and are excluded. ..

(Characteristics of Protein Encoded by Gene No. 12) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 22 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 948 of SEQ ID NO: 22, and b is 15 to 962.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 22, and b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No: 13 This gene was found to be expressed primarily in the following tissue / cDNA libraries: Human Ovarian Cancer Reexcis.
ion; and neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 23 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 929 of SEQ ID NO: 23, and b is 15 to 943.
, Both of which a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 23, and where b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No: 14 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Hemangiopericytomoma; and neutro.
pills control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 24 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1106 of SEQ ID NO: 24, and b is 15 to 11
Is an integer of 20, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 24, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 15) It was discovered that this gene is mainly expressed in neutrophils control.

A preferred epitope is the residue: Asp-22 to Cys in SEQ ID NO: 75.
An epitope including the sequence shown as -30 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 25 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 388 of SEQ ID NO: 25, and b is 15 to 402.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 25, and b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 16) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 26 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 200 of SEQ ID NO: 26, and b is 15 to 214.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 26, and b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 17) Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, accession number gi | 3135313 (
All information available via the listed accession numbers is hereby referred to as a sequence accessible through a "ORF3, splice variant [Home sapiens]", which is hereby incorporated by reference. Shared) with sequence homology. A partial alignment showing the observed homologies is presented immediately below.

[0064]

[Chemical 1] The segment of gi | 3135313, shown above as "S", is shown as SEQ ID NO: 111.

Preferred polypeptides of the present invention have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
It comprises a polypeptide having the amino acid sequence shown as SEQ ID NO: 112, which corresponds to the "Q" sequence.

This gene was found to be expressed primarily in the following tissue / cDNA libraries: neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 27 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 556 of SEQ ID NO: 27, and b is 15 to 570.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 27, and b is a + 14 or greater) and are excluded. .

Characterization of the Protein Encoded by Gene No: 18 This gene was found to be expressed primarily in the following tissue / cDNA library: neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 28 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1797 of SEQ ID NO: 28, and b is 15 to 18
1 is an integer of 11, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 28, and b is a + 14 or greater) It

Characterization of Protein Encoded by Gene No: 19 This gene was found to be expressed primarily in the following tissue / cDNA libraries: H. Male blader, adult; and neut
rophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 29 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 448 of SEQ ID NO: 29, and b is 15 to 462.
, Both of which a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 29, and where b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No. 20 This gene was found to be expressed primarily in the following tissue / cDNA library: neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 30 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 520 of SEQ ID NO: 30, and b is 15 to 534.
, Both of which a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 30, and where b is a + 14 or more) are excluded. .

(Characteristics of Protein Encoded by Gene No. 21) It was discovered that this gene is expressed mainly in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 31 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (where a is any integer between 1 and 997 of SEQ ID NO: 31 and b is between 15 and 10)
1 is an integer of 11, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 31, and b is a + 14 or more) are excluded. It

(Characteristics of Protein Encoded by Gene No. 22) It was discovered that this gene is mainly expressed in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 32 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, where a is any integer between 1-1651 of SEQ ID NO: 32 and b is 15-1
Is an integer of 665, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 32, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 23) It was discovered that this gene is mainly expressed in neutrophils control.

The disclosed cDNA is believed to reside on chromosome 22. Therefore, the polynucleotide related to the present invention is useful as a marker in the chromosome 22 linkage analysis.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 33 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 949 of SEQ ID NO: 33 and b is 15 to 96.
32 is an integer of 32, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 33, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of the Protein Encoded by Gene No. 24 It was discovered that this gene is expressed predominantly in neutrophils control.

A preferred epitope is represented by the residues in SEQ ID NO: 84: Pro-22 to Lys.
An epitope including the sequence shown as -30 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 34 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 1559 of SEQ ID NO: 34 and b is 15 to 1
An integer of 573, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 34, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of Protein Encoded by Gene No. 25) It was discovered that this gene is mainly expressed in neutrophils control.

A preferred epitope is represented by the residues: Ser-6 to Lys- in SEQ ID NO: 85.
17, Pro-37 to Glu-46, Lys-60 to Ala-66.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 35 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (where a is any integer between 1 and 1347 of SEQ ID NO: 35 and b is 15-1)
361 is an integer, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 35, and b is a + 14 or more) are excluded. It

Characterization of Protein Encoded by Gene No: 26 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Soares petal liver splene 1N.
FLS and, to a lesser extent, Bone Marrow Cell Lin
e (RS4,11); Stratagene colon (# 937204
); Osteoblasts; Primary Dendritic Cell
s, lib 1; Messangial cell, frac 2; Macro
page (GM-CSF treated); Human Gall Bl
adder; H Macrophage (GM-CSF treated),
re-excision; Soares melanocyte 2NbHM;
Activated T-Cell (12hs) / Thiouridine
labelledEco; CD34 depleted Buffy Coat
(Cord Blood), re-excision; Human Norm
al Cartage, Fraction I; Smooth Muscl
e Serum Treated, Norm; Healing grow w
sound-zero hr post-incision (control)
Human Colon Cancer, re-excision; Soar
es_multiple_sclerosis_2NbHMSP; Human
Colon, re-excision; Jurkat T-Cell, S ph
asse; Human Prostate; Human Primary Bree
ast Cancer Reexcision; Macrophage-oxL
DL; Soares_fetal_long_NbHL19W; Spinal
cord; Bone Marrow Stromal Cell, untrea
ted; Human Whole Six Week Old Embryo;
Stratagene lung (# 937210); Human Subs
tantia Nigra; Dendritic cells, pooled;
Human Neutrophil, Activated; Spleen, Ch
ionic lympholytic leukemia; Activated
T-cell (12h) / Thiouridine-re-excision
Soares_pregnant_uterus_NbHPU; Strata
gene colon (# 937204); LNCAP, different
ial expression; Leukocytes, normaliz
ed cot 5B; Human Activated T-Cells (I
I); CD34 + cells, II; Human Kidney Tumor;
Human Panoramic Langerhans; K562 + PMA
(36 hrs); LNCAP + o. 3nM R1881; Human To
nsil, Lib 3; Whole 6 Week Old Embryo; H
L-60, PMA 4H; Stratum Depression, su
bt; Human Colon Carcinoma (HCC) cell
line; Human Fetal Spleen; Human Neutro
pills, Activated, re-excision; Activate
d T-cells; Amniotic Cells-TNF induced
Breast Lymph node cDNA library; Apop
totic T-cell, re-excision; Stomach can
cer (human), re-excision; Human Amygda
la, re-excision; Stromal-Osteoclastoma
Stratagene placenta (# 937225); human
corpus colosum; Human endometrical st
local cells-treated with progresson
e; Ovarian Tumor 10-3-95; Jurkat T-cel
l G1 phase; wilm's tumor; Prostate BPH
H .; Lymph node breath Cancer; Brain F
general cortex, re-excision; Human Adul
t Small Instine; KMH2; Human Neutrop
hil; Human Chronic Synovitis; Fetal Li
ver, subtraction II; Human Feline Kidne
y; 12 Week Old Early Stage Human, II; H
human Uterine Cancer; Human Activated
T-Cells; Human Pancreas Tumor; T-Cell
PHA 24 hrs; Human Hypothalmus, Schizop
hrenia; Human umbilical vein endother
ial cells, IL-4 induced; Human Thymus;
Human Fetal Brain; Human Adrenal Glan
d Tumor; Macropage-oxLDL, re-excision
Resting T-Cell Library, II; Colon Car
cinoma; Human Tests Tumor; Colon Tumo
r II; Primary Dendritic cells, frac 2;
Human Federal Lung III; Human Adult Pul
monary, re-excision; Human Osteoclasto
ma; Human Testes; neutrophils control;
Stratagene pancreas (# 937208); Kerati
nocyte; Nine Week Old Early Stage Hum
an; and Soares placenta Nb2HP.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 36 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 1129 of SEQ ID NO: 36 and b is 15 to 1
143 is an integer of 143, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 36, and b is a + 14 or more) are excluded. It

(Characteristics of Protein Encoded by Gene No. 27) It was discovered that this gene is mainly expressed in neutrophils control.

A preferred epitope is represented by the residues: Met-1 to Thr-in SEQ ID NO: 87.
6, Cys-32 to Thr-39, Pro-56 to Ile-66.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 37 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (where a is any integer between 1 and 1099 of SEQ ID NO: 37 and b is 15-1)
Is an integer of 113, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 37, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of the Protein Encoded by Gene No. 28 This gene was found to be expressed primarily in the following tissue / cDNA library: neutrophils control.

Preferred epitopes include the residues: Trp-6 to Tyr- in SEQ ID NO: 88.
An epitope including the sequence shown as 11 is mentioned.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 38 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 481 of SEQ ID NO: 38 and b is 15 to 49.
5 is an integer of 5, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 38, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

FEATURES OF PROTEIN ENCODED BY GENE NO: 29 Using the computer algorithm BLASTX, the translation product of this gene can be found by way of non-limiting example under accession number gi | 288145 (registrations listed in the database below. All information available via numbers is hereby accessible through the sequence (which is herein referred to as the "putative ORF [Ho
described as "mo sapiens]"). A partial alignment showing the observed homologies is presented immediately below.

[0096]

[Chemical 2] The segment of gi | 288145, shown above as “S”, is SEQ ID NO: 1.
Shown as 13.

Preferred polypeptides of the present invention have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 114 corresponding to the "Q" sequence.

It was discovered that this gene is mainly expressed in neutrophils control.

When tested on sensory neuronal cells, supernatants removed from cells containing this gene activated the EGR1 assay. Therefore, this gene appears to activate sensory neuron cells through signaling pathways. Early proliferative response 1 (EGR1) is a promoter associated with specific genes that induce different tissues and cell types upon activation, leading to cell differentiation and proliferation.

Preferred epitopes include the residues in SEQ ID NO: 89: Ser-16 to Ser.
An epitope including the sequence shown as -22 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 39 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the present invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 1085 of SEQ ID NO: 39 and b is 15 to 1
Is an integer of 099, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 39, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of Protein Encoded by Gene No. 30 This gene was found to be expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 40 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 656 of SEQ ID NO: 40 and b is 15 to 67.
Is an integer of 0, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 40, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of the Protein Encoded by Gene No: 31 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Bone Caber, re-excision; CD34.
positive cells (Cord Blood); and neutr
opils control.

When tested against the Jurkat E and U937 cell lines, supernatants removed from cells containing this gene activated GAS. Therefore, this gene appears to activate T cells and myeloid cells through the Jak-STAT signaling pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes involved in the Jak-STAT pathway. This Jak-S
The TAT pathway is a large signaling pathway involved in cell differentiation and proliferation. Therefore, activation of the Jak-STATs pathway, reflected by binding of GAS elements, can be used to indicate proteins involved in cell proliferation and differentiation.

Preferred epitopes include the residues: Ala-8 to Gly-in SEQ ID NO: 91.
An epitope including the sequence shown as 20 is mentioned.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 41 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 571 of SEQ ID NO: 41 and b is 15 to 58.
5 is an integer of 5, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 41, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of the protein encoded by gene number 32) [0108] Using the computer algorithm BLASTX, the translation product of this gene is given as a non-limiting example, accession number gnl | PID | e49 in the database below.
A sequence accessible through 230 (all information available via listed accession numbers is incorporated herein by reference), which is herein referred to as "HC1.
(Described as ORF [Mus musculus] ”) was determined to share sequence homology. A partial alignment showing the observed homologies is presented immediately below.

[0109]

[Chemical 3] The segment of gnl | PID | e49230, shown above as "S", is
This is shown as SEQ ID NO: 115.

Preferred polypeptides of the present invention have the following features in the alignment shown above (where computer-introduced gaps are of course removed):
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 116 corresponding to the "Q" sequence.

This gene was found to be expressed predominantly in neutrophils control.

Preferred epitopes include the residues in SEQ ID NO: 92: Trp-60 to Trp.
-67, Gln-69 to Thr-78, Glu-97 to His-107, Gln.
An epitope including the sequence shown as -145 to Trp-152 is included.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 42 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 826 of SEQ ID NO: 42 and b is 15-84.
Is an integer of 0, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 42, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of the protein encoded by gene number 33) [0114] Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, accession number gi | 3769624 (
All information available through the listed accession numbers is hereby referred to as the "olfactory receptor [Rattus norvegicus]" accessible through a reference, which is incorporated herein by reference. ) To share sequence homology with. A partial alignment showing the observed homologies is presented immediately below.

[0115]

[Chemical 4] The segment of gi | 3769624, designated as "S" above, is SEQ ID NO: 1.
17 and SEQ ID NO: 119. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 118 and / or SEQ ID NO: 120 corresponding to the "Q" sequence.

This gene was found to be expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 43 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab, wherein a is any integer between 1 and 1576 of SEQ ID NO: 43 and b is between 15 and 1
Is an integer of 590, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 43, and where b is a + 14 or more) and one or more polynucleotides are excluded. It Characterization of Protein Encoded by Gene No. 34 This gene was found to be expressed primarily in the following tissue / cDNA libraries: Soares petal liver spleen 1N.
FLS, and to a lesser extent, Human Uterine Cancer
; And neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 44 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1-1000 of SEQ ID NO: 44, and b is 15-10.
14 is an integer of 14, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 44, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of the Protein Encoded by Gene No. 35 It was discovered that this gene is expressed predominantly in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 45 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 527 of SEQ ID NO: 45, and b is 15 to 541.
, Both of which a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 45, and where b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No. 36 It was discovered that this gene is expressed predominantly in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 46 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1277 of SEQ ID NO: 46, and b is 15 to 12
Is an integer of 91, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 46, and b is a + 14 or more) and one or more polynucleotides comprising It

(Characteristics of Protein Encoded by Gene No. 37) Using the computer algorithm BLASTX, the translation product of this gene is given as a non-limiting example, accession number gnl | PID | e13 in the database below.
08671 (all information available via listed accession numbers is incorporated herein by reference), which is referred to herein as "E
-Described as STOP protein [Rattus norvegicus])). A partial alignment showing the observed homologies is presented immediately below.

[0125]

[Chemical 5] The segment of gnl | PID | e1308671, designated above as "S", is designated as SEQ ID NO: 121. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention will have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 122 corresponding to the "Q" sequence.

This gene was found to be expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 47 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 568 of SEQ ID NO: 47, and b is 15 to 582.
, Both of which a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 47, and where b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No. 38 It was discovered that this gene is expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 48 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1205 of SEQ ID NO: 48, and b is 15 to 12
Is an integer of 19, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 48, and where b is a + 14 or more) and one or more polynucleotides comprising It

Characterization of Protein Encoded by Gene No. 39 It was discovered that this gene is expressed predominantly in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 49 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1529 of SEQ ID NO: 49, and b is 15 to 15
Is an integer of 43, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 49, and b is a + 14 or more) are excluded. It

Characterization of the Protein Encoded by Gene No. 40 This gene was found to be expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, 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 the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 803 of SEQ ID NO: 50, and b is 15 to 817.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 50, and b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No. 41 This gene was found to be expressed primarily in the following tissue / cDNA libraries: neutrophils control; and Brain.
-Medulloblastoma.

A preferred epitope is represented by the residues: Pro-23 to Gl in SEQ ID NO: 101.
An epitope is included which includes the sequence shown as y-30.

Many polynucleotide sequences (eg, 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 the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 815 of SEQ ID NO: 51, and b is 15 to 829.
, Both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 51, and b is a + 14 or more) are excluded. .

Characterization of the Protein Encoded by Gene No. 42 This gene was found to be expressed primarily in the following tissues / cDNA libraries: Neurophils control, re-excis.
ion; neutrophils control; and Weizmann
Life factory epithelium.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 52 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 564 of SEQ ID NO: 52, and b is 15 to 578.
, Both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 52, and b is a + 14 or greater) and are excluded. .

CHARACTERISTICS OF PROTEIN ENCODED BY GENE NO: 43 This gene was found to be expressed primarily in the following tissues / cDNA libraries: neutrophils control; and Human Synovial Sarcoma.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 53 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 1128 of SEQ ID NO: 53, and b is 15 to 11
42 is an integer of 42, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 53, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

FEATURES OF PROTEIN ENCODED BY GENE NO: 44 Using the computer algorithm BLASTX, the translation product of this gene is, by way of non-limiting example, accession number gi | 3493585 (
All information available through the listed accession numbers is accessible through a sequence (herein referred to as “cytoplast dynein intermediate chain 1 [Homo sapiens]”) accessible through Described as) and share sequence homology. A partial alignment showing the observed homologies is presented immediately below.

[0143]

[Chemical 6] The segment of gi | 3493585 referred to above as "S" is SEQ ID NO: 1.
Shown as 23. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 124 corresponding to the "Q" sequence.

This gene was found to be expressed primarily in the following tissue / cDNA libraries: neutrophils control; and Soare.
s infant brain 1 NIB.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 54 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 2464 of SEQ ID NO: 54, and b is 15 to 24.
Is an integer of 78, wherein both a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 54, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

(Characteristics of the protein encoded by gene No. 45) Using the computer algorithm BLASTX, the translation product of this gene is given as a non-limiting example, accession number gnl | PID | d10 in the database below.
26838 (all information available through the listed accession numbers is incorporated herein by reference), which is referred to herein as "sodium bicarbonate cotransporter 2 [Homo described as "sapiens]"). A partial alignment showing the observed homologies is presented immediately below.

[0148]

[Chemical 7] The segment of gnl | PID | d1026838, shown above as "S", is shown as SEQ ID NO: 125. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention have the following features in the alignment shown above (gaps introduced into the sequence by the computer are of course removed):
It comprises a polypeptide having the amino acid sequence shown as SEQ ID NO: 126, which corresponds to the "Q" sequence.

This gene was found to be expressed primarily in the following tissue / cDNA libraries: NTERA2, control; and neutrophil.
s control.

Many polynucleotide sequences (eg, 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 the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is any integer of 1 to 1078 of SEQ ID NO: 55, and b is 15 to 10
Is an integer of 92, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 55, and b is a + 14 or more) and one or more polynucleotides comprising It

CHARACTERISTICS OF PROTEIN ENCODED BY GENE NO: 46 This gene was found to be expressed primarily in the following tissue / cDNA library: neutrophils control.

A preferred epitope is represented by the residues: Met-1 to His in SEQ ID NO: 106.
-7, and an epitope containing a sequence shown as Pro-42 to Glu-49.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 56 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer of 1 to 616 of SEQ ID NO: 56, and b is 15 to 630.
, Both of which a and b correspond to the positions of the nucleotide residues set forth in SEQ ID NO: 56, and where b is a + 14 or more) are excluded. .

Characterization of Protein Encoded by Gene No. 47 This gene was found to be expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 57 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the described nucleotide sequence according to the general formula ab (
Here, a is an arbitrary integer of 1 to 2507 in SEQ ID NO: 57, and b is 15 to 25
21 is an integer of 21, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 57, and b is a + 14 or greater) and one or more polynucleotides are excluded. It

Characterization of the Protein Encoded by Gene No. 48 It was discovered that this gene is expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 58 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the described nucleotide sequence according to the general formula ab (
Here, a is an arbitrary integer of 1 to 1033 of SEQ ID NO: 58, and b is 15 to 10
47 is an integer of 47, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 58, and b is a + 14 or more) are excluded. It

Characterization of the Protein Encoded by Gene No. 49 It was discovered that this gene is expressed predominantly in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 59 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the described nucleotide sequence according to the general formula ab (
Here, a is an arbitrary integer of 1 to 1461 of SEQ ID NO: 59, and b is 15 to 14
Is an integer of 75, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 59, and b is a + 14 or more) and one or more polynucleotides are excluded. It

Characterization of Protein Encoded by Gene No. 50 It was discovered that this gene is expressed primarily in neutrophils control.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 60 and may have been publicly available prior to the idea of the invention. Preferably, such related polynucleotides are specifically excluded from the scope of the invention. Enumerating all relevant sequences is cumbersome. Therefore,
Preferably, according to the invention, the described nucleotide sequence according to the general formula ab (
Here, a is any integer of 1-1770 of SEQ ID NO: 60, and b is 15-17.
Is an integer of 84, wherein both a and b correspond to the position of the nucleotide residue set forth in SEQ ID NO: 60, and b is a + 14 or more) and one or more polynucleotides are excluded. It

[0163]

[Table 1] Table 1 summarizes the information corresponding to each "gene number" above. The nucleotide sequence identified as "nucleotide SEQ ID NO: X" is obtained from the "cDNA clone ID" identified in Table 1 and, in some cases, from additional related DNA clones. "Overlapping") sequences. Overlapping sequences are assembled into a single highly contiguous contiguous sequence (usually 3-5 overlapping sequences at each nucleotide site) and SEQ ID NO:
The final sequence identified as: X was obtained.

The cDNA clone ID was deposited on that date and is labeled "ATCC Accession No. Z
And the corresponding registration numbers listed under "Dates". 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 nucleotide sequence” refers to the total number of nucleotides in the contig identified by the “gene number”. The deposited clones may contain all or most of these sequences, which are found in SEQ ID NO: X "5 of clone sequences".
It is reflected by the position of the nucleotides designated as'nucleotide 'and'3' nucleotides of the clone sequence ". Putative start codon (methionine) SEQ
The nucleotide position of ID NO: X is "5 'nucleotide of start codon".
Identified as. Similarly, the nucleotide position of SEQ ID NO: X of the putative signal sequence is "5 'nucleotide of the first amino acid of the signal peptide".
Identified as.

[0166] The translated amino acid sequence begins with methionine and begins with "amino acid SEQ ID
Although identified as "NO: Y", other reading frames can also be readily translated using known molecular biology techniques. Polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The positions of the first and last amino acids of the SEQ ID NO: Y of the putative signal peptide are identified as “the first amino acid of the signal peptide” and “the last amino acid of the signal peptide”. The position of the putative first amino acid of SEQ ID NO: Y of the secretory part is identified as the "putative first amino acid of the secretory part." Finally, the amino acid position of SEQ ID NO: Y of the last amino acid in the open reading frame is identified as the “last amino acid of the ORF”.

SEQ ID NO: X (where X can be any polynucleotide sequence disclosed in the Sequence Listing) and translated SEQ ID NO: Y (where Y is disclosed in the Sequence Listing). It can be any polypeptide sequence) is sufficiently accurate and otherwise suitable for various applications well known in the art and as further described below. For example, SEQ ID NO: X
It is useful for designing nucleic acid hybridization probes that detect the nucleic acid sequences contained in SEQ ID NO: X or the cDNA contained in the deposited clone. These probes also hybridize to nucleic acid molecules in biological samples, thereby enabling the various forensic and diagnostic methods of the invention. Similarly, the polypeptide identified from SEQ ID NO: Y is
For example, it can be used to make antibodies that specifically bind to the proteins encoded by the cDNA clones identified in Table 1, including polypeptides and secreted proteins.

Nevertheless, the DNA sequences produced by the sequencing reaction may contain sequencing errors. This error, as a misidentified nucleotide,
Or as a nucleotide insertion or deletion in the generated DNA sequence. Incorrectly inserted or deleted nucleotides cause a frameshift in the reading frame of the deduced amino acid sequence. In these cases, even though the DNA sequence produced may be more than 99.9% identical to the actual DNA sequence (eg, a single base insertion or deletion in an open reading frame greater than 1000 bases), The deduced amino acid sequence differs from the actual amino acid sequence.

Therefore, for those applications requiring accuracy in the nucleotide or amino acid sequence, the present invention provides a generated nucleotide sequence identified as SEQ ID NO: X, and SEQ ID NO: Y as Not only the putative translated amino acid sequence identified, but also the ATC as described in Table 1.
Also provided is a sample of plasmid DNA containing the human cDNA of the invention deposited at C. The nucleotide sequence of each deposited clone can be readily determined by sequencing the deposited clone according to known methods. The deduced amino acid sequence can then be verified from such deposit. In addition, the amino acid sequence of the protein encoded by a particular clone may also be expressed by peptide sequencing or in a suitable host cell containing the deposited human cDNA, the protein collected, and the sequence determined. By determining, it can be determined directly.

The present invention also relates to the gene corresponding to SEQ ID NO: X, SEQ ID NO: Y, or the deposited clone. The corresponding gene can be isolated according to known methods using the sequence information disclosed herein. Such methods include the steps of preparing a probe or primer from the disclosed sequences and identifying or amplifying the corresponding gene from a suitable source of genomic material.

Also provided in the present invention are allelic variants, orthologs (or
and / or species homologues. SEQ ID NO: X, SEQ ID NO: Y or deposited clones using procedures known in the art and using the sequences disclosed herein or information from the deposited clones in the ATCC. Full length genes, allelic variants, splice variants, full length coding portions, orthologs, and / or species homologs of the corresponding gene can be obtained. For example, allelic variants and / or species homologs make suitable probes or primers from the sequences provided herein, and provide appropriate nucleic acid sources for the allelic variants and / or desired homologs. It can be isolated and identified by screening.

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or produced by a combination of these methods. Polypeptides that have been prepared. Means for preparing such polypeptides are well understood in the art.

The polypeptide can be in the form of a secreted protein (including the mature form) or can be part of a larger protein (eg, a fusion protein) (see below). It is often advantageous to include additional amino acid sequences including secretory or leader sequences, prosequences, sequences that aid in purification (eg, multiple histidine residues), or additional sequences for stability during recombinant production. Is.

The polypeptides of the present invention are preferably provided in isolated form and are preferably substantially purified. Recombinantly produced versions of polypeptides (including secreted polypeptides) may be derived from techniques described herein or otherwise known in the art (eg, Smith and John).
Son, Gene 67: 31-40 (1988) by a one-step method). The polypeptide of the present invention also comprises
For example, using antibodies described herein or other techniques known in the art, such as the antibodies of the invention raised against secreted proteins, natural sources, synthetic sources or It can be purified from recombinant sources.

The present invention provides a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: X, and / or the cDNA contained in ATCC Deposit Z. The present invention also provides a polypeptide comprising or consisting of the polypeptide sequence of SEQ ID NO: Y and / or the polypeptide encoded by the cDNA contained in ATCC Deposit Z. SEQ ID
NO: Y polypeptide sequence and / or cD contained in ATCC Deposit Z
Polynucleotides that encode polypeptides that include or consist of the polypeptide sequences encoded by NA are also included in the invention.

Signal Sequence The present invention also includes the mature form of the polypeptide having the polypeptide sequence of SEQ ID NO: Y and / or the polypeptide sequence encoded by the cDNA in the deposited clone. Also included in the invention are polynucleotides that encode the mature form (eg, such as the polynucleotide sequence of SEQ ID NO: X, and / or the polynucleotide sequence contained in the cDNA of the deposited clone). According to the signal hypothesis, once the transport of the growing protein chain across the rough endoplasmic reticulum is initiated, the protein secreted by mammalian cells is
It has a signal sequence or secretory leader sequence that is cleaved from the mature protein. Even most mammalian and insect cells cleave secreted proteins with the same specificity. However, in some cases the cleavage of the secreted protein is completely
Not homogenous, this results in more than one mature species of this protein. Furthermore, it has long been known that the cleavage specificity of secreted proteins is ultimately determined by the primary structure of the intact protein (ie, it is unique to the amino acid sequence of this polypeptide).

Methods are available for predicting whether a protein has a signal sequence, as well as a breakpoint for that sequence. For example, McGeoch, Vi
rus Res. 3: 271-286 (1985) uses information from a short N-terminal charged region followed by an uncharged region of the intact (uncleaved) protein. von Heinje, Nucleic Acids Res
． 14: 4683-4690 (1986) uses information from the residues surrounding the cleavage site (typically -13 to +2 residues), where +1 indicates the amino terminus of the secreted protein. . The accuracy of predicting breakpoints 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 putative breakpoint for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide is Si
A computer program called gnalP (Henrik Nielse
n et al., Protein Engineering 10: 1-6 (1997)).
Analyzed by This program predicts the location of proteins in cells based on their amino acid sequences. As part of the computer prediction of this localization, Mc
The method of Geoch and von Heinje is incorporated. Analysis of the amino acid sequences of the secreted proteins described herein by this program is shown in Table 1.
Provided the results shown in.

However, as will be appreciated by those of skill in the art, the cleavage site will occasionally vary from organism to organism and cannot be predicted with absolute certainty. Therefore, the present invention is
A secreted polypeptide is provided having the sequence shown in ID NO: Y, which has an N-terminus beginning within 5 residues of the putative breakpoint (ie, + or -5 residues). Similarly, it is also recognized that, in some cases, cleavage of the signal sequence from the secreted protein is not globally uniform, resulting in more than one secreted species. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.

Furthermore, the signal sequences identified by the above analysis may not necessarily be predictive of naturally occurring signal sequences. For example, the naturally occurring signal sequence can be further upstream from the putative signal sequence. However, the putative signal sequence likely directs the secreted protein to the ER. Nevertheless, the present invention provides SEQ ID NOS in mammalian cells (eg, COS cells as described above).
: X and / or the mature protein produced by expression of the polynucleotide sequence contained in the cDNA of the deposited clone. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.

(Polynucleotide and Polypeptide Variants) The present invention provides a modification of the polynucleotide sequence disclosed in SEQ ID NO: X, its complementary strand, and / or the cDNA sequence contained in the deposited clone. Regarding the body

The invention also includes variants of the polypeptide sequences disclosed in SEQ ID NO: Y and / or the polypeptide sequences encoded by the deposited clones.

“Variant” refers to a polynucleotide or polypeptide that differs from the polynucleotide or polypeptide of the invention, but retains their essential properties. In general, variants are wholly closely similar and, in many regions, identical to the polynucleotides or polypeptides of the invention.

The present invention also includes, for example, a sequence encoding a nucleotide of SEQ ID NO: X or its complementary strand, a sequence encoding a nucleotide contained in a deposited cDNA clone or its complementary strand, SEQ ID NO: Y. A nucleotide sequence encoding a polypeptide, a nucleotide sequence encoding a polypeptide encoded by the cDNA contained in the deposited clone, and / or a polynucleotide fragment of any of these nucleic acid molecules (eg, herein At least 80%, 85%, 90%, 95% relative to the described fragments)
, 96%, 97%, 98%, or 99% identical to a nucleic acid molecule that comprises or consists of a nucleotide sequence. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or less stringent conditions, polypeptides encoded by these polynucleotides are also encompassed by the invention.

The invention also includes, for example, the polypeptide sequence set forth in SEQ ID NO: Y, the polypeptide sequence encoded by the cDNA contained in the deposited clone, and / or any of these polypeptides. At least 80 for polypeptide fragments (eg, fragments described herein)
%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to or consisting of an amino acid sequence.

By virtue of a nucleic acid having a nucleotide sequence which is at least 95% “identical” to a reference nucleotide sequence of the present invention, the nucleotide sequence of the nucleic acid is 100 nucleotides each of the reference nucleotide sequence, the nucleotide sequence encoding a polypeptide. It is intended to be identical to a reference sequence, except that up to 5 point mutations can be included. In other words, at least 9 relative to the reference nucleotide sequence
Up to 5% of the nucleotides of the reference sequence can be deleted or replaced by another nucleotide in order to obtain a nucleic acid having a nucleotide sequence of 5% identity,
Alternatively, a large number of nucleotides, up to 5% of the total nucleotides in the reference sequence, can be inserted in the reference sequence. The query sequence can be the entire sequence shown in Table 1, the ORF (open reading frame), or any fragment identified as described herein.

As a practical matter, any particular nucleic acid molecule or polypeptide will be at least 80%, 85%, 90%, 95%, 96%, 9% relative to the nucleotide sequence of the invention.
Whether 7%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall compatibility (also referred to as global sequence alignment) between a query sequence (sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App
． Biosci. 6: 237-245 (1990)) based on the FASTDB computer program. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA
Sequences can be compared by converting U to T. The results of this global sequence alignment are expressed as percent identity (%). Preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch P.
energy = 1, Joining Penalty = 30, Randomiz
ation Group Length = 0, Cutoff Score = 1,
Gap Penalty = 5, Gap Size Penalty 0.05,
Windows Size = 500 or the length of the nucleotide sequence of interest (whichever is shorter).

If the subject sequence is shorter than the query sequence due to the 5'or 3'deletions (as opposed to due to internal deletions), a manual correction must be made to the results. This is because the FASTDB program does not consider 5'and 3'truncations of the subject sequence when calculating percent identity. For a subject sequence that is cleaved at the 5'or 3'end, the percent identity to the query sequence is the 5'and 3'of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence. Corrected by calculating the number of bases in. Whether the nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the bases outside the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5'end of the subject sequence, so the FASTDB alignment shows no match / alignment at the first 10 bases at the 5'end. 1
0 unpaired bases represent 10% of the sequence (number of bases at the non-matching 5'and 3'ends / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases were an exact match, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, so that there are no bases at the 5'or 3'of the subject sequence that are not matched / aligned with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5'or 3'bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

A polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence of the present invention, for example, the amino acid sequence of the subject polypeptide is such that the subject polypeptide sequence is each of the query amino acid sequences. It is intended to be identical to the query sequence, except that changes of up to 5 amino acids per 100 amino acids may be included. In other words, up to 5% of the amino acid residues in the sequence of interest are inserted, deleted, (indels) in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence.
Or it may be replaced by another amino acid. These modifications of the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, either individually between the residues in the reference sequence or the reference sequence. Interspersed in any of one or more consecutive groups within.

As a practical matter, any particular polypeptide may be encoded, for example, by the amino acid sequence shown in Table 1 (SEQ ID NO: Y) or by the amino acid encoded by the cDNA contained in the deposited clone. At least 80% of the sequence
, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical can be conventionally determined using known computer programs. The best overall match between the query sequence (the sequence of the invention) and the subject sequence (
The preferred method for determining (also referred to as the overall sequence alignment) is Bru
tlag et al. (Comp. App. Biosci. 6: 237-245 (1990).
)) Algorithm-based FASTDB computer program. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the above global sequence alignments are given in percent identity. FASTD
The preferred parameters used for the B amino acid alignment are: Matrix = PAM
0, k-tuple = 2, Mismatch Penalty = 1, Joini
ng Penalty = 20, Randomization Group Le
ngth = 0, Cutoff Score = 1, Window Size =
Sequence length, Gap Penalty = 5, Gap Size Penalty
= 0.05, Window Size = 500 or the length of the target amino acid sequence (whichever is shorter).

If the sequence of interest is shorter than the query sequence due to N- or C-terminal deletions (and not due to internal deletions), then manual corrections must be made to the results. This is because the FASTDB program does not consider N-terminal and C-terminal truncations of the subject sequence when calculating the overall percent identity. Regarding the target sequence shortened at the N-terminus and C-terminus, with respect to the query sequence,
Percent identity is corrected by calculating the number of residues in the query sequence that are N- and C-terminal to the subject sequence that are not matched / aligned with the corresponding subject residues as a percentage of the total bases of the query sequence. . Whether the residues are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using specific parameters to arrive at the final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only those residues N- and C-terminal to the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only the query residue is the furthest N in the subject sequence.
Located outside the terminal and C-terminal residues.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. Deletions occur at the N-terminus of the subject sequence, and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (mismatched N-terminus and C
Represents the number of residues at the end / total number of residues in the query sequence, and the result is FAST
10% is subtracted from the percent identity score calculated by the DB 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 to a 100 residue query sequence. In this case, the deletion is an internal deletion, so there are no residues at the N-terminus or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected.
No other manual correction is made for the purposes of the present invention.

Variants may include changes in the coding regions, non-coding regions, or both. Especially preferred are those that produce silent substitutions, additions or deletions,
Polynucleotide variants containing changes that do not alter the properties or activity of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Furthermore, 5 in any combination
Variants in which -10, 1-5, or 1-2 amino acids are substituted, deleted, or added are also preferred. Polynucleotide variants optimize codon expression for a variety of reasons (eg, codon expression for a particular host (codons in human mRNA are described in E.
for a preferred codon by a bacterial host such as E. coli)),
Can be generated.

Naturally occurring variants are referred to as “allelic variants” and refer to one of several alternative forms of a gene that occupy a given locus on the chromosome of an organism. (Genes II, Lewin, B., edited by John Wiley &
Sons, New York (1985)). These allelic variants are
It can vary at either the polynucleotide level and / or the polypeptide level and is 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 are
It can be made to improve or change the properties of the polypeptides of the invention. For example, 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. Ron et al. Biol. Ch
em. 268: 2984-2988 (1993) authors 3, 8, or 2
A modified KGF protein was reported that had heparin binding activity even after deletion of the 7 amino terminal amino acid residues. Similarly, interferon-gamma, after deleting 8-10 amino acid residues from the carboxy terminus of this protein,
It showed higher activity up to 0-fold (Dobeli et al., J. Biotechnol.
7: 199-216 (1988)).

Furthermore, a wealth of evidence demonstrates that variants often retain activities similar to those of naturally occurring proteins. For example, Gayle and co-workers (J. Biol. Chem 268: 22105-22111 (1
993)) performed an extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a variants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were tested at every potential amino acid position. The investigators found that "the majority of molecules can be altered with little effect on either [binding activity or biological activity]." (See summary). In fact, of the over 3,500 nucleotide sequences tested,
Only 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type.

In addition, deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide may result in modification or deletion of one or more biological functions, but not other biological functions. Activity can still be retained. For example, the ability of a deletion variant to induce and / or bind an antibody that recognizes a secreted form is the ability to remove less than the majority of the secreted form from the N-terminus or C-terminus. Seems to be retained. Whether a particular polypeptide lacking the N-terminal or C-terminal residues of a protein retains such immunogenic activity depends on conventional methods described herein, and so on. If not, it can be easily determined by a conventional method known in the art.

Accordingly, the present invention further includes polypeptide variants that exhibit substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art such that they have little effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions can be found in Bowie et al., Science 247: 1.
306-1310 (1990), where the authors point out that there are two main strategies for studying the tolerance of amino acid sequences to changes.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the course of evolution. Conserved amino acids can be identified by comparing amino acid sequences in different species. These conserved amino acids appear to be important for protein function. In contrast, the amino acid positions at which the substitutions were tolerated by natural selection indicate that these positions are not important for protein function. Thus, the positions that tolerate amino acid substitutions may be modified, yet still maintain the biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions in the cloned gene to identify regions important for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (
The introduction of one alanine mutation at each residue in the molecule) can be used. (Cuni
ngham and Wells, Science 244: 1081-1085 (
1989)). The resulting mutant molecule can then be tested for biological activity.

As the authors mention, these two strategies have revealed that the protein is surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be tolerated at particular amino acid positions in the protein. For example, amino acid residues that are mostly buried (within the tertiary structure of the protein) require non-polar side chains, but surface side chain characteristics are generally poorly conserved. In addition, tolerant conservative amino acid substitutions include the substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; the substitution of Ser and Thr for hydroxyl residues; the substitution of Asp and Glu for acidic residues; Amide residue Asn
And Gln, basic residues Lys, Arg, and His; aromatic residues Phe, Tyr, and Trp, and small-sized amino acids Ala, Ser, Thr, Met, and Gly. Including replacement.

In addition to conservative amino acid substitutions, variants of the invention include (i) substitution with one or more non-conservative amino acid residues, wherein the amino acid residue replaced is by the genetic code. It may or may not be the encoded amino acid residue, or (ii) substitution with one or more amino acid residues having a substituent, or (i
ii) fusion of the mature polypeptide with another compound (eg, a compound to increase the stability and / or solubility of the polypeptide (eg, polyethylene glycol)), or (iv) an additional amino acid (eg, IgG). Fc fusion region peptides, or fusions of polypeptides with leader or secretory sequences, or sequences which facilitate purification). Such variant polypeptides are considered to be within the skill of those in the art given the teachings herein.

For example, polypeptide variants that include amino acid substitutions of charged amino acids with other charged amino acids or neutral amino acids may have proteins with improved properties (eg, less aggregation). Can be generated. Aggregation of pharmaceutical formulations results in both reduced activity and increased clearance due to the immunogenic activity of the aggregate. (Pinckard et al., Clin. Exp. Immunol. 2: 331.
-340 (1967); Robbins et al., Diabetes 36: 838-.
845 (1987); Cleland et al., Crit. Rev. Therapeu
tic Drug Carrier Systems 10: 307-377 (
1993)).

A further embodiment of the invention comprises at least one amino acid substitution, but 50
Amino acids of the invention having an amino acid sequence of no more than amino acid substitutions, even more preferably no more than 40 amino acid substitutions, even more preferably no more than 30 amino acid substitutions, and even more preferably no more than 20 amino acid substitutions. A polypeptide comprising a sequence. Of course, in order of increasing preference, a peptide or polypeptide comprises at least one amino acid substitution, but
It is highly preferred to have an amino acid sequence which comprises the amino acid sequence of the invention not exceeding 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. In certain embodiments, the number of additions, substitutions, and / or deletions in the amino acid sequences of the invention or fragments thereof (eg, mature forms and / or other fragments described herein) is between 1 and 5, 5-10, 5-25, 5-50, 10-50, or 50
~ 150 and conservative amino acid substitutions are preferred.

Polynucleotides and Polypeptide Fragments The present invention also relates to polynucleotide fragments of the polynucleotides of the present invention.

[0208] In the present invention, "polynucleotide fragment" refers to a short polynucleotide having the following nucleic acid sequence: a polynucleotide contained in the deposited clone or encoded by the cDNA in the deposited clone. Part of the nucleic acid sequence encoding the peptide; part of the nucleic acid sequence shown in SEQ ID NO: X or their complements, or part of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: Y. is there. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, even 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. It has a length of 150 nt. For example, a fragment "at least about 20 nt in length" is intended to include 20 or more contiguous bases from the cDNA sequence contained in the deposited clone or the nucleotide sequence set forth in SEQ ID NO: X.
In this context, "about" means specifically stated values (at either or both termini a few (5, 4, 3, 2, or 1) nucleotides greater or less than this). Value) is included. These nucleotide fragments have uses, including but not limited to, use as diagnostic probes and primers, as discussed herein. Of course, larger fragments (eg 50, 150, 500, 600, 2000 nucleotides)
Is preferred.

Furthermore, as typical examples of the polynucleotide fragment of the present invention, for example,
Fragments that include or consist of sequences of the following approximate number of nucleotides are included: 1-50, 51-100, 101-150, 151-20.
0, 201-250, 251-300, 301-350, 351-400, 40
1-450, 451-500, 501-550, 551-600, 651-70
0, 701-750, 751-800, 800-850, 851-900, 90
1-950, 951-1000, 1001-1050, 1051-1100, 1
101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 13
00, 1301-1350, 1351-1400, 1401-1450, 145
1-1500, 1501-1550, 155-1600, 1601-1650
, 1651 to 1700, 1701 to 1750, 1751 to 1800, 1801
1850, 1851 to 1900, 1901 to 1950, 1951 to 2000, or 2001 to the end of SEQ ID NO: X, a complementary strand thereof, or a cDNA contained in the deposited clone. In this context, "about" means
Included are ranges specifically mentioned, and ranges larger or smaller by a few nucleotides (5, 4, 3, 2, or 1) at either or both ends. Preferably, these fragments encode a polypeptide having biological activity. More preferably, these polynucleotides may be used herein as probes or primers as discussed above. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or under lower stringency conditions are also included in the invention, as are the polypeptides encoded by these polynucleotides.

[0210] In the present invention, the "polypeptide fragment" refers to an amino acid sequence which is a part of the amino acid sequence encoded by the cDNA contained in SEQ ID NO: Y or contained in the deposited clone. A protein (polypeptide) fragment may be "free-standing," or within a larger polypeptide (wherein the fragment may be a portion or region, most preferably as a single continuous region). Form). Representative examples of polypeptide fragments of the present invention include, for example, fragments that contain or consist of the following approximate number of amino acids: 1-20, 21-40, 41-. 60, 61-80, 81-100, 10
2-120, 121-140, 141-160 or 161 to the end of the coding region. In addition, polypeptide fragments are about 20, 30, 40, 50, 6
0, 70, 80, 90, 100, 110, 120, 130, 140, or 15
It can be 0 amino acids in length. In this context, "about" means the range or value specifically mentioned and a few (5, 4, 3, 2, or 1) amino acids at either or both termini. Includes only large or small ranges or values. Polynucleotides encoding these polypeptides are also included in the invention.

Preferred polypeptide fragments include secreted proteins and mature forms. Further preferred polypeptide fragments include secreted proteins or mature forms that have a contiguous series of deleted residues from the amino terminus or the carboxy terminus or both. For example, any number of amino acids (range 1-60) can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids (range 1-30)
It may be deleted from the carboxy terminus of the secreted protein or mature form. further,
Any combination of the above amino-terminal and carboxy-terminal deletions is preferred.
Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Fragments of polypeptides and polynucleotides characterized by structural or functional domains (eg, α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn forming regions). ,
Coil and coil-forming region, hydrophilic region, hydrophobic region, α-amphipathic region, β-amphipathic region, flexible region, surface-forming region, substrate-binding region, and fragment containing high antigenicity indicator region) It is also preferable. Polypeptide fragments of SEQ ID NO: Y contained within the conserved domain are specifically contemplated by the present invention. In addition, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. A biologically active fragment is a fragment that exhibits similar activity but is not necessarily identical to that of the polypeptide of the invention. The biological activity of this fragment may include improved desired activity or reduced undesired activity. Polynucleotides encoding these polypeptide fragments are also
Included in the present invention.

Preferably, the polynucleotide fragment of the present invention encodes a polypeptide that exhibits functional activity. By a polypeptide that exhibits "functional activity" is meant a polypeptide that may exhibit one or more known functional activities associated with a full-length (complete) polypeptide of a protein of the invention. Such functional activities include biological activity, antigenicity [ability to bind (or compete with the polypeptide of the invention for binding) to antibodies to the polypeptide of the invention], immunogenicity ( Ability to generate antibodies that bind to the polypeptides of the invention), the ability to form multimers with the polypeptides of the invention, and the ability to bind receptors or ligands for the polypeptides of the invention, including but not limited to: Not done.

The functional activity of the polypeptides of the invention, and fragments, variants, derivatives and analogs thereof, can be assayed by a variety of methods.

For example, assaying for the ability of a polypeptide of the invention to bind to or compete with a full-length polypeptide of the invention for binding to an antibody, in one embodiment, various immunoassays known in the art. Can be used. Such assays include radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg colloidal gold, enzyme or Radioisotope labeling), western blot, precipitation reaction, agglutination assay (eg gel agglutination assay, hemagglutination assay), complement fixation assay,
Included, but not limited to, competitive and non-competitive assay systems using techniques such as immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. 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 immunoassays and are within the scope of the invention.

In another embodiment, if the ligand for the identified polypeptide of the invention, or the ability of the polypeptide fragment, variant, or derivative of the invention to multimerize, is assessed, binding is, eg, , Reduced and non-reduced gel chromatography, protein affinity chromatography, and affinity blotting can be assayed by means well known in the art.
Generally, Phizicky, E .; Et al., 1995, Microbiol. Rev
． 59: 94-123. In another embodiment, the physiological correlation (signal transduction) of binding of a polypeptide of the invention to its substrate can be assayed.

In addition, the assays described herein (see Examples) and other assays known in the art refer to polypeptides of the invention and fragments, variants, derivatives and analogs thereof. , Can be routinely applied to determine the ability to elicit (either in vitro or in vivo) the relevant biological activity related to that of the polypeptide of the invention. Other methods are known to those of skill in the art and are within the scope of this invention.

Epitopes and Antibodies The present invention hybridizes to an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: Y, or to the polynucleotide sequence contained in ATCC Deposit No. Z, or to the complement of the sequence of SEQ ID NO: X. An epitope of the polypeptide sequence encoded by the polynucleotide, or a polynucleotide sequence contained in ATCC Deposit No. Z under stringent or lower stringency hybridization conditions as defined above. Includes polypeptides that include or consist of epitopes of the encoded polypeptide sequence. The invention further provides a polynucleotide sequence that encodes an epitope of a polypeptide sequence of the invention (eg, the sequence disclosed in SEQ ID NO: X), a polynucleotide sequence of the complementary strand of a polynucleotide sequence that encodes an epitope of the invention, And a polynucleotide sequence that hybridizes to a complementary strand under stringent or lower stringency hybridization conditions as defined above.

The term “epitope” as used herein refers to that portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the invention includes a polypeptide that comprises an epitope as well as a polynucleotide that encodes this polypeptide. As used herein, an "immunogenic epitope" is defined as the portion of a protein that elicits an antibody response in an animal and can be any method known in the art (e.g., antibody production as described below). Method). (For example, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3.
998-4002 (1983)). As used herein,
The term "antigenic epitope" refers to a protein by which an antibody can immunospecifically bind to its antigen as measured by any method known in the art (eg, immunoassays described herein). Is defined as part of. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not necessarily require immunogenicity.

Fragments that function as epitopes can be generated by any conventional means. (For example, Houghten, Proc. Natl. Acad. Sci.
USA 82: 5131-5135 (1985) (U.S. Pat. No. 4,631,21).
1)).

In the present invention, the antigenic epitope is preferably at least 4, at least 5, at least 6, at least 7, more preferably at least 8,
At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and most It preferably comprises a sequence of between about 15 and about 30 amino acids. Preferred polypeptides are at least 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
It includes immunogenic or antigenic epitopes of amino acid residue 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 to this 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 target molecules in immunoassays. (For example, Wilson et al., Cell 37: 767-778 (19
84); Sutcliffe et al., Science 219: 660-666 (1).
983)).

Similarly, immunogenic epitopes can be used to induce antibodies, eg, according to methods well known in the art. (For example, Sutcliffe et al. (Supra); Wils
on et al. (supra); Chow et al., Proc. Natl. Acad. Sci. USA
82: 910-914; and Bittle et al., J. Am. Gen. Virol. 6
6: 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. A polypeptide comprising one or more immunogenic epitopes may be presented to elicit an antibody response against an animal system (eg rabbit or mouse) with a carrier protein (eg albumin), or the polypeptide may be If sufficiently long (at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids
It has been shown to be sufficient (e.g. in Western blotting) to raise antibodies capable of (at least) binding to the linear epitope of the denatured polypeptide.

Epitope-bearing polypeptides of the invention can be used to induce antibodies according to methods well known in the art. This method includes, but is not limited to, in vivo immunization, in vitro immunization, and phage display methods.
For example, Sutcliffe et al., Supra; Wilson et al., Supra; and Bitt.
le et al. Gen. Virol. , 66: 2347-2354 (1985). When in vivo immunization is used, animals can be immunized with free peptides; however, anti-peptide antibody titers are dependent on the macromolecule carrier (eg, keyhole limpet hemacyanin (KLH) or tetanus toxoid) peptide. Can be boosted by combining. For example, peptides containing cysteine residues can be linked to carriers using linkers such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be bound to the carrier using more common binding agents such as glutaraldehyde. Animals such as rabbits, rats, and mice use, for example, emulsions (about 100 μg of peptide or carrier protein and Freund's adjuvant or any known to stimulate an immune response) with either free peptide or carrier-bound peptide. Immunization by intraperitoneal injection (including other adjuvants) and / or intradermal injection. Several booster injections may be needed to provide a useful titer of anti-peptide antibody, for example, at intervals of about 2 weeks. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on the solid surface. Titer of anti-peptide antibody in serum from immunized animals is increased by selection of anti-peptide antibody (eg, by adsorption of peptide on solid support and elution of selected antibody according to methods well known in the art). You can

As will be appreciated by those in the art, and as discussed above, the polypeptides of the present invention comprising immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, the polypeptides of the present invention are immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C)
H2, CH3, or any combination thereof and portions thereof), resulting in a chimeric polypeptide. Such fusion proteins may facilitate purification and increase half-life in vivo. This has been shown for a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. For example, EP 394,827; Traunecker et al., Nature,
331: 84-86 (1988). Enhanced delivery of antigens across epithelial disorders to the immune system has been demonstrated for antigens bound to FcRn binding partners such as IgG or Fc fragments (eg insulin) (
See, for example, PCT publications WO 96/2024 and WO 99/04813). IgG fusion proteins having a disulfide-bonded dimer structure due to disulfide bonds of the IgG moiety are also more effective in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone Was found. For example, Fountoulakis et al. Biochem.
, 270: 3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (eg, a hemagglutinin (“HA”) tag or a flag tag) for detection and purification of the expressed polypeptide. Can assist. For example, Janknecht
The system described by et al. Allows easy purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Na.
tl. Acad. Sci. USA 88: 8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid so that the open reading frame of this gene is translationally fused to an amino-terminal tag consisting of 6 histidine residues. This tag serves as the substrate binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2 + nitriloacetic acid-agarose columns, and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

Further fusion proteins of the invention can be produced through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). D
NA shuffling can be utilized to modulate the activity of the polypeptides of the invention and this method can be used to produce polypeptides with modified activity, as well as agonists and antagonists of these polypeptides. Generally, US Pat. Nos. 5,605,793; 5,811,238; 5,83.
No. 0,721; No. 5,834,252; and No. 5,837,458,
And Patten et al., Curr. Opinion Biotechnol.
8: 724-33 (1997); Harayama, Trends Biote.
chnol. 16 (2): 76-82 (1998); Hansson et al., J. Am. M
ol. Biol. 287: 265-76 (1999); and Lorenzo
And Blasco, Biotechniques 24 (2): 308-13.
(1998), each of which is incorporated herein by reference in its entirety. In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: X and the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. D
NA shuffling involves assembling two or more DNA segments to produce changes in polynucleotide sequence by homologous or site-specific recombination. In another embodiment, the polynucleotide of the present invention or its encoded polypeptide is error-prone prior to recombination.
ne) It may be modified by subjecting it to random mutagenesis by PCR, random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention are one or more components, motifs, of one or more heterologous molecules. , Sections, parts, domains, fragments and the like.

Antibodies Furthermore, the polypeptides of the present invention may be polypeptides, polypeptide fragments of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). , Or a variant of SEQ ID NO: Y, and / or
Alternatively, an antibody that immunospecifically binds to an epitope and a T cell antigen receptor (T
CR). The antibody of the present invention is a polyclonal antibody, a monoclonal antibody,
Multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies (eg, (Including anti-Id antibodies against antibodies of the invention), and epitope binding fragments of any of the above, but not limited thereto. As used herein, the term "antibody" refers to
It refers to an immunologically active portion of an immunoglobulin molecule and an immunoglorin molecule, that is, a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the invention can be of any type of immunoglobulin molecule (eg, IgG, IgE, I
gM, IgD, IgA, and IgY), any class (eg, IgG1, I
gG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

Most preferably, the antibody is a human antigen-binding antibody fragment of the invention, which comprises Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (sc).
Fv), single chain antibodies, disulfide-bonded Fvs (sdFv) and VL or V
Examples include, but are not limited to, fragments that include any of the H domains. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or part of the following: hinge region, CH1 domain, C
H2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of variable and hinge regions, CH1 domains, CH2 domains and CH3 domains. Antibodies of the invention may be derived from any animal origin including birds and mammals. Preferably, the antibody is human, murine (eg, mouse and rabbit), donkey, ship rabbit (
ship rabbit), goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibody includes antibodies having the amino acid sequence of human immunoglobulin and is transgenic for a human immunoglobulin library or one or more human immunoglobulins and is endogenous. Antibodies isolated from animals that do not express sex immunoglobulin (as described below and, for example, in US Pat. No. 5,939,598 to Kucherlapati et al.).

Antibodies of the invention may be monospecific, bispecific, trispecific or of greater multispecificity. A multispecific antibody may be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Can be specific to. For example, PCT Publication WO 93/17715; WO 9
2/08802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat. No. 4
, 474,893, 4,714,681, 4,925,648,
No. 5,573,920, No. 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

The antibodies of the invention may be described or specified with respect to the epitopes or portions of the polypeptides of the invention to which they recognize or specifically bind. Portions of this epitope or polypeptide may be identified, for example, by N-terminal and C-terminal positions, by size in contiguous amino acid residues, as described herein, or as listed in the tables and figures. Can be transformed. Antibodies that specifically bind any epitope or polypeptide of the invention can also be excluded. Accordingly, the present invention includes antibodies that specifically bind the polypeptides of the present invention and allow for the exclusion of the polypeptides of the present invention.

The antibodies of the invention may also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the invention are included. 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 to the polypeptides of the present invention. Also included in the invention are antibodies that bind a polypeptide having (as calculated using methods known in the art and described herein). In certain embodiments, the antibodies of the invention cross-react with human, mouse, rat and / or rabbit homologs of human proteins and their corresponding epitopes. 9 for polypeptides of the invention
Less than 5%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, 6
Binds polypeptides having less than 5%, less than 60%, less than 55% and less than 50% identity (as calculated using methods known in the art and described herein) Antibodies that do not are also included in the invention. In certain embodiments, the above cross-reactivity is any monospecific antigenic or immunogenic polypeptide disclosed herein, or 2, 3, 4, 5 or more specific. It relates to a combination of antigenic and / or immunogenic polypeptides. Further included in the invention are antibodies that bind the polypeptide encoded by the polynucleotide that hybridizes to the polynucleotide of the invention under stringent hybridization conditions (as described herein). . The antibody of the present invention also comprises
Their binding affinities for the polypeptides of the invention may be described or specified. Preferred binding affinity is less than 5 × 10 ⁻² M, less than 10 ⁻² M, 5 ×
Less than 10 ^-3 M, less than 10 ^-3 M, less than 5 × 10 ^-4 M, less than 10 ^-4 M, 5 × 10 ^-5 M
Less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁶ M, less than 5 × 10 ⁻⁷ M, 1
Less than 0 ⁻⁷ M, less than 5 × 10 ⁻⁸ M, less than 10 ⁻⁸ M, less than 5 × 10 ⁻⁹ M, less than 10 ⁻⁹ M, less than 5 × 10 ⁻¹⁰ M, less than 10 ⁻¹⁰ M, 5 × Less than 10 ^-11 M, less than 10 ^-11 M,
Less than 5 × 10 ^-12 M, less than 10 ^-12 M, less than 5 × 10 ^-13 M, less than 10 ^-13 M, 5 ×
Included are affinities with dissociation constants or Kd's less than 10 ^-14 M, less than 10 ^-14 M, less than 5 × 10 ^-15 M or less than 10 ^-15 M.

The present invention also includes antibodies to epitopes of the invention, as determined by any method known in the art for determining competitive binding (eg, immunoassays described herein). Antibodies that competitively inhibit the binding of In a preferred embodiment, the antibody is 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% competitively bound to an epitope. Inhibit.

The antibodies of the invention can act as agonists or antagonists of the polypeptides of the invention. For example, the invention includes antibodies that disrupt the receptor / ligand interaction with a polypeptide of the invention either partially or completely. Preferably, the antibodies of the invention bind the antigenic epitopes disclosed herein, or portions thereof. The present invention features both receptor-specific and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie signal transduction) can be determined by the techniques described herein, or otherwise known in the art. For example, receptor activation can be determined by phosphorylation of the receptor (eg, tyrosine or serine / threonine), or immunoprecipitation followed by detection of its substrate by Western blot analysis (eg, as described above). . In certain embodiments, in the absence of this antibody, the ligand activity or receptor activity is at least 95% of its activity,
At least 90%, at least 85%, at least 80%, at least 75%,
Antibodies are provided that inhibit at least 70%, at least 60%, or at least 50%.

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, and, preferably, binds unbound receptor or unbound ligand. It has the characteristics of a receptor-specific antibody that does not recognize. Similarly, the present invention binds ligands and neutralizing antibodies that prevent the binding of ligands to receptors, and ligands, thereby preventing receptor activation, but not ligand binding to receptors. Contains antibodies. Further, the invention includes antibodies that activate the receptor. These antibodies may act as receptor agonists, ie enhance or activate either all or a subset of the biological activation of ligand-mediated receptor activation by, for example, inducing receptor dimerization. obtain. The antibody may be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of the peptides of the invention disclosed herein. The antibody agonist can be produced using a method known in the art. For example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-19.
88 (1998); Chen et al., Cancer Res. 58 (16): 366
8-3678 (1998); Harrop et al., J. Am. Immunol. 161 (4
): 1786-1794 (1998); Zhu et al., Cancer Res: 58.
(15): 3209-3214 (1998); Yoon et al., J. Am. Immunol
． 160 (7): 3170-3179 (1998); Prat et al. Cell
． Sci. 111 (Pt2): 237-247 (1998); Pittard et al.,
J. Immunol. Methods 205 (2): 177-190 (199).
7); Liautard et al., Cytokine 9 (4): 233-241 (1).
997); Carlson et al. Biol. Chem. 272 (17): 11
295-11301 (1997); Taryman et al., Neuron 14 (4).
): 755-762 (1995); Muller et al., Structure 6 (
9): 1153-1167 (1998); Bartunek et al., Cytokin.
e 8 (1): 14-20 (1996), all of the above references are incorporated herein by reference in their entireties.

Antibodies of the invention can be used, for example, but not limited to, to purify, detect, and target the polypeptides of the invention. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays for qualitatively and quantitatively measuring the levels of polypeptides of the invention in biological samples. For example, Harlo
w et al., Antibodies: A Laboratory Manual, (C
See Old Spring Harbor Laboratory Press, 2nd Edition, 1988), which is hereby incorporated by reference in its entirety.

As discussed in further detail below, the antibodies of the invention can be used either alone or in combination with other compounds. The antibody can further be recombinantly fused to the heterologous polypeptide at the N-terminus or C-terminus to the polypeptide or other compound, or can be chemically linked (including covalent and non-covalent). For example, the antibodies of the present invention can be recombinantly fused or conjugated to effector molecules such as molecules and heterologous polypeptides, agents, radionuclides, or toxins that are useful as labels in detection assays. For example, PCT publication WO92 / 0849
5; WO91 / 14438; WO89 / 12624; US Pat. No. 5,314,9
95; and EP 396,387.

The antibodies of the invention are modified (ie, covalently attached).
attachment), by covalent attachment of any type of molecule to the antibody such that the antibody does not prevent it from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylations, acetylations, pegylations, phosphorylations.
n), amidation, known blocking / blocking groups
Antibodies modified by derivatization with, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the invention may be produced by any suitable method known in the art.
Polyclonal antibodies to the antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the invention may be administered to a variety of host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. . Various adjuvants can be used to increase the immunological response, depending on the host species, and Freund's (complete and incomplete) mineral gels such as aluminum hydroxide (
mineral gel), surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
Keyhole limpet hemocyanin, dinitrophenol, and BCG (bacillus Calmette-Guerin) and corynebacterium parvum
Include, but are not limited to, potentially useful human adjuvants such as Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using the hybridoma technology known in the art, including: Har
Low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
, 2nd edition, 1988); Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), which is incorporated by reference in its entirety. 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 of 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 (eg, Example 16). In a non-limiting example, mice can be immunized with a polypeptide of the invention or cells expressing such a peptide. Once immune application is detected (eg, antibody specific for the antigen is detected in mouse serum)
The mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg cells from cell line SP20 available from the ATCC). Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies
) By immunizing mice with positive hybridoma clones,
Can be produced.

Accordingly, the invention provides a method of producing an antibody produced by a method comprising culturing a hybridoma cell secreting a monoclonal antibody and an antibody of the invention, wherein, preferably, this antibody is A hybridoma is a hybridoma clone that fuses myeloma cells with splenocytes isolated from a mouse immunized with the antigen of the present invention and then secretes an antibody capable of binding to the polypeptide of the present invention.
Produced by screening the hybridomas resulting from the fusion.

Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, Fab and F (ab ′) 2 fragments of the invention are (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce the b fragment) or pepsin (to produce the F (ab ') 2 fragment). 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 produced 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 certain embodiments, such phage can be utilized to display antigen binding domains expressed from leper trimer or combinatorial antibody libraries (eg, human or mouse). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with the antigen (eg, using labeled antibody or antigen bound or captured to a solid surface or bead). Phage used in these methods are typically phage having the antibody domain of Fab, Fv or disulfide-stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Is a filamentous phage containing the fd and M13 binding domains expressed from E. coli. Examples of phage display methods that can be used to generate the antibodies of the invention include those disclosed below: Brinkman et al. Immuno
l. Methods 182: 41-50 (1995); Ames et al., J. Am. Im
munol. Methods 184: 177-186 (1995); Kett.
leborough et al., Eur. J. Immunol. 24: 952-958 (
1994); Persic et al., Gene 187 9-18 (1997); Bu.
rton et al., Advances in Immunology 57: 191-.
280 (1994); PCT application number PCT / GB91 / 01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047.
WO 92/18619; WO 93/11236; WO 95/15982.
WO 95/20401; and US Pat. No. 5,698,426;
, 223,409; 5,403,484; 5,580,717;
No. 5,427,908; No. 5,750,753; No. 5,821,04.
No. 7; No. 5,571,698; No. 5,427,908; No. 5,516.
No. 5,637, No. 5,780,225; No. 5,658,727; No. 5,
733, 743 and 5,969, 108, each of which is hereby incorporated by reference in its entirety.

As described in the above references, after phage selection, the region encoding the antibody derived from the phage may be cloned to produce whole antibodies, including human antibodies, or any other desired antigen binding fragment. It can be separated and used and expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, techniques for recombinantly producing Fab, Fab ′ and F (ab ′) 2 fragments can also be used with methods known in the art, such methods being disclosed below. R: PC
T Publication WO 92/22324; Mullinax et al., BioTechniq.
es 12 (6): 864-869 (1992); and Sawai et al., AJR.
I 34: 26-34 (1995); and Better et al., Science.
240: 1041-1043 (1998) (these references are incorporated by reference in their entirety).

Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston.
Et al., Methods in Enzymology 203: 46-88 (19.
91); 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, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species (for example, an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region). Methods for producing chimeric antibodies are known in the art. For example, Morris
on, Science 229: 1202 (1985); Oi et al., BioTec.
hnies 4: 214 (1986); Gillies et al., (1989) J.
． Immunol. Methods 125: 191-202; US Pat. No. 5,
807,715; 4,816,567; and 4,816397, which are hereby incorporated by reference in their entireties.
. A humanized antibody is an antibody molecule from a non-human species antibody that binds to a desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues in the human framework regions will be replaced with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, eg, C to identify framework residues important for antigen binding.
By modeling the interaction of DR and framework residues, and sequence comparisons to identify unusual framework residues at specific positions. (For example,
Queen et al., US Pat. No. 5,585,089; Riechmann et al., Na.
see tur 332: 323 (1988), which are hereby incorporated by reference in their entireties. ) Antibodies can be humanized using various techniques known in the art including the following: eg, CDR-grafted (European Patent No. 239).
, 400; PCT Publication WO 91/09967; U.S. Patent No. 5,225,53.
9; 5,530,101 and 5,585,089), veneering or resurfacing.
g) (European Patent Nos. 592,106; 519,596; Padlan, M
molecular Immunology 28 (4/5): 489-498 (
1991); Studnicka et al., Protein Engineerin.
g 7 (6): 805-814 (1994); Roguska et al., PNAS 9
1: 969-973 (1994)), and chain shuffling (chai).
n shuffling) (US Pat. No. 5,565,332).

Fully 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. U.S. Pat. Nos. 4,444,887, 4,716,111; and PCT Publication W.
O 98/46645, WO 98/50433, WO 98/24893, W
See also O 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region interfere with endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried in the transgenic mouse is B
It rearranges during cell differentiation and then undergoes class switching and somatic mutation. Thus, the use of such techniques allows for the production of therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, eg, PCT Publications WO98 / 24893; WO92 / 01047; WO96 / 340.
96; WO96 / 33735; European Patent No. 0 598 877; US Patent No. 5,
No. 413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806.
No. 5,814,318; No. 5,885,793; No. 5,916.
771; and 5,939,598, which are hereby incorporated by reference in their entireties. In addition, Abgenix, Inc. (F
Companies such as Reemont, CA) and Genpharm (San Jose, CA) may be engaged in providing human antibodies to selected antigens using techniques similar to those described above.

Human antibodies that fully recognize the selected epitope were "guided (gui).
ed) selection ”. In this approach,
The selected non-human monoclonal antibody (eg, mouse antibody) is used to guide the selection of human antibodies that fully recognize the same epitope. (Jespers
Et al., Bio / technology 12: 899-903 (1988)).

Furthermore, antibodies against the polypeptides of the present invention may in turn be utilized to produce anti-idiotypic antibodies that “mimic” the polypeptides of the present invention using techniques well known to those of skill in the art. (Eg Greenspan and Bona, FASEB
J. 7 (5): 437-444; (1989) and Nissinoff, J.
． Immunol. 147 (8): 2429-2438 (1991). ) For example, binding and multimerization of polypeptides.
cation) and / or an antibody that competitively inhibits binding of the polypeptide of the invention to a ligand is used to produce an anti-idiotype that "mimics" the multimerization domain and / or binding domain of the polypeptide. Obtain and, as a result, bind and neutralize the polypeptide and / or its ligand.
Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to bind its ligand / receptor, thereby blocking its biological activity.

Polynucleotide Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also provides an antibody (eg, as defined above) under stringent or less stringent hybridization conditions, preferably which 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 polynucleotide may be obtained by any method known in the art and the nucleotide sequence of the polynucleotide determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be constructed from chemically synthesized oligonucleotides (eg, Kutmeie).
r. et al., BioTechniques 17: 242 (1994)), which briefly includes the following steps: Synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody. , Annealing and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, the polynucleotide encoding the antibody can be generated from nucleic acid from a suitable source. Although clones containing nucleic acid encoding a particular antibody are not available, if the sequence of the antibody molecule is known, the immunoglobulin-encoding nucleic acid can be chemically synthesized or, alternatively, a suitable source ( For example, an antibody cDNA library, or a cDNA library produced from any tissue or cell expressing an antibody (eg, a hybridoma cell selected for expression of an antibody of the invention), or a nucleic acid isolated therefrom. PCR amplification using synthetic primers hybridizable to the 3'and 5'ends of the sequence, for example to identify a cDNA clone from a cDNA library encoding the antibody (preferably poly A + RNA). Or by using an oligonucleotide probe specific for a particular gene sequence. It may be obtained by cloning. P
The amplified nucleic acid produced by CR can then be cloned into a replicable cloning vector using any method known in the art.

Once the antibody nucleotide sequence and corresponding amino acid sequence are determined,
Nucleotide sequences of antibodies may be prepared using methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL.
Laboratory Manual, 2nd Edition, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. Both are incorporated herein by reference in their entirety. )) Can be engineered to produce antibodies with different amino acid sequences, eg, to make amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the variable domains of the heavy and / or light chains are identified by methods well known in the art for identification of the sequences of complementarity determining regions (CDRs) (eg sequence Other heavy chains to determine hypervariable regions,
And by comparing the variable region of the light chain with a known amino acid sequence). Using conventional recombinant DNA techniques, one or more CDRs may be inserted within the framework regions as described above (eg, into the human framework regions for humanizing non-human antibodies). . The framework regions can be naturally occurring or consensus framework regions, and preferably human framework regions (eg, for the listed human framework regions, Chothia et al., J. Mol. Biol. 278: 457-479 (1
998)). Preferably, the polynucleotide produced by the combination of the framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods may generate antibody molecules lacking one or more intrachain disulfide bonds,
It can be used to make amino acid substitutions or deletions of cysteine residues in one or more variable regions involved in intrachain disulfide bonds. Other changes to the polynucleotide are encompassed by the present invention and in the art.

Furthermore, by splicing a gene derived from a mouse antibody molecule of appropriate antigen specificity with a gene derived from a human antibody molecule of appropriate biological activity,
Techniques developed to produce "chimeric antibodies" (Morrison et al., Proc
． Natl. Acad. Sci. 81: 851-855 (1984); Neub.
erger et al., Nature 312: 604-608 (1984); Take.
da et al., Nature 314: 452-454 (1985)) may be used. As mentioned above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from the constant regions of mouse mAb and human immunoglobulin (eg, humanized antibody). .

Alternatively, described techniques for the production of single chain antibodies (US Pat. No. 4,946,46)
778; Bird, Science 242: 423-42 (1988); H.
uston et al., Proc. Natl. Acad. Sci. USA 85: 587.
9-5883 (1988); and Ward et al., Nature 334: 544.
-54 (1989)) can be adapted to the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bridges, yielding single chain polypeptides. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Sk
erra et al., Science 242: 1038-1041 (1988)).

Methods of Producing Antibodies The antibodies of the invention may be produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis or, preferably, by recombinant expression techniques. obtain.

Antibodies of the invention, or fragments, derivatives or analogs thereof (eg,
Recombinant expression of a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once the polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of the antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) is obtained, production of the antibody molecule Vectors for can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods 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. Accordingly, the invention provides a replicable vector 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. I will provide a.
Such a vector may be a constant region of an antibody molecule (eg PCT Publication WO86 /
05807; PCT Publication WO 89/01036; and US Pat. No. 5,122.
, 464), and the variable domain of the antibody can be cloned into such a vector for total expression of heavy or light chains.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. Accordingly, the invention includes a host cell 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 a preferred embodiment for expression of double-chain antibodies, the vectors encoding both heavy and light chains are co-expressed in a host cell for expression of entire immunoglobulin molecules, as detailed below. Can be done.

A variety of host expression vector systems may be utilized to express the antibody molecule of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest may be produced and subsequently purified, but also in situ when transformed or transfected with sequences encoding the appropriate nucleotides. 2 represents a cell capable of expressing an antibody molecule of the invention. These include, but are not limited to: bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing sequences encoding the antibody (eg, E. coli, Microorganisms such as B. subtilis; yeast transformed with recombinant yeast expression vectors containing antibody-encoding sequences (eg, Sacc.
harmomyces, Pichia); insect cell lines infected with a recombinant viral expression vector (eg, baculovirus) containing antibody-encoding sequences; recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) Or a plant cell line transformed with a recombinant plasmid expression vector (eg, Ti plasmid) containing a sequence encoding an antibody; or a promoter derived from the genome of a mammalian cell (
Mammalian cell lines (eg, COS, CHO, BHK) carrying recombinant expression constructs containing, for example, a metallothionein promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter). 293, 3T3 cells). Preferably Esch
Bacterial cells such as E. coli, and more preferably eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major immediate early gene promoter element from human cytomegalovirus are effective expression systems for antibodies. (Föcking et al., Gene 45: 101 (1986); Cock.
ett et al., Bio / Technology 8: 2 (1990)).

In bacterial systems, many expression vectors may be advantageously selected depending upon the use intended for the expression of the antibody molecule. For example, if large quantities of such proteins are to be produced, a vector that directs high levels of expression of the easily purified fusion protein product may be desired for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to: The antibody-encoding sequence, in frame with the lacZ-encoding region, in the vector.
e) can be separately ligated, so that a fusion protein is produced in E. coli
Expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1
983)); pIN vector (Inouye & Inouye, Nucleic
Acids Res. 13: 3101-3109 (1985); Van Hee.
ke & Schuster, J .; Biol. Chem. 24: 5503-5509
(1989)) and so on. The pGEX vector can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site 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 heterologous genes. This virus grows in Spodoptera frugiperda cells. The antibody coding sequences can be individually cloned into non-essential regions of the virus (eg the polyhedrin gene) and placed under control of the AcNPV promoter (eg the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems are available. When an adenovirus is used as an expression vector, the sequence encoding the antibody of interest may be linked to the adenovirus transcription / translation control complex, such as the late promoter and a three-part leader sequence. Then
This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in nonessential regions of the viral genome (eg, E1 or E3 regions) result in recombinant viruses that are viable and capable of expressing antibody molecules in infected hosts (eg, Logan & Shenk).
, Proc. Natl. Acad. Sci. USA 81: 355-359 (1
984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals are ATG
It includes a start codon and adjacent sequences. In addition, this initiation codon ensures that translation of the entire insert is ensured by the reading frame of the desired coding sequence (
must be in phase with the reading frame). These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., Methods in E).
nzymol. 153: 51-544 (1987)).

In addition, a host cell line 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 (eg glycosylation) and processing of protein products (
Cleavage, for example) can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for 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 can be used that have the cellular machinery for the precise processing of primary transcription, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and especially, for example, BT48
3, breast cancer cell lines such as Hs578T, HTB2, BT20 and T47D, and normal breast cell lines such as, for example, CRL7030 and Hs578Bst.

Long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using an expression vector containing a viral origin of replication, the host cell may choose suitable expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.).
, And a DNA controlled by a selectable marker. Following the introduction of the heterologous DNA, the engineered cells can be grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection, and the cell stably integrates the plasmid into its chromosome and proliferates to form a foci of cells that can now be cloned. , Can be extended to cell lines. This method can be advantageously used to engineer cell lines that express antibody molecules. 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 herpes simplex virus thymidine kinase (Wigler et al. Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & S.
zybalski, Proc. Natl. Acad. Sci. USA 48: 2
02 (1992)), and adenine phosphoribosyl transferase (L
owy et al., Cell 22: 817 (1980)), but without limitation, these genes can be used in tk-, hgprt- or aprt- cells, respectively. Antimetabolite resistance can also be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate (W
igler 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 is the aminoglycoside G-.
Conferring resistance to 418 (Clinical Pharmacy 12: 4)
88-505; Wu and Wu, Biotherapy 3: 87-95 (
1991); Tolstoshev, Ann. Rev. Pharmacol. T
oxicol. 32: 573-596 (1993); Mulligan, Sc.
ience 260: 926-932 (1993); and Morgan an.
d Anderson, Ann. Rev. Biochem. 62: 191-21
7 (1993); May 1993, TIB TECH 11 (5): 155-2.
15); as well as hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Recombinant DNA
Methods well known in the art can be routinely applied to select the desired recombinant clones, and such methods are described below: eg Aus.
ubel et al. (ed.), Current Protocols in Molcu
lar Biology, John Wiley & Sons, NY (1993)
Kriegler, Gene Transfer and Expressi
on, A Laboratory Manual, Stockton Pres
s, NY (1990); and Chapters 12 and 13, Dracopoli et al.
Ed), Current Protocols in Human Geneti
cs, John Wiley & Sons, NY (1994); Colberre
-Garapin et al. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (for review,
Bebington and Hentschel, The use of ve
ctors based on gene amplification fo
r the expression of cloned genes in
mammalian cells in DNA cloning, Vol. Three
． (See Academic Press, New York, 1987). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Since the augmented region is linked to the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257.
(1983)).

A host cell may be co-transfected with two expression vectors of the invention, a first vector encoding a polypeptide from the heavy chain and a second vector encoding a polypeptide from the light chain. . 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 expresses both heavy and light chain polypeptides. In this situation,
To avoid excess toxic free heavy chains, the light chain should be placed before the heavy chain (Proudfoot, Nature 322: 52 (1986); Kohle.
r, Proc. Natl. Acad. Sci. USA 77: 2197 (198).
0)). The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

Once the antibody molecule of the present invention is produced by an animal, chemically synthesized, or recombinantly expressed, any of the art-known methods for the purification of immunoglobulin molecules. Methods such as chromatography (eg, ion exchange,
It can be purified by affinity (particularly by affinity for protein A followed by specific antigen), and size column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.
Furthermore, 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 provides a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 of this polypeptide,
Or 100 amino acids), recombinantly fused or chemically linked (including both covalent and non-covalent bonds) to produce a fusion protein. The fusion need not be direct, but can occur via a linker sequence. The antibody is a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70,
(80, 90, or 100 amino acids). For example, by fusing or conjugating a polypeptide of the invention to an antibody specific for a particular cell surface receptor, either in vitro or in vivo, the polypeptide of the invention is directed against a particular cell type. To target
Antibodies can be used. Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, eg, Harbor et al., Supra, and PCT Publication WO93.
/ 21232; EP 439,095; Naramura et al., Immunol. L
ett. 39: 91-99 (1994); US Patent No. 5,474,981; G.
illies et al., PNAS 89: 1428-1432 (1992); Fell.
Et al., J. Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The invention further includes compositions comprising a polypeptide of the invention fused or linked to a domain other than the variable region of an antibody. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The part of this antibody fused to the polypeptide of the invention comprises the constant region, hinge region, CH1 domain, C
It may include the H2 domain, and the CH3 domain, or any combination of whole domains or portions thereof. These polypeptides can also be fused or conjugated to portions of the antibody described above to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. A higher degree of multimeric morphology allows the polypeptide to be IgA
And a portion of IgM. Methods for fusing or conjugating the polypeptides of the invention to antibody moieties are known in the art. For example, US Pat. Nos. 5,336,603; 5,622,929;
No. 5,359,046; No. 5,349,053; No. 5,447,85.
No. 1; No. 5,112,946; EP 307,434; EP 367,16.
6; PCT publication WO96 / 04388; WO91 / 06570; Ashkena
zi et al., Proc. Natl. Acad. Sci. USA 88: 10535-.
10539 (1991); Zheng et al. Immunol. 154: 559
0-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89: 113337-11341 (1992), the above references are incorporated by reference in their entireties.

A polypeptide corresponding to the polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y, as discussed above, is for increasing the in vivo half-life of this polypeptide, or It can be fused or conjugated to the antibody moieties described above for use in immunological assays using methods known in the art. In addition, the polypeptide corresponding to SEQ ID NO: Y can be fused or linked to the antibody portion described above to facilitate purification. One reported example describes a chimeric protein 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 (198).
8)). The polypeptides of the invention, fused or linked to an antibody having a disulfide-linked dimeric structure (due to IgG), also bind to other molecules than monomeric secreted proteins or protein fragments alone and It may be more efficient at neutralizing (Funtoulakis et al., J. Biochem. 27).
0: 3958-3964 (1995)). In many cases, the Fc portion of fusion proteins may be beneficial in therapy and diagnosis, thus resulting, for example, in improved pharmacokinetic properties (EP A 232,262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. For example, in drug discovery,
Human proteins such as hIL-5 have been fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5 (Bennett et al., J. Molecular Recognitio.
n 8: 52-58 (1995); Johnson et al., J. Am. Biol. Che
m. 270: 9459-9471 (1995)).

Further, the antibodies of the invention or fragments thereof may be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats.
The tag, provided by W. Worth, CA, 91311), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc
． Natl. Acad. Sci. Hexa-histidine provides convenient purification of fusion proteins, as described in USA 86: 821-824 (1989). Another peptide tag useful for purification is the "HA" tag (Wilson et al., Ce, which corresponds to an epitope derived from the influenza hemagglutinin protein.
ll37: 767 (1984)), and "flag" tags.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically, for example, to monitor tumor development or progression as part of a clinical testing procedure (eg, to 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 substances, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. , Can be mentioned. This detectable substance is an antibody (or fragment thereof)
, Either directly or indirectly, via a mediator using techniques known in the art, such as a linker known in the art, or linked. See, eg, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β
-Galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin /
Examples include biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol. Examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc.

Further, the antibody or fragment thereof may be used in therapeutic moieties (eg cytotoxins (eg cytostatic or cytotoxic agents)), therapeutic agents or radioactive metal ions (eg α-emitters (eg 213Bi)). Etc.). Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D,
Ethidium bromide, emetine, mitomycin, etoposide, teniposide (ten
oposide), vincristine, vinblastine, colchicine (colch)
icin), doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol,
And puromycin, and analogs or homologues thereof. The therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine (5-f
luourouracyl decarbazine), an alkylating agent (eg,
Mechlorethamine, thioepa chlorambucil, melphalan,
Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracycline (eg, daunorubicin (eg, daunorubicin). Formerly daunomycin) and doxorubicin), antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents such as vincristine and vinblastine. ), But
It is not limited to them.

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or drug moiety is not construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that has the desired biological activity. Such proteins include, for example, toxins (eg, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin); proteins (eg,
Tumor necrosis factor, a-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg, TNF-α, TNF-β, AIM I (International Publication No. WO97 / 33899).
No.), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6).
: 1567-1574 (1994)), VEGI (International Publication No. WO99 / 231).
05)), thrombotic or anti-angiogenic agents (eg, angiostatin or endostatin); or biological response modifiers (eg, lymphokines, interleukin-1 (“IL-1”)). , Interleukin-2 (“IL
-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CSF ").
), Or other growth factors, etc.).

The antibody can also be attached to a solid support, which is particularly useful for immunoassay 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 coupling such therapeutic moieties to the antibody are well known, eg Arnon.
Et al., "Monoclonal Antibodies For Immuno.
targeting Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The
rapid, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Lis.
s, Inc. 1985); Hellstrom et al., "Antibodies F."
or Drug Delivery "Controlled Drug Del
Ivery (2nd edition), Robinson et al. (eds.), pp. 623-53 (Marc.
el Dekker, Inc. 1987); Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Can
cer Therapy: A Review ”Monoclonal Anti
bodies'84: Biological And Clinical Ap
applications, Pinchera et al. (eds.), pp. 475-506 (198).
5); "Analysis, Results, And Future Pros
selective Of The Therapeutic Use Of Ra
dilabelled Antibody In Cancer Therap
y ”Monoclonal Antibodies For Cancer D
inspection And Therapy, Baldwin et al. (eds.), 303
-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Prop.
parties Of Antibody-Toxin Conjugates "
, Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a secondary antibody and the antibody heteroconjugate as described by Segal in US Pat. No. 4,676,980, which is incorporated by reference herein in its entirety. A body (heteroconjugate) may be formed.

Antibodies, with or without a therapeutic moiety attached to the antibody, administered alone or in combination with cytotoxic factors and / or cytokines, can be used as therapeutic agents.

Immunophenotyping The antibodies of the invention can be utilized for immunophenotyping cell lines and biological samples. The translation products of the genes of the present invention are useful as cell-specific markers, or more particularly as cell markers that are differentially expressed at various stages of differentiation and / or maturation of particular cell types. obtain. Monoclonal antibodies directed against specific epitopes, or combinations of epitopes, allow screening of cell populations expressing markers. Various techniques can be utilized with monoclonal antibodies to screen for cell populations that express markers, and include magnetic separation using antibody-coated magnetic beads, solid matrices (ie, "Panning" using antibodies attached to plates, as well as flow cytometry (eg US Pat. No. 5,985,6).
60; and Morrison et al., Cell, 96: 737-49 (1999.
) Refer to)).

These techniques apply to hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And allows screening of specific populations of cells as may be found with "non-self" cells in transplantation to prevent graft versus host disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem cells and progenitor cells that may undergo proliferation and / or differentiation as may be found in human cord blood.

Assays for Antibody Binding The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include Western blots, radioimmunoassays, to name just a few.
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A Included, but not limited to, competitive and non-competitive assay systems that use techniques such as immunoassays. Such assays are routine and well known in the art (eg, Ausubel et al. Eds., 1994, Current Protocol), which is incorporated by reference herein in its entirety.
ls in Molecular Biology, Volume 1, John Wil
ey & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1, 1% NP-40, supplemented with protein phosphatase inhibitors and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate). % Sodium deoxycholate, 0.1%
SDS, 0.15M NaCl, 0.01M sodium phosphate (pH 7.2)
) Lysing a population of cells in a lysis buffer such as 1% Transylol), adding the antibody of interest to the cell lysate, and incubating at 4 ° C. for a period of time (eg 1-4 hours) Adding protein A sepharose beads and / or protein G sepharose beads to the cell lysate, incubating at 4 ° C. for about 1 hour or more, washing the beads in lysis buffer, and SDS / sample buffer Resuspending the beads in. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assayed, for example, by Western blot analysis. One of skill in the art will be able to discern parameters that can be modified to increase binding of antibody to antigen and to reduce background (eg, preclearing cell lysates with Sepharose beads). . For further discussion on immunoprecipitation protocols, see, eg, Ausub.
el et al., 1994, Current Protocols in Molec.
ural Biology, Volume 1, John Wiley & Sons, Inc.
． , New York, 10.16.1.

Western blot analysis generally involves the steps of preparing a protein sample, polyacrylamide gel (eg 8% -20% SDS-depending on the molecular weight of the antigen).
Electrophoresis of the protein sample in PAGE), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking solution (eg 3% BSA or PBS with nonfat dry milk). Blocking the membrane in a washing buffer (eg PB
Washing the membrane in S-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer , A secondary antibody (recognizing the primary antibody, eg, an anti-human antibody) bound to an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, 32P or 125I) diluted in blocking buffer With the step of blocking the membrane, washing the membrane in wash buffer, and detecting the presence of the antigen. One of ordinary skill in the art will be aware of parameters that can be modified to increase the signal detected and to reduce background noise. For further discussion on Western blot protocols, see, eg, Ausu.
Bel et al., 1994, Current Protocols in Mole.
polar Biology, Volume 1, John Wiley & Sons, In
c. , New York, 10.8.1.

ELISA is for the purpose of preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, binding to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). Antibody to the wells and incubating for a period of time, and detecting the presence of the antigen. In the ELISA, the antibody of interest need not be bound to the detectable compound; instead, a second antibody (which recognizes the antibody of interest) bound to the detectable compound can be added to the wells. . Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added subsequent to the addition of the antigen of interest to the coated wells. One of ordinary skill in the art will be aware of parameters that can be modified to increase the signal detected, and other variations of the ELISA known in the art. EL
For further discussion on ISA, see, eg, Ausubel et al., Eds., 199.
4, Current Protocols in Molecular Bio
LOGY, Volume 1, John Wiley & Sons, Inc. , New Yo
See rk, 11.2.1.

[0287] The binding affinity of the antibody for the antigen and the off-rate of the antibody-antigen interaction (of
f-rate) can be determined by competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which involves labeling with a labeled antigen (eg, 3H or 125I), incubation with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and labeling. Includes detection of antibody bound to antigen. The affinity of the antibody of interest for a particular antigen, and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with the second antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest bound to a labeled compound (eg, 3H or 125I) in the presence of increasing amounts of unlabeled second antibody.

Therapeutic Uses The present invention further relates to antibody-based therapies for treating one or more of the disclosed diseases, disorders, or conditions in animals, preferably mammals, and most Preferably, the step of administering the antibody of the present invention to a human patient is included. Therapeutic compounds of the invention include antibodies of the invention (including fragments, analogs and derivatives thereof, as described herein) and nucleic acids encoding the antibodies of the invention (described herein). (Including, but not limited to, fragments, analogs and derivatives thereof and anti-idiotypic antibodies). Antibodies of the invention include diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the invention (including any one or more of the diseases, disorders, or conditions described herein). Can be used to treat, inhibit or prevent). Aberrant expression of the polypeptide of the invention and /
Alternatively, treating and / or preventing a disease, disorder or condition associated with activity includes, but is not limited to, ameliorating the symptoms associated with the disease, disorder or condition. The antibodies of the present invention are known in the art or can be provided in a pharmaceutically acceptable composition as described herein.

A summary of how the antibodies of the invention may be used therapeutically is locally or systemically in the body, or mediated (eg, by complement (CDC), or by effector cells (ADCC)). By direct binding of the polynucleotide or polypeptide of the invention by the direct cytotoxicity of the antibody. Some of these approaches are described in more detail below. Given the teachings provided herein, one of ordinary skill in the art would be able to determine, without undue experimentation, how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes. Recognize.

Antibodies of the invention may be other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg, IL-2, IL-2, IL-2, IL-2, IL-2, IL-3 and IL
(Such as −7).

The antibodies of the invention can be administered alone or in combination with other types of treatments such as radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents.
Generally, it is preferred to administer a species-sourced or species-reactive product that is the same species as the patient's species (in the case of antibodies). Thus, in a preferred embodiment, human antibodies,
The fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

A high affinity and / or affinity for a polypeptide or polynucleotide of the invention, both for immunoassays for the polynucleotides or polypeptides of the invention (including fragments thereof) and for the treatment of disorders associated therewith. Or potent in vivo inhibitory and / or neutralizing antibodies, fragments thereof,
Alternatively, it is preferable to use those areas. Such antibodies, fragments or regions are preferably polynucleotides or polypeptides of the invention (
(Including its fragments). The preferred binding affinity is 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M, 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁻⁷ M, 5 × 10 ⁻⁸ M, 10 ⁻⁸ M, 5 × 10 ⁻⁹ M, 10 ^{− 9 M, 5 × 10 -10 M} , 10 - 10 M, 5 × 10 -11 M, 10 -11 M, 5 × 10 -12 M, 10 -12 M, 5 × 10 -13 M
Included are binding affinities with dissociation constants or Kd less than 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, and 10 ⁻¹⁵ M.

Gene Therapy In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof treats a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention, Administered for the purpose of gene therapy to inhibit or prevent. Gene therapy refers to a therapy that is performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

[0294] Any method available in the art for gene therapy can be used in accordance with the present invention. An exemplary method is described below.

For a general review of methods of gene therapy, see Goldspiel et al., Cli.
Nal Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstos.
hev, 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, TIB.
See TECH 11 (5): 155-215 (1993). Methods generally known in the recombinant DNA art that can be used are described in Ausubel.
Et al., Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Express.
n, A Laboratory Manual, Stockton Press
, NY (1990).

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, said nucleic acid sequence being an expression vector expressing the antibody, or fragment or chimeric protein thereof, or heavy or light chain thereof, in a suitable host. Is part of. In particular, such nucleic acid sequences have a promoter 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 that flank the region encoding the antibody and any other desired sequences that facilitate homologous recombination at the desired site in the genome, thus rendering the antibody Provides intrachromosomal expression of the encoding nucleic acid (Koller and Smithies, Proc. N.
atl. Acad. Sci. USA 86: 8932-8935 (1989);
Zijlstra et al., Nature 342: 435-438 (1989)).
In a particular embodiment, the expressed antibody molecule is a single chain antibody; or the nucleic acid sequence comprises sequences encoding both the heavy and light chains of the antibody or fragments thereof.

Delivery of the nucleic acid to the patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cell is first transfected with the nucleic acid in vitro). Converted and then transplanted to the patient). These two approaches are known as in vivo gene therapy or as ex vivo gene therapy, respectively.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This is accomplished by a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector and constructing them (eg, defective or attenuated retrovirus or other viral vectors (US Pat. 4,980,286) (by infection with cells) or by direct injection of naked DNA, or by microparticle bombardment (eg gene gun; Biol).
istic, Dupont), or by coating with lipids or cell surface receptors, or by transfecting agents, or by encapsulating them in liposomes, microparticles, or microcapsules, or By administering to a peptide that is known to enter, by administering to a ligand that undergoes receptor-mediated endocytosis (eg, Wu and Wu, J. Biol. Chem.
． 262: 4429-4432 (1987)), which can be used to target cell types that specifically express the receptor. In another embodiment, nucleic acid-ligand complexes can be formed where the ligand disrupts endosomes fusogen.
ic) contains a viral peptide, allowing this nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO92 / 06180; WO92 / 226).
No. 35; No. WO92 / 20316; No. WO93 / 14188, No. WO
93/20221). Alternatively, the nucleic acid can be introduced inside the cell and incorporated into the host cell DNA for expression by homologous recombination (
Koller and Smithies, Proc. Natl. Acad. Sci
． USA 86: 8932-8935 (1989); Zijlstra et al., Na.
true 342: 435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acid sequences that encode the antibodies of the invention are used. For example, a retroviral vector can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993).
checking). These retroviral vectors contain the necessary components for correct packaging of the viral genome and correct integration into host cell DNA. The nucleic acid sequence encoding the antibody used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into patients. For more details on retroviral vectors, see Boesen et al., Bi.
others, 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Can be issued. Other references describing the use of retroviral vectors in gene therapy are: Clowes et al., J. Am. Clin. Invest. 93: 644-651 (19
94); Kiem et al., Blood 83: 1467-1473 (1994); S.
almons and Gunzberg, Human Gene Therapy
4: 129-141 (1993); and Grossman and Wils.
on, Curr. Opin. in Genetics and Devel. Three
: 110-114 (1993).

Adenovirus is another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenovirus naturally infects respiratory epithelia where it causes mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, Curr
ent Opinion in Genetics and Developopm
ent 3: 499-503 (1993) provides an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3.
-10 (1994) demonstrated the use of adenoviral vectors to transfer genes to the rhesus monkey respiratory epithelium. Other examples of the use of adenovirus in gene therapy are described by Rosenfeld et al., Science 252: 431-434 (19).
91); Rosenfeld et al., Cell 68: 143-155 (1992).
Mastrangeli et al., J .; Clin. Invest. 91: 225-2
34 (1993); PCT Publication No. WO 94/12649; 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); US Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves transfer of the selectable marker to the cells. The cells are then subjected to selection to isolate those cells that take up and express the transferred gene.
The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art, including but not limited to: transfection, electroporation, microinjection, viruses including nucleic acid sequences. Infection with vector or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Many techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Met.
h. Enzymol. 217: 599-618 (1993); Cohen et al., M.
eth. Enzymol. 217: 618-644 (1993); Cline,
Pharmac. Ther. 29: 69-92m (1985)),
And if the necessary developmental and physiological functions of the recipient cells are not disrupted, they can be used according to the invention. This technique should provide for stable transfer of the nucleic acid to the cell so that the nucleic acid is expressible by the cell, preferably heritable and expressible by the progeny of the cell.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient condition, etc. and can be determined by one of skill in the art.

Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type, including but not limited to: epithelial cells, endothelial cells,
Keratinocytes, fibroblasts, muscle cells, hepatocytes; T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, blood cells such as granulocytes; various stem cells or progenitor cells, In particular, hematopoietic stem cells or hematopoietic progenitor cells (eg cells such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver, etc.).

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments in which recombinant cells are used in gene therapy, the nucleic acid sequences encoding the antibody are introduced into the cells such that the nucleic acid sequences are expressible by the cells or their progeny, and then the recombinant cells are , Administered in vivo for therapeutic effects. In a specific embodiment, stem cells or progenitor cells are used. Any stem and / or progenitor cells that can be isolated in vitro and maintained in vitro can potentially be used according to this embodiment of the invention (
For example, PCT Publication No. WO 94/08598: Temple and Ande.
rson, Cell 71: 973-985 (1992); Rheinwald.
, Meth. Cell Bio. 21A: 229 (1980); and Pit
telkow and Scott, Mayo Clinic Proc. 61: 7
71 (1986)).

In a particular embodiment, the nucleic acid to be introduced for the purpose of gene therapy contains an inducible promoter operably linked to the coding region so that expression of the nucleic acid is controlled by the appropriate transcription inducer. Can be controlled by controlling the presence or absence of.

Demonstration of Therapeutic or Preventive Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro prior to use in humans and then in vivo for the desired therapeutic or prophylactic activity. To be done. For example, in vitro assays to demonstrate therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on cell lines and / or tissue samples can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cytolytic assays. In vitro assays that can be used in accordance with the present invention to determine if administration of a particular compound is indicated include in vitro cell culture assays, in which a patient tissue sample is grown in culture, The compound is then exposed or otherwise administered and the effect of such compound on the tissue sample is observed.

Therapeutic / Prophylactic Administration and Compositions The present invention provides for 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. To provide a method. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably
Animals such as but not limited to cows, pigs, horses, chickens, cats, dogs, and the like, and preferably mammals, and most preferably humans.

Formulations and methods of administration that can be used when the compound comprises nucleic acids or immunoglobulins are described above; additional suitable formulations and routes of administration are selected from among those described herein below. Can be done.

Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compound). , Receptor-mediated endocytosis (
For example, Wu and Wu, J .; Biol. Chem. 262: 4429-443
2 (1987)), the construction of nucleic acids as part of retroviruses or other vectors). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining, such as the oral, rectal and intestinal mucosa, and other It can be administered with a biologically active agent. Administration can be systemic or local. Further, the pharmaceutical compounds or compositions of the invention may be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection may be for example, intraventricular injection attached to a reservoir, such as the Ommaya reservoir. Introduction to the central nervous system by means of a catheter). Pulmonary administration may also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In specific embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is for example, but not limited to, Local injection during surgery, topical application (eg, in combination with post-surgical wound dressing), injection, catheter, suppository, or implant (the implant being a membrane such as a sialastic membrane). Or a porous, non-porous, or glue-like material, including fibers). Preferably, in administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

In another embodiment, the compound or composition can be delivered into vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (
1990); Treat et al., Liposomes in the Therap.
y of Infectious Disease and Cancer, L
opez-Berestein and Fidler (ed.), Liss, New
York, pp. 353-365 (1989); Lopez-Berestein,
Ibid, pages 317-327; see 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 (Lang.
er, (supra); Sefton, CRC Crit. Ref. Biomed. E
ng. 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 may be used (Medical Applications of Control).
led Release, Langer and Wise (ed.), CRC Pre
s. , Boca Raton, Florida (1974); Control
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
1 (eds.), Wiley, New York (1984); Ranger and P.
eppas, J .; Macromol. Sci. Rev. Macromol. Ch
em. 23:61 (1983); Levy et al., Science 2
28: 190 (1985); During et al., Ann. Neurol. 25: 3
51 (1989); Howard et al., J. Am. Neurosurg. 71: 105 (
1989) also). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, thus requiring only a portion of the systemic dose (eg, Goodson, Medical Applicat).
ions of Controlled Release, (supra), Volume 2,
115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

In a specific embodiment, wherein the compound of the invention is a nucleic acid encoding a protein, the nucleic acid constitutes it as part of a suitable nucleic acid expression vector and is such that it is intracellular. By administration (eg, by use of retroviral vectors (see US Pat. No. 4,980,286), or by direct injection, or microparticle bombardment (eg, gene gun; Biolis).
Tic, Dupont) or by coating with a lipid or cell surface receptor or transfecting agent, or in combination with a homeobox-like peptide known to enter the nucleus (eg,
Joliot et al., Proc. Natl. Acad. Sci. USA 88:18
64-1868 (see 1991)) and the like, so as to enhance the expression of the encoded protein in vivo. Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for homologous recombination for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" has been approved by the Federal Regulatory Authority or the U.S. Government for use in animals, and more particularly in humans, or in the United States Pharmacopeia or other Means that it is listed in the generally accepted pharmacopoeia.
The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Can be. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, and aqueous dextrose and glycerol solutions can also be used 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, skim milk powder, glycerol, propylene. , Glycol, water, ethanol and the like. The composition can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, 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 formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E.I. W. "Reming" by Martin
ton's Pharmaceutical Sciences ". Such a composition comprises a therapeutically effective amount of the compound, preferably in purified form,
It is included with a suitable amount of carrier 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. If desired, the composition can also include a solubilizer and a local anesthesia such as lignocaine to relieve pain at the site of injection. Generally, the components are either separately or mixed together in a single dosage form, eg, in ampoules or sachets (s) showing a fixed amount of active agent.
supplied as a lyophilized powder or water-free concentrate in a sealed container such as an achette). 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, the ingredients may be mixed prior to administration,
Ampoules of sterile water for injection or saline can be provided.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
Mention may be made of salts formed with cations such as those derived from 2-ethylaminoethanol, histidine, procaine and the like.

The amount of a compound of the invention that is effective in this treatment (suppression and prevention of diseases or disorders associated with aberrant expression and / or activity of the polypeptides of the invention) will be determined by standard clinical techniques. Can be done. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. Precise dosages to be used in the formulation will also depend on the route of administration, and the severity 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 to 100 mg per kg patient body weight. Preferably, the dosage administered to a patient is between 0.1 mg and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, 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, low dosages and infrequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies of the invention may be altered (eg,
It can be reduced by enhancing antibody uptake by lipidation and the like and tissue penetration (eg into the brain).

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Notifications in the form prescribed by governmental agencies regulating the manufacture, use or sale of pharmaceutical or biological products may optionally be accompanied by such containers. This notice
It reflects the agency's approval of manufacture, use or sale for human administration.

Diagnostic and Imaging Labeled antibodies that specifically bind to the polypeptide of interest, as well as derivatives and analogs thereof, have been used for diagnostic purposes to aberrate expression and abnormal expression of the polypeptides of the invention. Diseases and / or disorders associated with activity can be detected, diagnosed or monitored. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which comprises (a) using one or more antibodies specific for the polypeptide of interest to target in a cell or body fluid of an individual. Assaying the expression of the polypeptide of
And (b) comparing the level of gene expression to the level of standard gene expression, whereby increasing or decreasing the level of assayed polypeptide gene expression compared to the standard level of expression. Shows abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, which assay comprises (
a) assaying the expression of the polypeptide of interest in cells or fluids of an individual using one or more antibodies specific for the polypeptide of interest, and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. With respect to cancer, the presence of relatively high amounts of transcripts 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 onset of actual clinical symptoms. Can be provided. A more definitive diagnosis of this type may allow health professionals to use preventative measures or early aggressive treatment, thereby preventing the development or further progression of cancer.

The antibodies of the invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those of skill in the art (eg, Jalk).
anen et al. Cell. Biol. 101: 976-985 (1985);
Jalkanen et al. Cell. Biol. 105: 3087-3096 (
1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and are enzymatic labels (eg glucose oxidase); radioisotopes (eg iodine (125I, 1, 1
21I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg luminol); and fluorescent labels (eg fluorescein and rhodamine), and Examples include biotin.

One aspect of the present invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the polypeptide of interest in animals, preferably mammals, and most preferably humans. In one embodiment, diagnosis is a) administering to a subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds to a polypeptide of interest. B) to allow the labeled molecule to preferentially concentrate at the site in the subject where the polypeptide is expressed (and to remove unbound labeled molecule to background levels) A) waiting for a time interval after administration; c) determining the background level; and d) detecting the labeled molecule in the subject, so that the label is above this background level. Detection of the activating molecule 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 a variety of methods, including comparing the amount of labeled molecule detected to standard values previously determined for a particular system.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, the amount of radioactivity injected for a human subject will usually be about 5
It is in the range of 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein.
In vivo tumor imaging is described by S. W. Burchiel et al., "Immunophar
macokinetics of Radiolabeled Antibod
ies and Their Fragments "(Tumor Image
ng: The Radiochemical Detection of Ca
ncer, Chapter 13, S. W. Burchiel and B.I. A. Rhodes, Masson Publishing Inc. (1982).

Depending on a number of variables, including the type of label used and the mode of administration, the labeled molecule will preferentially concentrate at the site of the subject and will not be bound. The time interval after administration is 6-48 hours or 6-24 hours or 6-12 hours, which allows the C. to be cleared to background levels.
In another embodiment, the post-administration time interval is 5-20 days or 5-10 days.

In one embodiment, monitoring a disease or disorder comprises repeating the method for diagnosing the disease or disorder (eg, 1 month after the first diagnosis, 6 months after the first diagnosis, the first diagnosis). 1 year later).

The presence of labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art can determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the invention include, but are not limited to, computer tomography (CT), whole body scanning such as position emission tomography (PET), magnetic resonance imaging (MRI). , And ultrasonic diagnostic methods.

In certain embodiments, the molecule is radioisotope-labeled and detected in a patient using a radiation-responsive surgical instrument (Thurston et al., US Pat. 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 patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).

(Kit) The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody of the invention (preferably a purified antibody) in one or more containers. In certain embodiments, the kits of the invention comprise a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies included in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another specific embodiment, a kit of the invention provides for the binding of an antibody to a polypeptide of interest (e.g., the antibody has a detectable substrate (e.g., the antibody is a fluorescent compound, an enzyme substrate, a radioactive compound or The second antibody, which may be conjugated to a luminescent compound) or which recognizes the first antibody, may be conjugated to a detectable substrate).

In another particular embodiment of the invention, the kit is for diagnostic use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. It's a kit. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen that comprises an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such kits are for detecting the binding of the antibody to an antigen (eg, the antibody can be conjugated with a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means are provided. In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of this kit can also be attached to a solid support.

In a more particular embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is attached. Such a kit also comprises a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further embodiment, the invention comprises a diagnostic kit for use in screening serum containing antigens of the polypeptides of the invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody is
Attached to a solid support. In a particular embodiment, the antibody may be a monoclonal antibody. The detection means of this kit may include a second labeled monoclonal antibody. Alternatively, or additionally, the detection means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having surface bound antigen obtained by the method of the invention. After binding the specific antigen antibody with this reagent and removing the unbound serum components by washing, in order to bind the reporter with this reagent, depending on the amount of anti-antigen antibody bound on the solid support, This reagent is reacted with a reporter-labeled anti-human antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

The solid surface reagents in the above assay are used to convert protein material to solid support material (
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96-well plates or filtration material). These methods of attachment generally include non-specific adsorption of proteins to the support or chemically active groups (eg, activated carboxyl groups, hydroxyl groups, or aldehyde groups) on the solid support. Covalent attachment of proteins (typically via free amine groups). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

The invention thus provides an assay system or kit for carrying out this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins Any polypeptide of the invention can be used to produce a fusion protein. For example, the polypeptides of the present invention can be used as an antigenic tag when fused to a second protein. Antibodies raised against the polypeptides of the invention can be used to indirectly detect a second protein by binding to the polypeptide. Moreover, since secreted proteins target cell locations based on transport signals, the polypeptides of the invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused with the polypeptides of the invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily have to be direct, but can occur via a linker sequence.

In addition, fusion proteins can also be engineered to improve the characteristics of the polypeptides of the invention. For example, additional amino acids, especially regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and durability during purification from host cells 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. Addition of peptide moieties to facilitate handling of polypeptides is a conventional technique well known in the art.

Furthermore, the polypeptides (including fragments, and especially epitopes) of the present invention include immunoglobulin (IgA, IgE, IgG, IgM) constant domains or portions thereof (CH1, CH2, CH3, and any thereof). Combination of (
(Including both whole domains and parts thereof)) to give chimeric polypeptides. These fusion proteins facilitate purification and exhibit increased half-life in vivo. One reported example is human CD4
A chimeric protein consisting of the first two domains of the polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins has been described (EP A 394,827; Traunecker et al. Nature 33).
1: 84-86 (1988)). Fusion proteins with a disulfide-linked dimeric structure (due to IgG) may also be more efficient at binding and neutralizing other molecules than monomeric secretory proteins or protein fragments alone (Funtoulakis et al. , J. Biochem. 270: 395.
8-3964 (1995)).

Similarly, EP-A-O 464 533 (Canadian patent 2045869).
No.) discloses fusion proteins comprising various parts of the constant regions of immunoglobulin molecules together with another human protein or parts thereof. In many cases, the Fc portion of fusion proteins may be beneficial in therapy and diagnosis, thus resulting, for example, in improved pharmacokinetic properties (EP-A 0232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 have been fused to Fc moieties for the purpose of high throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al. J. Molecular Reco
gnition 8: 52-58 (1995); Johnson et al.
Biol. Chem. 270: 9459-9471 (1995)).
.

Furthermore, the polypeptides of the present invention can be fused to a marker sequence (eg, a peptide that facilitates purification of the fused polypeptide). In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg p
QE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, CA, 91311)),
Many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1
989), hexa-histidine provides 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 (Wi.
Lson et al., Cell 37: 767 (1984)).

Thus, any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention.

Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotides of the present invention, host cells, and the production of polypeptides by recombinant techniques. For example, the vector can be a phage vector, a plasmid vector, a viral vector, or a retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation generally will be complete (complementary).
only occurs in host cells.

The polynucleotide may be ligated to a vector containing a selectable marker for propagation in the host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

[0349] The polynucleotide insert may include any suitable promoter (eg, phage λPL promoter, E. coli lac promoter, trp promoter, phoA promoter and tac promoter, SV40 early promoter and SV40 late promoter, and retrovirus, to name but a few). Should be operably linked to the LTR promoter). Other suitable promoters are known to those of skill in the art. The expression construct further comprises sites for transcription initiation, transcription termination,
And in the transcribed region, it contains a ribosome binding site for translation. The coding portion of the transcript expressed by this construct is preferably a translation initiation codon initially, and a termination codon (U) appropriately located at the end of the polypeptide to be translated.
AA, UGA or UAG).

As shown, the expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance genes for eukaryotic cell culture, as well as E. coli. It contains a tetracycline, kanamycin or ampicillin resistance gene for cultivation in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells (eg E. c.
oli, Streptomyces and Salmonella typhim
urium cells); fungal cells (eg yeast cells); insect cells (eg Dros)
Ophilia S2 and Spodoptera Sf9 cells); animal cells (
CHO cells, COS cells, 293 cells and Bowes melanoma cells); and plant cells, but are not limited thereto. Appropriate culture media and conditions for the above-described host cells are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE6.
0 and pQE-9 (available from QIAGEN, Inc.); pBluesc
ript vector, Phagescript vector, pNH8A, pNH16
a, pNH18A, pNH46A (Stratagene Cloning S
systems, Inc. And ptrc99a, pKK223.
-3, pKK233-3, pDR540, pRIT5 (Pharmacia B
iotech, Inc. Available from). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (S
(available from tratagene); and pSVK3, pBPV, pMSG
And pSVL (available from Pharmacia). Preferred expression vectors for use in yeast systems include pYES2, pYD1, pTEF1
/ Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph,
pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.
5K, pPIC9K, and PAO815 (all from Invitrogen, C
(available from Arlbad, CA), but is not limited thereto. Other suitable vectors will be readily apparent to those of skill in the art.

Introduction of the construct into the host cell is carried out by calcium phosphate transfection, DE
It can be provided by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described by Davis et al., Basic Metho.
It is described in many standard laboratory manuals such as ds In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by host cells lacking the recombinant vector.

The polypeptides of the present invention may be ammonium sulfate precipitated or ethanol precipitated, acid extracted, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectins. It can be recovered from recombinant cell culture and purified by well known methods, including chromatography. Most preferably high performance liquid chromatography (“HPLC”)
Is used for purification.

The polypeptides of the invention, and preferably secreted forms, may also be recovered from: purified from natural sources, including body fluids, tissues and cells, whether directly isolated or cultured. A product of a chemical synthesis procedure; and
Products recombinantly produced from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host used in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the present invention may also, in some cases, include an initial modified methionine residue as a result of host-mediated processes. Therefore, it is well known in the art that, in general, the N-terminal methionine encoded by the translation initiation codon is highly efficiently removed from any post-translational protein in all eukaryotic cells. N-terminal methionine is also effectively removed in most prokaryotes in most proteins, but for some proteins this prokaryotic removal process depends on the nature of the amino acids to which the N-terminal methionine is covalently attached. And is inefficient.

In one embodiment, the yeast Pichia pastoris is used to express a polypeptide of the invention in a eukaryotic cell line. Pichi
a pastoris is a methyltrophic yeast that can metabolize with methanol as its sole carbon source. The major step in the methanol metabolism pathway is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pas
toris produces high levels of alcohol oxidase, due in part to the relatively low affinity of alcohol oxidase for O ₂ . Consequently, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active in growth media that relies on methanol as the major carbon source. In the presence of methanol, the alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of total soluble protein in Pichia pastoris. Ellis, S .; B. Et al., Mol. Cell.
Biol. 5: 1111-21 (1985); Koutz, P .; J. Et al, Yea
st 5: 167-77 (1989); Tschopp, J .; F. Nucl
． Acids Res. 15: 3859-76 (1987). Therefore, AOX1
Heterologous coding sequences under transcriptional regulation of all or part of the regulatory sequences, such as the polynucleotides of the invention, are expressed at exponentially high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K was transformed into "Pichia P
rotocols: Methods in Molecular Biolog
y ", D.I. R. Higgins and J.M. Edited by Cregg (The Humana
Press, Totowa, NJ, 1998), and is used to express DNA encoding a polypeptide of the invention as shown herein in the Pichea yeast system, essentially as described. This expression vector is a Pichia pastoris located upstream of the multiple cloning site.
The strong AOX1 promoter linked to the alkaline phosphatase (PHO) secretion signal peptide (ie leader) allows expression and secretion of the proteins of the invention.

Many other yeast vectors (eg pYES2, pYD1, pTEF1 / Ze
o, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pP
IC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K
, And PAO815) are arranged appropriately such that transcription, translation, secretion (if desired), etc., including an in-frame AUG when the proposed expression construct is required, as will be readily appreciated by those of skill in the art. Can be used in place of pPIC9K as long as it provides a good signal.

In another embodiment, high level expression of a heterologous coding sequence (eg, a polynucleotide of the invention, etc.) involves cloning the heterologous polynucleotide of the invention into an expression vector (eg, pGAPZ or pGAPZalpha). , And can be achieved by growing the yeast culture in the absence of methanol.

[0359] In addition to including host cells containing the vector constructs discussed herein, the present invention also includes primaries of vertebrate origin, particularly mammalian origin.
y) Includes host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, coding sequences) and / or genetic material operably linked to a polynucleotide of the invention (eg, A heterologous polynucleotide sequence) and activates, alters, and / or amplifies the endogenous polynucleotide. For example, techniques known in the art can be used to operably link heterologous control regions (eg, promoters and / or enhancers) and endogenous polynucleotide sequences via homologous recombination, such that Form a new transfer unit (eg, US Pat. No. 5,641,670 issued June 24, 1997; US Pat. No. 5,7,31 issued March 31, 1998).
33,761; International Publication No. WO 96/2, published September 26, 1996.
9411; International Publication No. WO 94/1265, published on August 4, 1994.
No. 0; Koller et al., Proc. Natl. Acad. Sci. USA 86
: 8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989). Each of these disclosures
Incorporated as a reference in their entirety).

In addition, the polypeptides of the invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Stru).
cultures and Molecular Principles, W.C. H.
Freeman & Co. , N .; Y. And Hunkapiller et al., Na
Tur., 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide sequence of the present invention can be synthesized by using 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, the common amino acid D isomers, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4
-Aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline , Homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
Phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. In addition, the amino acids can be D (dextrorotatory) or L (levorotatory).

The present invention may be used during or after translation, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular agents. It includes polypeptides that are differentially modified, such as by binding to a ligand. Any of numerous chemical modifications, including the following may be carried out by known, but not limited to techniques: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage by NaBH _4; acetylation, Formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Further post-translational modifications included by the present invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone. Attachment, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label such as an enzymatic label, a fluorescent label, an isotope label or an affinity label to allow detection and isolation of the protein.

The present invention also provides chemically modified derivatives of the polypeptides of the present invention that may provide additional advantages such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. (See US Pat. No. 4,179,337). The chemical moieties for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three or more attached chemical moieties.

The polymer can be a polymer of any molecular weight and can be branched or unbranched. For polyethylene glycol, a preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term "about" refers to some molecules in the preparation of polyethylene glycol Heavy, and some indicate lighter than the indicated molecular weight). The desired therapeutic profile (eg, desired duration of sustained release, effect on biological activity in some cases, ease of handling, degree of antigenicity or lack of antigenicity, and therapeutic protein or analog). Other sizes may be used, depending on other known effects of polyethylene glycol).

The polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domains of this protein. There are numerous coupling methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF to P, incorporated herein by reference).
EG)), Malik et al., Exp. Hematol. 20: 1028-
1035 (1992) (pegylation of GM-CSF with tresyl chloride).
See also report)))). For example, polyethylene glycol can be covalently attached via a amino acid residue by a reactive group such as a free amino or carboxyl group. Reactive groups are groups to which activated polyethylene glycol molecules can be attached. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Residues may be mentioned. Sulfhydryl residues can also be used as a reactive group to attach the polyethylene glycol molecule. Preferred for therapeutic purposes is a bond at the amino group, eg at the N-terminus or at the lysine group.

Proteins that are chemically modified at the N-terminus may be specifically desired. Using polyethylene glycol as an illustration of the composition of the invention, from various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, the pegs to be performed. The type of oxidization reaction and the method of obtaining the selected N-terminal pegylated protein can be selected. The method of obtaining the N-terminal PEGylated preparation (ie, separating this moiety from other mono-PEGylated moieties, if necessary) is performed by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. The selective protein chemically modified by N-terminal modification is
It can be achieved by reductive alkylation, which takes advantage of the differential reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in a particular protein. Under appropriate reaction conditions, substantially selective derivatization of proteins at the N-terminus with a carbonyl group-containing polymer is achieved.

Polypeptides of the invention can be monomers or multimers (ie, dimers,
Trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomeric and multimeric polypeptides of the invention, their preparation and compositions (preferably therapeutic agents) containing them. In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further embodiment, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the present invention can be homomers or heteromers. As used herein, the term homomer refers to the polypeptides encoded by the cDNAs corresponding to the amino acid sequence of SEQ ID NO: Y or contained in the deposited clone (these polypeptides described herein. A multimer containing only fragments (including fragments, variants, splice variants and fusion proteins) corresponding to peptides. These homomers may include polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers that only include polypeptides that have the same amino acid sequence. In another particular embodiment, the homomers of the invention are multimers that include polypeptides with different amino acid sequences. In certain embodiments, the multimers of the invention are homodimers (eg, containing polypeptides having the same or different amino acid sequences) or homotrimers (eg, containing polypeptides having the same and / or different amino acid sequences). .. In a further embodiment, the homomeric multimers of the invention are at least homodimers, at least homotrimers or at least homotetramers.

As used herein, the term heteromer refers to a multimer that includes one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the polypeptide of the invention. In a particular embodiment, the multimers of the invention are heterodimers, heterotrimers or heterotetramers. In a further embodiment, the heteromeric multimers of the invention are at least heterodimers, at least heterotrimers or at least heterotetramers.

The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent attachment and / or may be indirectly linked, eg by liposome formation. Thus, in one embodiment, multimers of the invention (eg, homodimers or homotrimers) are formed when the polypeptides of the invention contact each other in solution. In another embodiment, the heteromultimer of the invention (
(Eg, heterotrimers or heterotetramers) forms when a polypeptide of the invention is contacted in solution with an antibody to a polypeptide of the invention, including an antibody to a heterologous polypeptide sequence in a fusion protein of the invention. To be done. In other embodiments, the multimers of the invention are formed by covalent attachment to and / or between polypeptides of the invention. Such a covalent bond may include one or more amino acid residues contained in a polypeptide sequence (eg, a polypeptide sequence described in the sequence listing or contained in a polypeptide encoded by a deposited clone). May be included. In one example, this covalent bond is
A bridge between cysteine residues that are present within a polypeptide sequence that interact in a native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of chemical or recombinant manipulation. Alternatively, such a covalent bond may comprise one or more amino acid residues contained in the heterologous polypeptide sequence in the fusion protein of the invention.

In one example, the covalent bond is between the heterologous sequences contained in the fusion protein of the invention (see, eg, US Pat. No. 5,478,925). In a particular example, this covalent bond is between the heterologous sequences contained in the Fc fusion protein of the invention (as described herein). In another particular example, the covalent attachment of the fusion protein of the invention is between heterologous polypeptide sequences from another protein, such as osteoprotegerin, which is capable of forming a covalently linked multimer (eg, osteoprotegerin). , International Publication No. WO 98/49305, the contents of which are incorporated herein by reference in their entirety). In another embodiment, two or more polypeptides of the invention are linked through a peptide linker. Examples include the peptide linkers described in US Pat. No. 5,073,627, which is incorporated herein by reference. Proteins comprising multiple polypeptides of the invention separated by a peptide linker can be produced using conventional recombinant DNA techniques.

Another method for preparing the multimeric polypeptides of the invention involves the use of the polypeptides of the invention fused to a leucine zipper polypeptide sequence or an isoleucine zipper polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. The leucine zipper originally had some DN
Identified in A-binding proteins (Landschulz et al., Science)
240: 1759, (1988)), and since then in a variety of different proteins. Particularly known leucine zippers are naturally occurring peptides and their derivatives that form dimers or trimers. Examples of leucine zipper domains suitable for producing the soluble multimeric proteins of the present invention include PCT application WO 94/10, incorporated herein by reference.
The leucine zipper domain described at 308. Recombinant fusion proteins comprising a polypeptide of the invention fused to a dimer- or trimer-forming polypeptide sequence in solution are expressed in a suitable host cell, and the resulting soluble multimeric fusion proteins are Are recovered from the culture supernatant using techniques known in.

The trimer polypeptides of the present invention may provide the advantage of enhanced biological activity. Preferred leucine zipper and isoleucine moieties are those that preferentially form trimers. One example is Ho, which is incorporated herein by reference.
Leucine zipper derived from pulmonary surfactant protein D (SPD) as described in ppe et al. (FEBS Letters 344: 191, (1994)) and US patent application Ser. No. 08 / 446,922. Other peptides derived from naturally occurring trimer proteins can be used in the preparation of the trimer polypeptides of the invention.

In another example, a protein of the invention associates through an interaction between Flag® polypeptide sequences contained in a fusion protein of the invention that includes a Flag® polypeptide sequence. In a further embodiment, the protein of the invention is associated by interaction between the heterologous polypeptide sequence contained in the Flag® fusion protein of the invention and the anti-Flag® antibody.

The multimers of the present invention can be produced using chemical techniques known in the art. For example, the polypeptides desired to be included in the multimers of the present invention can be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (eg, herein See US Pat. No. 5,478,925, which is incorporated by reference in its entirety). In addition, the multimers of the invention can be generated using techniques known in the art to create one or more intermolecular bridges between cysteine residues present within the sequence of the polypeptide desired to be included in the multimer. Can be formed (eg, US Pat. No. 5,656, incorporated herein by reference in its entirety).
478,925). Furthermore, the polypeptides of the present invention can 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 are known to include one or more of these modified polypeptides. It can be applied to generate multimers containing peptides (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, techniques known in the art can be applied to generate liposomes containing the polypeptide components desired to be included in the multimers of the invention (eg, incorporated herein by reference in its entirety). US Pat. No. 5,4
78,925).

Alternatively, the multimers of the invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptides included in the multimers of the invention are recombinantly produced using fusion protein technology described herein or otherwise known in the art (eg, herein). See US Pat. No. 5,478,925, which is incorporated by reference in its entirety). In certain embodiments, a polynucleotide encoding a homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention linked to a sequence encoding a linker polypeptide, and then further from the original C-terminus to the N-terminus. Produced by ligating to a synthetic polynucleotide (lacking a leader sequence) that encodes the translation product of the polypeptide in the opposite direction to that of (eg, US Pat. See Japanese Patent No. 5,478,925). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to include a transmembrane domain (or hydrophobic peptide or signal peptide),
And produce a recombinant polypeptide of the invention that can be incorporated into liposomes by membrane reconstitution techniques (eg, incorporated herein by reference in its entirety,
See U.S. Pat. No. 5,478,925).

Uses of Polynucleotides Each of the polynucleotides identified herein can be used as reagents in a number of ways. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. Chromosome marking reagents based on actual sequence data (repeated polymorphisms) are currently scarcely available, so there remains a need to identify new chromosomal markers. Each polynucleotide of the present invention can be used as a chromosomal marker.

Briefly, the sequence can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp) from the sequence shown in SEQ ID NO: X. Primers can be selected using computer analysis so that they do not span more than one predicted exon in genomic DNA. These primers are then used to bind P of somatic cell hybrids containing individual human chromosomes.
Used for CR screening. Only the hybrid containing the human gene corresponding to SEQ ID NO: X produces an amplified fragment.

[0380] Similarly, somatic cell hybrids carry the polynucleotide P to a particular chromosome.
It provides a rapid method for CR mapping. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotide can be accomplished using a panel of specific chromosomal fragments. Other gene mapping strategies that may be used are in situ hybridization, labeled flow sorted.
flow sorted) pre-screening on the chromosome and pre-selection by hybridization to construct a chromosome-specific cDNA library.

The exact chromosomal location of the polynucleotide is also determined by the metaphase chromosomal spread (spr
ead) fluorescence in situ hybridization (FISH). This technique uses polynucleotides as short as 500 or 600 bases; however, 2,000-4,000 bp polynucleotides are preferred. For a review of this technology, see Verma et al., “Human Chromoso.
mes: a Manual of Basic Technologies ", P
See ergamon Press, New York (1988).

For chromosome mapping, polynucleotides may be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (to mark multiple sites and / or multiple chromosomes). Can be done. Preferred polynucleotides correspond to the non-coding regions of cDNA. Because coding sequences tend to be more conserved within gene families, which increases the chance of cross-hybridization during chromosome mapping.

Once the polynucleotide has been mapped to a precise chromosomal location, the physical location of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between the chromosomal location and the presentation of a particular disease. (Disease mapping data is described in, for example, V. McKusick, Mendel.
ian Inheritance in Man (Johns Hopkins
(Available online through the University Welch Medical Library)). Given 1 megabase mapping resolution and 1 gene per 20 kb, the cDNA precisely localized to the chromosomal region associated with disease could be one of 50-500 potential causative genes.

Thus, once co-inheritance is established, differences in polynucleotides and corresponding genes between affected and unaffected individuals can be tested. First, visible structural changes in the chromosome (eg, deletions or translocations) are examined in chromosome spreads or by PCR. In the absence of structural changes, the presence of point mutations is confirmed. Mutations observed in some or all affected individuals but not in normal individuals indicate that this mutation may cause the disease.
However, complete sequencing of polypeptides and the corresponding genes from some normal individuals is required to distinguish mutations from polymorphisms. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

In addition, increased or decreased expression of genes in affected individuals compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these changes (
Altered expression, chromosomal rearrangements, or mutations) can be used as a diagnostic or prognostic marker.

Accordingly, the present invention also provides a diagnostic method useful during the diagnosis of disorders, which method comprises the step of measuring the expression level of the polynucleotide of the present invention in cells or body fluids from an individual, and Comprising a step of comparing the measured gene expression level to a standard level of polynucleotide expression level, whereby an increase or decrease in gene expression level relative to a standard is indicative of a disorder.

In yet another embodiment, the invention comprises a kit for analyzing a sample for the presence of proliferative and / or cancerous polynucleotides from a test subject. In a general embodiment, the kit comprises at least one polynucleotide probe containing a nucleotide sequence that specifically hybridizes to a polynucleotide of the invention, and a suitable container. In this particular embodiment, the kit comprises two polynucleotide probes defining an internal region of a polynucleotide of the invention, wherein each probe comprises a 31 'mer end internal to this region. Has one chain. In a further embodiment, this probe may be useful as a primer for polymerase chain reaction amplification.

The present invention is useful as a prognostic indicator when the diagnosis of a disorder has already been made by conventional methods, whereby patients exhibiting enhanced or suppressed polynucleotide expression of the present invention are closer to standard levels. Experience poor clinical outcome compared to patients who express this gene at levels.

By “measuring the expression level of the polynucleotide of the present invention”, the level of the polypeptide of the present invention or the mRNA encoding this polypeptide is directly measured in the first biological sample. (Eg, by determining or assessing absolute protein or mRNA levels) or relative (
For example, it is intended to be qualitatively or quantitatively measured or assessed (either by comparison with polypeptide levels or mRNA levels in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or evaluated and compared to a standard polypeptide level or mRNA level, the standard being from an unimpaired individual. Determined by averaging the levels obtained from the second biological sample obtained or from a population of unimpaired individuals. As will be appreciated in the art, once standard polypeptide or mRNA levels are known, this 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 that contains a polypeptide or mRNA of the invention. As shown, the biological sample is capable of expressing a body fluid containing the polypeptide of the invention (eg, semen, lymph, serum, plasma, urine, synovial fluid and spinal fluid), and the polypeptide of the invention. Includes other tissue sources found. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, tissue biopsy is a preferred source.

The methods provided above may preferably be applied to diagnostic methods and / or kits in which the polynucleotides and / or polypeptides are bound to a solid support. In one exemplary method, the support is described in US Pat. Nos. 5,837,832, 5,874,219 and 5,856,174, "
It can be a "gene chip" or a "biological chip". Further, such gene chips to which the polynucleotides of the invention are attached can be used to identify polymorphisms between this polynucleotide sequence and polynucleotides isolated from the test subject. Knowledge of such polymorphisms (ie their location as well as their presence) is useful in identifying disease loci for many disorders, including cancerous diseases and conditions. Such methods are described in US Pat. Nos. 5,858,659 and 5,856,104. The United States patents referenced above are incorporated herein by reference in their entireties.

The present invention includes polynucleotides of the present invention that are chemically synthesized (ie, reproduced as peptide nucleic acid (PNA)) or synthesized according to other methods known in the art. The use of PNA serves as the preferred form when the polynucleotide is incorporated into a solid support, ie a gene chip. For the purposes of the present invention, peptide nucleic acid (PNA) is a DNA analogue of the polyamide type, and monomer units for adenine, guanine, thymine and cytosine are commercially available (Perceptice Biosystems). DNA
Specific components of P, such as phosphorus, phosphorus oxide or deoxyribose derivatives, are
Not present in NA. P. E. Nielsen, M .; Egholm, R.A. H. B
erg and O.D. Buchardt, Science 254, 1497 (19
97); Egholm, O.I. Buchardt, L .; Christ
ensen, C.I. Behrens, S .; M. Freier, D.F. A. Drive
r, R. H. Berg, S .; K. Kim, B.H. Norden and P.M. E. Ni
As disclosed by Elsen, 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 does DNA itself. This is probably due to the absence of electrostatic repulsion between the two chains and also the more flexible polyamide backbone. by this,
PNA / DNA duplexes bind under a wider range of stringency conditions than DNA / DNA duplexes, making it easier to perform multiplex hybridization. Due to the strong binding, smaller probes can be used than with DNA. Furthermore, perhaps single base mismatches can be determined using PNA / DNA hybridizations. Because the single mismatch in the 15-mer PNA / DNA is 4 ° C to 16 ° C for the 15-mer DNA / DNA duplex.
On the other hand, the melting point (T.sub.m) is lowered by 8 to 20 ° C. Also, the absence of charged groups in the PNA means that hybridization can be done at low ionic strength and reduce possible interference by salts during the analysis.

The present invention is useful for the detection of cancer in mammals. In particular, the invention is useful during the diagnosis of pathological cell proliferative neoplasia, including but not limited to: acute myeloid leukemia (acute monocytic leukemia, acute myeloblastic leukemia, acute Including promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia); and chronic myelogenous leukemia (chronic myelomonocytic leukemia, chronic granulocytic leukemia) Including leukemia). Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Especially preferred is human.

Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes (
Gelmann, E .; P. Et al., “The Etiology of Accuet”
Leukemia: Molecular Genetics and Vir
al Oncology ”, Neoplastic Diseases of
the Blood, Volume 1, Wiernik, P .; H. Et al., 161-182
(1985)). Neoplasia is now from qualitative alterations of the normal cellular gene product, by the insertion of viral sequences into the chromosome, chromosomal translocations of genes into more actively transcribed regions, or by some other mechanism, or of gene expression. It is believed to result from quantitative modification (Gelmann et al., Supra). Mutated or altered expression of certain genes appears to be involved in the pathogenesis of some leukemias, among other tissues and other cell types (Gelmann et al., Supra). In fact, human counterparts of some animal neoplastic oncogenes have been amplified or translocated in some cases of human leukemia and cancer (Gelmann et al., Supra). For example, c-my
c expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. H
The levels of c-myc are found to be downregulated when L-60 cells are chemically induced to stop amplification (International Publication No. WO 91/1.
5580). However, exposure of HL-60 cells to a DNA construct that is complementary to the 5'end of c-myc or c-myb was associated with c-myc protein or cm
It has been shown to block translation of the corresponding mRNA that downregulates expression of the yb protein, causing arrest of cell growth and differentiation of treated cells (International Publication No. WO 91/15580; Wickstrom et al., Proc.
． Natl. Acad. Sci. 85: 1028 (1988); Anfossi.
Et al., Proc. Natl. Acad. Sci. 86: 3379 (1989)).
However, one of ordinary skill in the art will find the utility of the present invention in view of the large number of cells and cell types of various origins known to exhibit a proliferative phenotype, proliferative diseases, disorders of hematopoietic cells and tissues. And / or recognize that treatment of the condition is not limited.

In addition to the above, the polynucleotide may be triple helix formed or antisense DN.
It can be used to control gene expression through A or RNA. Antisense technology is described in, for example, Okano, J. et al. Neurochem. 56: 560 (1
991), "Oligodeoxynucleotides as Antis.
Sense Inhibitors of Gene Expression ",
CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, in Lee et al., Nucleic Acids Re.
search 6: 3073 (1979); Cooney et al., Science.
241: 456 (1988); and Dervan et al., Science 251.
: 1360 (1991). Both methods rely on the binding of the polynucleotide to complementary DNA or RNA. For these techniques, the preferred polynucleotides are usually oligonucleotides 20-40 bases in length, and the gene regions involved in transcription (triple helix-Lee et al., Nucl. A).
cids Res. 6: 3073 (1979); Cooney et al., Science.
e 241: 456 (1988); and Dervan et al., Science 2
51: 1360 (1991)) or the mRNA itself (antisense-Okano, J. Neurochem. 56: 560 (1991); Olig.
odeoxy-nucleotides as Antisense Inhi
bits of Gene Expression, CRC Press,
Boca Raton, FL (1988)). Triple helix formation optimally results in the blockage of RNA transcription from DNA, whereas antisense RNA hybridization blocks translation of mRNA molecules into polypeptides. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an attempt to treat disease.

The 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 that has a defective gene in an attempt to correct the genetic defect. The polynucleotide disclosed in the present invention is
It provides a means to target such genetic defects in a highly accurate manner. Another goal is to insert new genes that were not present in the host genome, thereby creating new traits in the host cell.

Polynucleotides are also useful for identifying an individual from a small biological sample. For example, the United States military is considering the use of restriction fragment length polymorphisms (RFLPs) to identify its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed for Southern blots to generate unique bands to identify staff. This method does not suffer from the current limitations of the "Dog tag," which can be difficult to positively identify by being lost, exchanged, or stolen. The polynucleotide of the present invention can be used as an additional DNA marker for RFLP.

The polynucleotides of the present invention may also be used as an alternative to RFLP by determining the actual base-by-base DNA sequence of selected portions of the genome of an individual. These sequences can be used to prepare PCR primers to amplify and isolate such selected DNA, which can then be sequenced. Individuals can be identified using this technique because each individual has a unique set of DNA sequences. Once a unique ID database is established for an individual, whether that individual is alive or dead, positive identification of that individual can be made from a very small tissue sample.

Forensic biology will also benefit from the use of DNA-based identification techniques as disclosed herein. Tissue (eg hair or skin) or body fluid (eg blood, saliva, semen, synovial fluid, amniotic fluid, milk, lymph, lung sp (pulmonary sp)
DNA) obtained from a very small biological sample, such as urium) or surfactant, urine, fecal material, etc.) can be amplified using PCR.
In one prior art, gene sequences amplified from polymorphic loci such as the DQa class II HLA gene are used in forensic biology to identify individuals. (Errich, H., PCR Technology, Freem.
an and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes. This results in an identified set of bands for Southern blots probed with DNA corresponding to the DQa class II HLA gene. Similarly, the polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents that can identify the source of a particular tissue. Such a need arises in forensic medicine, for example, if tissue of unknown origin is provided. Suitable reagents may include, for example, DNA probes or primers specific to a particular tissue prepared from the sequences of the invention. Such a panel of reagents may identify tissues by species and / or organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contaminants.

At a minimum, the polynucleotides of the invention “subtract from known sequences in the process of discovering novel polynucleotides, as molecular weight markers in Southern gels, as diagnostic probes for the presence of specific mRNAs in specific cell types. As a probe for "selecting and making oligomers for attachment to a" gene chip "or other support, for raising anti-DNA antibodies using DNA immunization techniques, and for eliciting an immune response. Can be used as

Uses of Polypeptides Each of the polypeptides identified herein can be used in a number of ways. The following description should be considered exemplary and utilizes known techniques.

The polypeptides of the present invention can be used to assay protein levels in biological samples using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistochemistry (Jalka).
nen, 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 enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include, for example, enzyme labels such as glucose oxidase, as well as iodine (125I, 121I), carbon (14I).
C), sulfur (35S), tritium (3H), indium (112In), and radioisotopes such as technetium (99mTc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in biological samples, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of proteins include those detectable by X-ray radiography, NMR or ESR. For x-ray radiography, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not overtly harmful to the subject. NM
Suitable markers for R and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into antibodies by labeling the nutrients for the relevant hybridoma. .

Protein-specific, labeled with a suitable detectable imaging moiety such as a radioisotope (eg, 131I, 112In, 99mTc), radiopaque material, or material detectable by nuclear magnetic resonance. The antibody or antibody fragment is introduced into a mammal (eg, parenterally, subcutaneously, or intraperitoneally). It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety,
For human subjects, the amount of radioactivity injected is usually about 5-20 mTc.
It is in the millimeter range. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein. In vivo tumor imaging is described by S. W. Burchiel et al., “Immunopharmacokine
tics of Radiolabeled Antibodies and
Their Fragments. "(Tumor Imaging: The
The first of Radiochemical Detection of Cancer
Chapter 3, S. W. Burchiel and B.I. A. Rhodes, Masson
Publishing Inc. (1982)).

Accordingly, the invention provides a method of diagnosing a disorder, which method comprises (a) assaying the expression of a polypeptide of the invention in cells or body fluids of an individual; (b) the level of expression of this gene. Is compared to a standard gene expression level, whereby an increase or decrease in the gene expression level of the assayed polypeptide relative to the standard expression level is indicative of a disorder. For cancer, may the presence of relatively high abundance of transcripts in biopsied tissue from an individual predispose to the development of this disease?
Alternatively, it may provide a means to detect this disease prior to the onset of actual clinical symptoms.
A more definitive diagnosis of this type may allow health professionals to use earlier preventative measures or aggressive treatments, thereby preventing the onset or further progression of cancer.

Furthermore, the polypeptides of the present invention may be used to treat diseases, prophylaxis and / or diseases. For example, in a patient, replacing the absence or reduced level of a polypeptide (eg, insulin) with a different polypeptide (eg, hemoglobin B).
Absent or reduced levels of hemoglobin S, SOD, catalase, DNA repair proteins) against, inhibition of the activity of the polypeptide (eg, oncogene or tumor suppressor), activity of the polypeptide (eg,
Activating (by binding to a receptor), a free ligand (eg,
A soluble TNF receptor used in reducing inflammation) by reducing the activity of the membrane-bound receptor by competing with the membrane-bound receptor,
Alternatively, the polypeptides of the invention may be administered in addressing the production of a desired response (eg, inhibition of vascular growth, enhancement of immune response to proliferating cells or tissues).

Similarly, antibodies against the polypeptides of the present invention may also be used to treat, prevent and / or diagnose disease. For example, administration of an antibody to a polypeptide of the invention may bind to the polypeptide and reduce overproduction of the polypeptide. Similarly, administration of an antibody may activate that polypeptide, for example, by binding to a membrane-bound polypeptide (receptor).

At least the polypeptides of the invention are SDS-P using methods well known to those of skill in the art.
It can be used as a molecular weight marker in AGE gel or molecular sieve gel filtration columns. Polypeptides can also be used to raise antibodies, which are then used to measure protein expression from recombinant cells as a method of assessing transformation of host cells. In addition, the polypeptides of the invention can be used to test the following biological activities.

(Gene Therapy Method) Another aspect of the present invention relates to a gene therapy method for treating disorders, diseases, and conditions. This gene therapy method involves introducing nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the invention. This method requires a polynucleotide encoding a polypeptide of the invention operably linked to a promoter and any other genetic element required for expression of this polypeptide by the target tissue. Techniques for such gene therapy and delivery are known in the art, see, eg, WO 90/11092, incorporated herein by reference.

Thus, for example, cells from a patient may be prepared by ex vivo inducing a polynucleotide (DNA or R
NA) and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art.
For example, Belldegrun et al. Natl. Cancer Inst. ，
85: 207-216 (1993); Ferrantini et al., Cancer.
Research, 53: 107-1112 (1993); Ferrantin.
i et al. Immunology 153: 4604-4615 (1994);
Kaido, T .; Et al., Int. J. Cancer 60: 221-229 (19
95); Ogura et al., Cancer Research 50: 5102-5.
106 (1990); Santodonato et al., Human Gene Th.
erase 7: 1-10 (1996); Santodonato et al., Gene.
See Therapy 4: 1246-1255 (1997); and Zhang et al., Cancer Gene Therapy 3: 31-38 (1996). These documents are incorporated herein by reference. In one embodiment, the engineered cells are arterial cells. The arterial cells can be reintroduced into the patient by direct injection into the arteries, tissues surrounding the arteries, or by catheter injection.

As discussed in more detail below, the polynucleotide constructs may be delivered to any method of delivering an injectable substance to cells of an animal, such as tissue (heart, muscle, skin,
Injection into the interstitial space of the lungs, liver, etc.). The polynucleotide construct can 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 any delivery vehicle (that acts to assist, facilitate or facilitate entry into cells.
(Including viral sequences, viral particles, liposomal formulations, lipofectin, precipitants, etc.). However, the polynucleotides of the invention can also be delivered in liposomal formulations, and lipofectin formulations and the like can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972 and 5,589, incorporated herein by reference.
, 466 and 5,580,859.

The polynucleotide vector constructs of the invention used in the gene therapy method are preferably not integrated into the host genome and do not contain sequences that allow replication.
It is a construct. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; Phar
pSVK3, pBPV, pMSG and pSVL available from Macia; and pEF1 / V5, pcDNA3.1 available from Invitrogen.
, And pRc / CMV2. Other suitable vectors will be readily apparent to those of skill in the art.

Any strong promoter known to those of skill in the art can be used to drive the expression of the polynucleotide sequences of the present invention. Suitable promoters include adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV) promoter);
RS virus (RSV) promoter; inducible promoter (eg MMT promoter, metallothionein promoter); heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg herpes simplex thymidine kinase promoter); retrovirus LTR; b-actin promoter; and human growth hormone promoter. The promoter can also be the native promoter for the polynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for production of the desired polypeptide for a period of up to 6 months.

The polynucleotide construct of the present invention is used in tissues (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, Includes intestines, testes, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue)
Can be delivered to the interstitial space of the. This interstitial space is the space between the reticulated fibers of organ tissue,
Liquid mucopolysaccharide matrix between cells, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrotic tissue, or connective tissue sheath muscle cells (connective cells).
Includes the same matrix within the issue enheasing muscle cell) or in the lacunae of the bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space
Preferred for reasons discussed below. The polynucleotide construct of the present invention comprises
It can be conveniently delivered by injection into the tissue containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression may be in undifferentiated cells or cells that are not fully differentiated ( (Eg, blood stem cells or dermal fibroblasts). Muscle cells in vivo take up a polynucleotide,
And it is particularly qualified in its ability to develop.

For naked nucleic acid sequence injection, the effective dosage of DNA or RNA is about 0.05.
It ranges from mg / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary depending on the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily 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, including, for example, inhalation of aerosol formulations, especially for delivery to lung or bronchial tissues, throat or mucous membranes of the nose, among others. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in this procedure.

Naked polynucleotides can be any method known in the art including, but not limited to, direct needle injection at the site of delivery, intravenous injection, topical administration, catheter injection, and so-called “gene gun”. Delivered by. These methods of delivery are known in the art.

The constructs can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofectins, precipitants and the like. Such delivery methods are known in the art.

In a particular embodiment, the polynucleotide construct of the present invention is complexed in a liposome preparation. Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because they can form a robust charge complex between the cationic liposomes and the polyanionic nucleic acid. Cationic liposomes, in functional form, are plasmid DNA (Felgner et al., Proc. Natl. Acad. S, incorporated herein by reference).
ci. USA, 84: 7413-7416 (1987)); mRNA (Malone et al., Proc. Natl. Acad., Incorporated herein by reference).
Sci. USA, 86: 6077-6081 (1989)); and purified transcription factors (Debs et al., J. Biol. C, incorporated herein by reference).
hem. , 265: 10189-10192 (1990)) has been shown to mediate intracellular delivery.

Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTM
A) Liposomes are particularly useful, and are available under the trademark Lipofectin® from GIBCO BRL, Grand Island, N.F. Y. (Felgner et al., Proc. Natl. Acad. Sc, incorporated herein by reference.
i. USA, 84: 7413-7416 (1987)). Other commercially available liposomes include transfectase (t
transface) (DDAB / DOPE) and DOTAP / DOPE
(Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. DOTAP (1,2-bis (oleoyloxy)-
For a description of the synthesis of 3- (trimethylammonio) propane) liposomes, see, eg, PCT Publication No. WO 90/11092, which is incorporated herein by reference. The preparation of DOTMA liposomes has been described in the literature and is described, for example, in Felgner et al., Proc. Na
tl. Acad. Sci. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic and neutral liposomes can be prepared, for example, by Avant.
It is readily available from i Polar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials.
Such substances include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC).
), Dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These substances also
It can be mixed with DOTMA starting material and DOTAP starting material in suitable proportions. Methods of using these materials to form liposomes are well known in the art.

For example, commercially, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) were used in various combinations with cholesterol added. Even without, conventional liposomes can be produced. Therefore,
For example, DOPG / DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonicated vial. The sample is placed under vacuum pump overnight and hydrated the next day with deionized water. This sample was then placed in a capped vial using a Heat Systems Model 350 sonicator equipped with an inverted cup (bath type) probe at maximum setting while the bath was circulating at 15 EC. Sonicate for 2 hours. Alternatively, negatively charged vesicles can be prepared without sonication to generate multilamellar vesicles or extruded through nucleopore membranes to separate unilamellar vesicles of different sizes. Can be generated. Other methods are known and available to those of skill in the art.

The liposomes can include multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), with SUVs being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example, Straubinge, which is incorporated herein by reference.
r et al., Methods of Immunology, 101: 512-527.
(1983). For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipids on the walls of glass tubes and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs, producing a homogeneous population of unilamellar vesicles. The substance to be encapsulated is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, dry lipid membranes should be treated with sterile water or 10
Resuspend in a suitable solution, such as an isotonic buffer solution such as mM Tris / NaCl, sonicate, then mix the preformed liposomes directly with the DNA. Due to the binding of the positively charged liposomes to the cationic DNA, the liposomes and DNA form a very stable complex. SUVs find use with small nucleic acid fragments. LUVs are prepared by many methods well known in the art. A commonly used method is Ca ²⁺ -EDTA chelation (Pap
ahadjopoulos et al., Biochim. Biophys. Acta, 3
94: 483 (1975); Wilson et al., Cell, 17:77 (1979).
)); Ether injection (Deamer et al., Biochim. Biophys. Ac.
Ta, 443: 629 (1976); Ostro et al., Biochem. Biop
hys. Res. Commum. 76: 836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348 (1979).
)); Detergent dialysis (Enoch et al., Proc. Natl. Acad. Sci.
． USA, 76: 145 (1979)); and reverse phase evaporation (REV).
) (Fraley et al., J. Biol. Chem., 255: 10431 (198).
0); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:
145 (1978); Schaefer-Ridder et al., Science, 2.
15: 166 (1982)), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes is from about 10: 1 to about 1:10. Preferably, the ratio is about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

US Pat. No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with cationic liposome carriers into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466.
No. 5,693,622, No. 5,580,859, No. 5,703.
055 and WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. No. 5,589,466,
No. 5,693,622, No. 5,580,859, No. 5,703,05
No. 5 and WO 94/9469, which are incorporated herein by reference, provide methods of delivering DNA-cationic lipid complexes to mammals.

[0430] In a particular embodiment, an RN comprising a sequence encoding a polypeptide of the invention.
The retroviral particles containing A are used to engineer cells ex vivo or in vivo. Examples of the retrovirus capable of inducing a retrovirus plasmid vector include Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus,
Harvey sarcoma virus, chicken leukemia virus, gibbon leukemia virus,
Human immunodeficiency virus, myeloproliferative sarcoma virus, and breast cancer virus are included, but are not limited to.

This retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaging cell lines that can be transfected include Miller, Human Gene Therapy, 1: 5-14 (199), which is incorporated herein by reference in its entirety.
0) PE501, PA317, R-2, R-AM, PA1 as described in
2, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP +
Examples include, but are not limited to, E-86, GP + envAm12 and DAN cell lines. The vector may transduce the packaging cells by any means known in the art. Such means, electroporation, the use of liposomes, and CaPO ₄ precipitation include, but are not limited to. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or attached to lipids and then administered to a host.

This producer cell line produces infectious retroviral vector particles which contain a polynucleotide encoding a polypeptide of the invention. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cell expresses a polypeptide of the invention.

[0433] In certain other embodiments, the polynucleotides of the invention contained in adenovirus vectors are used to engineer cells ex vivo or in vivo. Adenovirus can be engineered so that it encodes and expresses the polypeptide of the invention, while at the same time being inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, resulting in less worry about insertional mutagenesis. Furthermore, adenovirus has been used for many years as a live vaccine for the intestine with excellent safety aspects (Schwartzet et al., Am. Re.
v. Respir. Dis. , 109: 233-238 (1974)). Finally, adenovirus-mediated gene transfer has been demonstrated in many cases, including the transfer of α-1-antitrypsin and CFTR into the lungs of cotton rats (Ro.
senfeld et al., Science, 252: 431-434 (1991); R.
osenfeld et al., Cell 68: 143-155 (1992)). Moreover, extensive research seeking to establish adenovirus as the causative agent in human cancers was uniformly negative (Green et al. Proc. Natl. Acad. S).
ci. USA, 76: 6606 (1979)).

Suitable adenovirus vectors useful in the present invention are, for example, Koz.
arsky and Wilson, Curr. Opin. Genet. Devel
． 3: 499-503 (1993); Rosenfeld et al., Cell, 68.
: 143-155 (1992); Engelhardt et al., Human Gen.
et. Ther. 4: 759-769 (1993); Yang et al., Nature.
e Genet, 7: 362-369 (1994); Wilson et al., Natu.
Re, 365: 691-692 (1993); and US Pat. No. 5,652,2.
24, which are hereby incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the adenovirus E1 region and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene deleted from this vector. . In addition to Ad2, a variety of other adenoviruses (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

[0435] Preferably, the adenovirus used in the present invention is replication defective. Replication-defective adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest operably linked to a promoter, but is unable to replicate in most cells. A replication-defective adenovirus can be deleted at one or more of the following genes: E1a, E1b, E3, E4, E2a or all or part of L1 to L5.

[0436] In certain other embodiments, adeno-associated virus (AAV) is used to engineer the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (
Muzyczka, Curr. Topics in Microbiol. Im
munol. , 158: 97 (1992)). AAV is also one of the few viruses that can integrate its DNA into cells that are not dividing. 300
Vectors containing AAV as small as base pairs can be packaged and can integrate, but space for foreign DNA is limited to about 4.5 kb. Methods of generating and using such AAV are known in the art. For example, US Pat.
, 139,941, 5,173,414, 5,354,678,
No. 5,436,146, No. 5,474,935, No. 5,478,74
5 and 5,589,377.

For example, an AAV vector suitable for use in the present invention contains all the sequences necessary for DNA replication, encapsidation, and host cell integration. Sam
Brook et al., Molecular Cloning: A Laborator.
y Manual, Cold Spring Harbor Press (19
A polynucleotide construct containing a polynucleotide of the invention is inserted into this AAV vector using standard cloning methods, such as the method found in 89). This recombinant AAV is then isolated using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
The vector is transfected into packaging cells infected with helper virus. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV viral particles that include the polynucleotide constructs of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cell contains the polynucleotide construct integrated into its genome and expresses the desired gene product.

Another method of gene therapy is homologous recombination (see, eg, US Pat. No. 5,641,6).
70, published June 24, 1997; International Publication No. WO 96/29411, 19
Published September 26, 1996; International Publication No. WO94 / 12650, August 4, 1994
Published: Koller et al., Proc. Natl. Acad. Sci. USA, 8
6: 8932-8935 (1989); and Zijlstra et al., Nature.
e, 342: 435-438 (1989)) operably linking a heterologous regulatory region and an endogenous polynucleotide sequence (eg, a sequence encoding a polypeptide sequence of interest). including. The method involves activation of a gene that is present in the target cell but is normally not expressed in that cell or is expressed at a lower level than desired.

Polynucleotide constructs are made using standard techniques known in the art. This construct contains a promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is located sufficiently close to the 5'end of the desired endogenous polynucleotide sequence so that upon homologous recombination the promoter is operably linked to the endogenous sequence.

The promoter and targeting sequence can be amplified using PCR. Preferably, the amplified promoter contains additional restriction enzyme sites at the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence contains the 3'end of the amplified promoter. Includes the same restriction sites as the termini. The amplified promoter and targeting sequences are digested and ligated together.

Liposomes, viral sequences, viral particles, whole viruses, lipofections, either as naked polynucleotides or as described in more detail above.
The promoter-targeting sequence construct is delivered to cells, either with a transfection facilitating agent such as a precipitating agent. Direct needle injection, intravenous injection,
The P promoter-targeting sequence may be delivered by any method, including local administration, catheter infusion, particle accelerators and the like. This method is described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination occurs between this construct and the endogenous sequence, so that the endogenous sequence is placed under the control of this promoter. This promoter then drives the expression of the endogenous sequence.

Polynucleotides encoding the polypeptides of the present invention can be administered with other polynucleotides encoding other angiogenic proteins. Angiogenic proteins include acidic and basic fibroblast growth factor, VEGF-1, VEG
F-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor α and β, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor α,
Hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor, and nitric oxide synthase, but are not limited thereto.

Preferably, the polynucleotide encoding the polypeptide of the present invention comprises a secretory signal sequence that promotes secretion of the protein. Typically, the signal sequence is located in the coding region of the polynucleotide, expressed towards or at the 5'end of the coding region of the polynucleotide. The signal sequence may be homologous or heterologous to the polynucleotide of interest and homologous or heterologous to the transfected cells. In addition, the signal sequence can be chemically synthesized using methods known in the art.

Any dosage form of any of the above polynucleotide constructs may be used, so long as the dosage form expresses one or more molecules in an amount sufficient to provide a therapeutic effect. . This includes direct needle injections, systemic injections, catheter injections, biolistic injectors, particle accelerators (ie "gene guns"), gel foam sponge depots, and other commercial depots.
pot) substances, osmotic pumps (eg Alza minipumps), solid (tablets or pills) pharmaceutical formulations for oral or suppository, and intra-operative decanting or topical application. For example, direct injection of calcium phosphate-precipitated naked plasmid into rat liver and rat spleen or direct injection of protein-coated plasmid into the portal vein resulted in gene expression of foreign genes in rat liver (Ka
neda et al., Science, 243: 375 (1989)).

The preferred method of local administration is by direct injection. Preferably, the recombinant molecule of the present invention complexed with the delivery vehicle is administered by direct injection within the arterial region or locally within the arterial region. Topical administration of the composition within the arterial region refers to injection of the composition into the artery several centimeters, preferably several millimeters.

Another method of topical administration is to contact the polynucleotide construct of the invention within or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the tissue surface inside the wound or the construct can be injected into the tissue area inside the wound.

Therapeutic compositions useful for systemic administration include a recombinant molecule of the present invention complexed with a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use in systemic administration include liposomes containing ligands that target the vehicle to a particular site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injections may be performed using methods standard in the art. Aerosol delivery may also be performed using methods standard in the art (eg, Stribling, incorporated herein by reference).
Et al., Proc. Natl. Acad. Sci. USA, 189: 11277-1.
1281 (1992)). Oral delivery can be accomplished by complexing the polynucleotide constructs of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include:
Mention may be made of plastic capsules or tablets, such as those known in the art. Topical delivery may be accomplished by mixing a lipophilic reagent (eg DMSO) that is capable of passing into the skin with a polynucleotide construct of the present invention.

Determining the effective amount of a substance to be delivered can be determined, for example, by the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition requiring treatment and its severity, and administration. It may depend on a number of factors, including the pathway. The frequency of treatment is
It depends on many factors such as the amount of polynucleotide construct administered per dose and the health and medical history of the subject. The exact amount, frequency of dosing and timing of dosing will be determined by the attending physician or veterinarian. The therapeutic composition of the present invention comprises
It can be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, especially humans.

Biological Activity Polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides show activity in a particular assay, it is likely that these molecules may be involved in diseases associated with their biological activity. Accordingly, the polynucleotides or polypeptides, or agonists or antagonists, can be used to treat associated diseases.

(Immune Activity) The polynucleotide or polypeptide, or agonist or antagonist of the present invention activates or inhibits the proliferation, differentiation, or mobilization (chemotactic) of immune cells, whereby diseases of the immune system, It may be useful in the treatment, prevention and / or diagnosis of disorders and / or conditions. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, erythrocytes, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune diseases, disorders and / or conditions can be genetic, somatic (eg, cancer or some autoimmune disorder), acquired (
For example, due to chemotherapy or toxins) or infectious. In addition, the polynucleotides or polypeptides of the invention, or agonists or antagonists, are markers or detectors of a particular immune system disease or disorder (det
vector).

The polynucleotides or polypeptides, or agonists or antagonists of the invention, may be useful in the treatment, prevention and / or diagnosis of hematopoietic cell diseases, disorders and / or conditions. The polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat pluripotent stem cells in an attempt to treat or prevent diseases, disorders and / or conditions associated with the reduction of certain (or many) types of hematopoietic cells. Can be used to increase the differentiation and proliferation of hematopoietic cells, including. Examples of immunodeficiency syndromes include disorders of blood proteins (eg, agammaglobulinemia, hypogammaglobulinemia), ataxia-telangiectasia, unclassified immunodeficiency, Di George syndrome, HIV infection, HTL.
V-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCID), Viscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria. , But not limited to them.

In addition, the polynucleotides or polypeptides of the invention, or agonists or antagonists, may also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic activity or thrombolytic activity, a polynucleotide or polypeptide of the invention,
Or using agonists or antagonists, the disease, disorder and / or condition of blood coagulation (eg fibrinogenemia, factor deficiency), disease of blood platelets,
A disorder and / or condition (eg, thrombocytopenia) or a wound resulting from trauma, surgery or other causes may be treated or prevented. Alternatively, a polynucleotide or polypeptide of the invention, or an agonist or antagonist that can reduce hemostatic or thrombolytic activity, can be used to inhibit or lyse blood clots. These molecules may be important in the treatment of heart attack (infarction), stroke or scarring.

The polynucleotides or polypeptides of the invention, or agonists or antagonists, may also be useful in the treatment, prevention and / or diagnosis of autoimmune diseases, disorders and / or conditions. Many autoimmune disorders result from inappropriate recognition of self as foreign substances by immune cells. This improper recognition triggers an immune response that results in the destruction of host tissue. Therefore, the immune response,
Administration of a polynucleotide or polypeptide of the invention, or an agonist or antagonist, which specifically inhibits T cell proliferation, differentiation or chemotaxis, may be an effective treatment in the prevention of autoimmune diseases, disorders and / or conditions. .

Examples of autoimmune disorders that can be treated, prevented and / or diagnosed or detected by the present invention include Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, Glomerulonephritis, Goodpasture syndrome,
Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmitis, bullous pemphigoid,
Pemphigus vulgaris, multiple endocrine disorders, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, gian-
Barley syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease include, but are not limited to.

Similarly, asthma (particularly allergic asthma) or other respiratory problems,
Allergic reactions and conditions can also be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention, or agonists or antagonists. In addition, these molecules can be used to treat anaphylaxis, hypersensitivity or blood group incompatibility to antigenic molecules.

The polynucleotides or polypeptides of the invention, or agonists or antagonists, may also be used to treat, prevent and / or diagnose organ rejection or graft versus host disease (GVHD). Organ rejection occurs by the destruction of transplanted tissue by host immune cells via the immune response. Similarly, the immune response is also involved in GVHD, where foreign transplanted immune cells destroy host tissue. Administration of a polynucleotide or polypeptide of the invention, or an agonist or antagonist that inhibits an immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective treatment in preventing organ rejection or GVHD.

Similarly, the polynucleotides or polypeptides of the present invention, or agonists or antagonists, may also be used to modulate inflammation. For example, the polypeptide or polynucleotide, or agonist or antagonist, may inhibit proliferation and differentiation of cells involved in the inflammatory response. Using these molecules, infection-related inflammation (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury-related inflammation, endotoxin lethal-related inflammation, Inflammation associated with arthritis, inflammation associated with complement-mediated hyperacute rejection,
Resulting from inflammation associated with nephritis, inflammation associated with cytokine or chemokine-induced lung injury, inflammation associated with inflammatory bowel disease, inflammation associated with Crohn's disease or overproduction of cytokines (eg TNF or IL-1) Inflammatory conditions, both chronic and acute, including inflammation, may be treated, prevented and / or diagnosed.

Hyperproliferative Disorders Polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat, prevent and / or diagnose hyperproliferative diseases, disorders, including neoplasms. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may inhibit the growth of this disorder via direct or indirect interactions. Alternatively, the polynucleotides or polypeptides of the invention, or agonists or antagonists, can proliferate other cells that can inhibit hyperproliferative disorders.

For example, by increasing the immune response, particularly by increasing the antigenic quality of hyperproliferative disorders, or by expanding, differentiating, or recruiting T cells.
Hyperproliferative diseases, disorders and / or conditions may be treated, prevented and / or diagnosed. This immune response can be augmented by either enhancing an existing immune response or initiating a new immune response. Alternatively, reducing the immune response can also be a method of treating, preventing and / or diagnosing hyperproliferative diseases, disorders and / or conditions, such as chemotherapeutic agents.

Examples of hyperproliferative diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention, or agonists or antagonists include abdomen, bone, breast, digestive system. , Liver, pancreas,
Peritoneum, endocrine glands (adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid), eyes,
Head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen,
It includes, but is not limited to, the rib cage, as well as neoplasms located in the genitourinary organs.

Similarly, other hyperproliferative diseases, disorders and / or conditions may also be treated, prevented and / or diagnosed with a polynucleotide or polypeptide of the invention, or an agonist or antagonist. Examples of such hyperproliferative diseases, disorders and / or conditions include hypergammaglobulinemia, lymphoproliferative disorders, disorders and / or conditions, paraproteinemia, purpura, sarcoidosis, Sezary. Syndromes, Waldenström macroglobulinemia, Gaucher's disease, histiocytosis, and any other hyperproliferative disorders, as well as neoplasms located in the organ systems listed above. Not limited to.

One preferred embodiment utilizes the polynucleotide of the invention to utilize the invention,
And / or gene therapy with protein fusions or fragments thereof inhibits abnormal cell division.

The invention thus provides a method for treating or preventing a cell proliferative disease, disorder and / or condition by inserting a polynucleotide of the invention into an abnormally proliferating cell. . Here, the polynucleotide suppresses the expression.

Another embodiment of the invention provides a method of treating or preventing a disease, disorder and / or condition of cell proliferation in an individual, the method comprising the step of: Administering one or more active gene copies. In a preferred embodiment, the polynucleotide of the present invention is a DNA construct containing a recombinant expression vector effective in expressing the DNA sequence encoding the above polynucleotide. In another preferred embodiment of the invention, a DNA construct encoding a polynucleotide of the invention is inserted into a cell and treated with a retrovirus (or, more preferably, an adenovirus vector). G J. Nabel et al., PNAS 1999 96: 3244-, incorporated herein by reference.
326). In the most preferred embodiment, the viral vector is deficient and does not transform non-proliferating cells, only proliferating cells. Further, in a preferred embodiment, the polynucleotide of the invention inserted into proliferating cells, alone or in combination with or in fusion with other polynucleotides, is then subjected to external stimulation (i.e., Magnetic properties, certain small molecules, chemicals, or drug administration, etc.). This stimulus is
Acts on a promoter upstream of the above polynucleotide to induce expression of the encoded protein product. Thus, the beneficial therapeutic effects of the present invention are
It can be clearly regulated (ie, to increase, decrease, or inhibit expression of the polynucleotides of the invention) based on the external stimuli described above.

The polynucleotides of the present invention may be useful in suppressing the expression of oncogenic genes or antigens. By "suppressing the expression of oncogenic genes", suppression of gene transcription, degradation of gene transcript (pre-message RNA), inhibition of splicing, messenger RNA destruction, and prevention of post-translational modification of proteins , Intended to destroy a protein, or inhibit the normal function of a protein.

For topical administration to abnormally proliferating cells, the polynucleotides of the invention may be administered by any method known to those of skill in the art, including transfection, electroporation, cellular Microinjection, including, but not limited to, lipofectin, or naked polynucleotides in vehicles such as liposomes, or any other method described throughout this specification. The polynucleotides of the present invention can be used in known gene delivery systems (eg, retroviral vectors (Gilboa,
J. Virology 44: 845 (1982); Hocke, Nature.
320: 275 (1986); Wilson et al., Proc. Natl. Aca
d. Sci. USA. 85: 3014); vaccinia virus system (Chakra)
barty et al., Mol. Cell Biol. 5: 3403 (1985)) or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (
1985))). These references are exemplary only and incorporated herein by reference. Retroviruses or adenoviruses known to those of skill in the art to specifically deliver to, or transfect, and to leave non-dividing cells in cells that are abnormally proliferating (eg, in the art or It is preferred to utilize a delivery system (as described herein). Since host DNA replication requires retroviral DNA to integrate, this retrovirus cannot self-replicate due to the lack of the required retroviral genes for its life cycle. For the polynucleotides of the invention, such retroviral delivery systems are utilized to target the above genes and constructs to aberrantly proliferating cells and leave non-dividing normal cells.

The polynucleotides of the present invention are used to direct cell proliferative disorders in internal organs, body cavities, etc. by the use of imaging devices that are used to guide the injection needle directly to the site of disease.
It can be delivered directly to the disease site. The polynucleotides of the present invention may also be administered to the disease site during surgical intervention.

By “cell proliferative disorder” is meant that any human or animal disease or disorder affects any one or any combination of organs, cavities, or body parts. The disease, benign or malignant, is characterized by a single or multiple localized abnormal growth of cells, groups of cells, or tissues.

Any amount of a polynucleotide of the invention can be administered so long as the amount has a biologically inhibitory effect on the growth of treated cells. Moreover, more than one polynucleotide of the invention can be administered at the same site at the same time. "
By “biologically inhibitory” is meant partial or total growth inhibition as well as a decrease in the rate of proliferation or growth of cells. A biologically inhibitory dose may be used to assess the effect of a polynucleotide of the invention on the growth of abnormally proliferating cells in the target malignant or tissue culture, tumor growth in animals and cell culture, or It can be determined by any other method known to those skilled in the art.

The present invention further relates to antibody-based therapy, the method comprising treating a mammalian patient, preferably a human, to treat, prevent and / or diagnose one or more of the diseases, disorders and / or conditions mentioned above. Patient) with an anti-polypeptide antibody and an anti-polynucleotide antibody. Methods for producing anti-polypeptide 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.

An overview of the ways in which the antibodies of the invention may be used therapeutically is given, for example, complement (CDC).
Or conjugating a polynucleotide or polypeptide of the invention locally or systemically in the body or by direct cytotoxicity of the antibody, as mediated by effector cells (ADCC). Some of these approaches are described in more detail below. Given the teachings provided herein, one of skill in the art would know how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the invention have or develop the disease, disorder and / or condition of cell proliferative and / or differentiation as described herein. It is useful for treating, preventing and / or diagnosing a subject. Such treatment involves administering a single dose or multiple doses of the antibody, or a fragment, derivative, or conjugate thereof.

The antibodies of the invention may be combined with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (eg, which act to increase the number or activity of effector cells that interact with the antibody. ) And can be used advantageously.

Using a high affinity and / or potent in vivo inhibitory and / or neutralizing antibody against a polypeptide or polynucleotide of the invention, fragment or region thereof, may be used to modify the polynucleotide or polynucleotide of the invention. peptide(
(Including fragments thereof) and immunoassays for diseases, disorders and / or conditions and their treatment. Such antibody, fragment, or region preferably has an affinity for the polynucleotide or polypeptide, including fragments thereof. Preferred binding affinity is 5x
10 ^-6 M, 10 ^-6 M, ^{5x10 -7} M, 10 ^-7 M, ^{5x10 -8} M, 10 ^-8 M, ^5x
10 ^-9 M, 10 ^-9 M, 5 × 10 ^-10 M, 10 ^-10 M, 5 × 10 ^-11 M, 10 ^-11 M,
^{5 × 10 -12 M, 10 -12} M, 5 × 10 -13 M, 10 -13 M, 5 × 10 -14 M, 10 - 14 M, 5 × 10 -15 M, and 10 less than ^-15 M Includes affinity with dissociation constant or Kd.

Furthermore, the polypeptides of the present invention may be used alone, as a fusion protein, or directly or indirectly in combination with other polypeptides, as described elsewhere herein. Either of which is useful in inhibiting angiogenesis of proliferating cells or tissues. In the most preferred embodiment, the anti-angiogenic effect described above can be achieved indirectly, for example through the inhibition of hematopoietic tumor-specific cells (eg tumor-associated macrophages) (incorporated herein by reference). Will be J
oseph IB et al., J Natl Cancer Inst, 90 (21):
1648-53 (1998)). Antibodies to the polypeptides or polynucleotides of the invention may also result in direct or indirect inhibition of angiogenesis (Wite L. et al., Cancer, incorporated herein by reference).
Metastasis Rev. 17 (2): 155-61 (1998)).

The polypeptides of the present invention (including protein fusions), or fragments thereof, may be useful in inhibiting proliferating cells or tissues through the induction of apoptosis. The polypeptides described above may be used, for example, to induce apoptosis of proliferative cells and tissues either directly or indirectly, eg, death domain receptors (eg, tumor necrosis factor (TNF) receptor 1, CD95 (Fas). / APO-
1), TNF receptor-related apoptosis-mediating protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 (
Schulze-Osthoff K, et al., Incorporated herein by reference,
Eur J Biochem 254 (3): 439-59 (1998))). Further, in another preferred embodiment of the present invention, the above-mentioned polypeptide is mediated by other mechanisms (for example, in the activation of other proteins that activate apoptosis) or by expressing the protein alone or as a small molecule drug. Alternatively, apoptosis can be induced either by stimulation with either an adjuvant (eg, apoptonin, galectin, thioredoxin, anti-inflammatory protein) (eg, Mutat Res, incorporated herein by reference). 400 (1-2): 447-55 (1
998), Med Hypotheses. 50 (5): 423-33 (199
8), Chem Biol Interact. Apr 24; 111-112.
23-34 (1998)), J Mol Med. 76 (6): 402-12.
(1998), Int J Tissue React; 20 (1): 3-15.
(1998)).

The polypeptides of the present invention (including protein fusions thereto), or fragments thereof, are useful in inhibiting metastasis of proliferating cells or tissues. Inhibition can be as a direct result of the step of administering the polypeptide or an antibody to said polypeptide, as described elsewhere herein, or indirectly (eg, known to inhibit metastasis). A protein (eg, activating expression of α4 integrin) can occur (eg, Cur, incorporated herein by reference).
r Top Microbiol Immunol 1998; 231: 125.
See -41). Such therapeutic effects of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention comprises a composition comprising a polypeptide of the invention (eg, a composition comprising a polypeptide antibody, heterologous nucleic acid, toxin, or prodrug related to the polypeptide or heterologous polypeptide). There is provided a method of delivering an article to a targeted cell expressing a polypeptide of the invention. Polypeptides or polypeptide antibodies of the invention can be attached to heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs through hydrophobic, hydrophilic, ionic and / or covalent interactions.

Polypeptides of the invention, protein fusions thereto, or fragments thereof, may be used directly against proliferative antigens and immunogens (eg, when the polypeptide of the invention is "vaccinated") as described above. An immune response to respond to an antigen and an immunogen of E. coli or indirectly (eg, in activating the expression of proteins known to enhance the immune response (eg, chemokines)). And are useful in enhancing the immunogenicity and / or antigenicity of proliferating cells or tissues.

Cardiovascular Disorders Polynucleotides or polypeptides of the invention, or agonists or antagonists are used to treat cardiovascular diseases, disorders and / or conditions, including peripheral arterial disease such as limb ischemia. It can be prevented and / or diagnosed.

Cardiovascular disorders include arterio-arterial fistulas.
stula), arteriovenous fistula, cerebral arteriovenous birth defects, congenital heart defects (congeni)
cardiovascular abnormalities such as tal heart defects, pulmonary valve atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, tricentric heart, coronary vessel anomalies
, Cross heart, right thoracic heart, patent ductus arteri
oss), Ebstein's anomaly, Eisenmenger complex, left ventricular underdevelopment syndrome, left thoracic heart, tetralogy of Fallot, transposition of the great arteries, bilateral right ventricle origin, tricuspid valve closure, residual arterial canal , And heart septal deficiency
ts) (e.g., aortopulmonary separation)
eptal defect, endocardial bed deficiency, Luthambache syndrome, trilogy of Fallot, ventricular heart septal defect (ventricular heart sep
tal defects)).

Cardiovascular diseases, disorders and / or conditions also include arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocardium. Inflammation (including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, hydrocephalus, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction Post-infarction heart rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial effusion, pericarditis (including infarct and tuberculosis), pericarditis, Postpericardiotomy syndrome, right heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications (cardiovascul)
ar pregnancy competitions), Scimitar syndrome,
Cardiovascular syphilis and cardiovascular tuberculosis
heart diseases such as ulosis).

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature contraction, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long).
QT syndrome), collateral contraction, Lone-Ganning-Levine syndrome, Maheme-type pre-excita
tion syndrome), Wolff-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. As tachycardia, paroxysmal tachycardia,
Supraventricular tachycardia, ventricular intrinsic rhythm promotion, atrioventricular nodal reentry tachycardia (atrioventri
circular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial nodal reentry tachycardia
dal reentry tachycardia), sinus tachycardia, torsade
De pointe, and ventricular tachycardia.

Heart valve diseases include aortic insufficiency, aortic stenosis, and murmur (hea).
r murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve dysfunction, mitral valve stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Includes atresia, tricuspid dysfunction, and tricuspid stenosis.

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis (, subvalvular pulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, heart Intimal fibroelasticity, endocardial myocardial fibrosis, Keens syndrome, myocardial reperfusion injury,
And myocarditis.

Myocardial ischemia includes angina, coronary aneurysm, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunnin.
g) such as coronary artery disease.

Cardiovascular diseases also include aneurysms, angiogenesis, hemangiomatosis, bacterial hemangiopathies, Hippel-Lindau Disease.
), Klipel-Tornone-Weber syndrome, Sturgi-Weber syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Leuriche syndrome,
Arterial occlusion disease, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disease, disorders and / or conditions, diabetic vasculopathy, diabetic retinopathy, embolism, thrombosis, apex Pain, hemorrhoids, hepatic vein occlusion disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasia, ataxia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, dilated meandering vein , Varicose ulcers, vasculitis, and vascular diseases such as venous insufficiency.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms. Is mentioned.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and occlusive thrombosis. Sexual vasculitis.

Cerebrovascular diseases, disorders and / or conditions include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral artery disease. Embolism and thrombosis, carotid thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (sub)
araxhnoid hemorrhage), cerebral infarction, cerebral ischemia (including transient), subclavian artery stealing syndrome, periventricular leukomalacia (periventricula)
r leukomalacia), vascular headaches, cluster headaches, migraine headaches, and vertebrobasilar dysfunction.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, toe cyanosis syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. As thrombosis, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
Includes sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, and partition syndrome (compartme).
nt syndrome, anterior compar
ment syndrome), myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet.
t) Syndrome, Churg-Strauss Syndrome, Mucocutaneous Lymph Node Syndrome, Obstructive Thrombotic Vasculitis, Hypersensitivity Vasculitis, Schoenline-Henoho Purpura (Schoenl)
ein-Henoch purpura), allergic cutaneous vasculitis and Wegener's granulomatosis.

The polynucleotides or polypeptides, or agonists or antagonists of the invention are particularly effective for the treatment of dangerous limb ischemia and coronary artery disease.

The polypeptide may be administered using any method known in the art including direct needle injection at the site of delivery, intravenous injection, topical administration,
Catheter injection, biolistic injection, particle accelerators, gel foam sponge depots, other commercial depot materials, osmotic pumps, solid pharmaceutical formulations for oral or suppository, intraoperative decanting or topical application, aerosol delivery. But is not limited to these. Such methods are known in the art.
The polypeptides of the present invention are described in more detail below in therapeutic agents (Theraps).
eutic). Methods of delivering the polynucleotides of the invention are described in more detail herein.

Anti-Angiogenic Activity The naturally occurring equilibrium between an endogenous stimulator and an inhibitor of angiogenesis is the equilibrium in which the inhibitory effects predominate. Rastinejad et al., Cell 5
6: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryogenesis, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal. It is specified. Conditions for pathological angiogenesis (eg, characterizing solid tumor growth)
Below, there is no control over these adjustments. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of ocular diseases, disorders and / or conditions, and psoriasis. See, eg, Moses et al., Biotech. 9: 630-634 (
1991); Folkman et al., N. et al. Engl. J. Med. 333: 175
7-1763 (1995); Auerbach et al. Microvasc. R
es. 29: 401-411 (1985); Folkman, Advances.
in Cancer Research, Klein and Weinhouse
e, Academic Press, New York, pp. 175-203 (
1985); Patz, Am. J. Opthalmol. 94: 715-743
(1982); and Folkman et al., Science 221: 719-7.
25 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated that suggests that solid tumor growth depends on angiogenesis. Folkman and Klagsbrun, Science 235: 442-447 (1987).
).

The present invention provides treatment of diseases, disorders and / or conditions associated with neovascularization by the administration of the polynucleotides and / or polypeptides of the present invention and the agonists or antagonists of the present invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the invention, or agonists or antagonists, include malignant diseases, solid tumors, and cancers described herein, as well as others known in the art. (For a review of such disorders,
Fishman et al., Medicine, Second Edition, J. Am. B. Lippincott
Co. , Philadelphia (1985)), but is not limited thereto. Accordingly, the present invention provides a method of treating, preventing and / or diagnosing angiogenesis-related diseases and / or disorders, the method comprising a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and of the invention. And / or the agonist is administered to an individual in need of the treatment. For example,
The polynucleotides, polypeptides, antagonists and / or agonists can be utilized in a variety of additional ways to therapeutically treat or prevent cancer or tumors. Cancers that can be treated, prevented and / or diagnosed with polynucleotides, polypeptides, antagonists and / or agonists include prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver Solid tumors including cancer, parotid cancer, bile duct cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer; primary tumors and metastases; melanoma; Glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancy; and tumors of blood origin (eg, leukemia), but are not limited thereto. For example, the polynucleotides, polypeptides, antagonists and / or agonists can be delivered locally to treat or prevent cancers such as skin cancer, head and neck tumors, breast tumors and Kaposi's sarcoma.

In yet another aspect, the polynucleotides, polypeptides, antagonists and / or agonists can be utilized to treat superficial forms of bladder cancer, eg, by intravesical administration. Polynucleotides, polypeptides, antagonists and / or agonists can be delivered directly to the tumor or near the tumor site via injection or catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary with the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and / or agonists are useful in treating, preventing and / or diagnosing cancer, as well as other diseases, disorders and / or conditions, including angiogenesis. Can be. These diseases, disorders and / or conditions include, but are not limited to, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas); atherosclerotic plaques. Angiogenic diseases of the eye (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, posterior lens fibrosis, rubeosis, retinoblastoma, uveitis and eye) Wing (Pter)
ygia) (abnormal blood vessel growth)); rheumatoid arthritis; psoriasis; delayed wound healing;
Endometriosis; Angiogenesis; Granulation; Hyperplastic scar (keloid); Pseudoarthritis fracture; Scleroderma; Trachoma; Vascular adhesion; Myocardial angiogenesis; Coronary coronary
cerebral collaterals; arteriovenous malformations; ischemic extremity angiogenesis; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, in one aspect of the invention, treating hyperplastic scars and keloids comprising the step of administering the polynucleotides, polypeptides, antagonists and / or agonists of the invention to hyperplastic scars or keloids. , Prevention and /
Alternatively, a method for diagnosing is provided.

In one embodiment of the invention, the polynucleotides, polypeptides, antagonists and / or agonists are directly injected into hyperplastic scars or keloids to prevent the progression of these lesions. This treatment is of particular value in the prophylactic treatment of conditions known to result in the development of hypertrophic scars and keloids (eg burns), and preferably at the time the proliferative phase progresses (of the initial injury). About 14 days later) but before the onset of hyperplastic scars or keloids. As mentioned above, the present invention also provides neovascularization disorders of the eye (eg,
Methods for treating, preventing and / or diagnosing corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post lens fibroplasia and macular degeneration).

Furthermore, the polynucleotides and polypeptides of the invention (agonists and / or
Or an antagonist), ocular diseases, disorders and / or conditions associated with neovascularization include, but are not limited to, neovascular glaucoma, diabetic retinopathy, retinoblastoma. Cellular tumors, post lens fibroplasia, uveitis, immature retinopathy, macular degeneration, corneal transplant neovascularization, as well as other inflammatory diseases of the eye, eye tumors, and choroidal or iris neovascularization disease. For example, Waltman et al., Am. J. Ophthal. 85: 704-7
10 (1978) and Gartner et al., Surv. Ophthal. 22:
See review by 291-312 (1978).

Accordingly, in one aspect of the invention, corneal neovascularization (comprising the step of administering to the patient a therapeutically effective amount of a compound (described above) to the cornea such that blood vessel formation is inhibited. Methods for treating or preventing neovascularization disorders of the eye, including corneal transplant neovascularization) are provided. Briefly, the cornea is a tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries may extend from the limbal pericorneal plexus to the cornea. When the cornea becomes vascularized, it also becomes clouded, resulting in diminished vision of the patient. Vision loss can be complete when the cornea is completely opacitated. A wide variety of disorders can result in corneal neovascularization, including, for example: corneal infections (eg trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (eg graft rejection). And Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (for any cause), toxic and nutritional deficiencies, and as a complication of wearing contact lenses.

In a particularly preferred embodiment, the present invention may be prepared for topical administration in saline (in combination with any preservatives and antibacterial agents commonly used in ophthalmic formulations), and It can be administered in the form of eye drops. Solutions or suspensions may be prepared in pure form and administered several times daily. Alternatively, the anti-angiogenic composition prepared as described above can also be administered directly to the cornea. In a preferred embodiment, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to the cornea. In a further embodiment, the anti-angiogenic factor or anti-angiogenic composition can be utilized as an adjunct to conventional steroid therapy. Topical treatment may also be prophylactically useful in corneal lesions that are known to have a high potential to induce an angiogenic response (eg, chemical burns). In these cases, treatment (possibly combined with steroids) may be started immediately to help prevent subsequent complications.

In another embodiment, the above compounds may be injected directly into the corneal stroma by an ophthalmologist under the guidance of a microscope. Although the preferred site of injection may vary in the form of individual lesions, the goal of administration is to place the composition on the advancing surface of the vasculature (ie, dispersed between blood vessels and normal corneas). It is). In most cases this is a peril to "protect" the cornea from advancing vessels.
imbic) Cornea injection is included. This method can also be utilized immediately after corneal injury to prevent corneal neovascularization prophylactically. In this situation, the substance may be injected between the corneal lesion and its undesired potential blood supply limbus and injected into the perilimbal cornea. Such methods can also be utilized in a similar fashion to prevent capillary infiltration of the transplanted cornea. In the sustained release form, the infusion is 2 to 1 year.
It may only be needed three times. Steroids may also be added to the infusion solution to reduce inflammation resulting from the injection itself.

In another aspect of the invention, angiogenesis, comprising the step of administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye so as to inhibit the formation of blood vessels. Methods for treating or preventing glaucoma are provided. In one embodiment, the compounds can be administered topically to the eye to treat or prevent the early forms of neovascular glaucoma. In other embodiments, the compound may be implanted by injection into the area of the anterior chamber angle. In other embodiments, the compound can also be placed in any location such that the compound is continuously released into the aqueous humor. In another aspect of the invention, a proliferative diabetic, comprising the step of administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist so that the formation of blood vessels is inhibited. Methods are provided for treating or preventing retinopathy.

In a particularly preferred embodiment of the present invention, proliferative diabetic retinopathy is the treatment of aqueous humor or the vitreous to increase local concentrations of polynucleotides, polypeptides, antagonists and / or agonists in the retina. It can be treated by injection. Preferably, this treatment should be initiated prior to the acquisition of the severe disease requiring photocoagulation.

In another aspect of the invention, a lens comprising administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye such that blood vessel formation is inhibited. Methods are provided for treating or preventing post-fibroproliferative disorders. The compounds may be administered locally via intravitreal injection and / or via intraocular implantation.

In addition, diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: hemangiomas. , Arthritis, psoriasis, angiofibromas, atherosclerotic plaque, delayed wound healing, granulation, hemophilic joints, hyperplastic scars, pseudoarticular fractures, Osler-Weber
er) syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesion.

Further, diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: solid tumors. , Blood borne tumors (eg leukemia), tumor metastases, Kaposi's sarcoma, benign tumors (eg hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas), rheumatoid arthritis, psoriasis , Ocular angiogenic disorders (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, rubeosis, retinoblastoma, and uvitis) ),
Delayed wound healing, endometriosis, angiogenesis, granulation, hyperplastic scars (keloids),
Nonunion fracture, scleroderma, trachoma, vascular adhesion, myocardial angiogenesis, coronary collateral (
coronary collaterals), cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Ausler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilia, angiofibromas, fibromuscular formations Abnormalities, wound granulation, Crohn's disease, atherosclerosis, birth control agents (by controlling menstruation, by preventing angiogenesis necessary for embryo implantation), pathogenic consequences (eg cat scratching) Disease (Rochele minalia quintosa), ulcer (H
diseases with angiogenesis, such as E.coli pylori), Bartonellosis and bacterial hemangiomas).

In one aspect of the birth control method, the amount of the compound sufficient to block embryo implantation is administered before or after sexual intercourse and fertilization occur, thus providing an effective method of birth control, perhaps "post-treatment." (Morning after) method. Polynucleotides, polypeptides, agonists and / or agonists can also be used in controlling menstruation or can be administered either as peritoneal lavage fluid in the treatment of endometriosis or for peritoneal transplantation.

The polynucleotides, polypeptides, agonists and / or agonists of the invention can be incorporated into surgical sutures to prevent stitch granulomas.

The polynucleotide, polypeptide, agonist and / or agonist is
It can be utilized in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (eg, in the form of a spray or film) is used to separate normal surrounding tissue from malignant tissue and / or prevent the spread of disease to surrounding tissue. In order to do so, it can be utilized to coat or spray the area prior to tumor removal. In another aspect of the invention, the composition (eg, in the form of a spray) is delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. obtain. In yet another aspect of the invention, a surgical mesh coated with an anti-angiogenic composition of the invention can be utilized in any procedure in which a surgical mesh can be utilized. For example, in one embodiment of the invention, a surgical mesh laden with an anti-angiogenic composition.
en) may be utilized during abdominal cancer resection surgery (eg, after colectomy) to provide support for the structure and to release certain amounts of anti-angiogenic factors.

In a further aspect of the invention, polynucleotides, polypeptides, agonists and / or agonists are added to the tumor margin after resection so that local recurrence of cancer and formation of new blood vessels at the site are inhibited. Methods for treating a tumor excision site are provided that include administering. In one embodiment of the invention, the anti-angiogenic compound is administered directly to the site of tumor resection (eg, smearing, brushing, or otherwise ablated the tumor with the anti-angiogenic compound). Applied by coating). Alternatively, the anti-angiogenic compound is
It can be incorporated into known surgical pastes prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy and neurosurgery for malignancy.

[0516] In one aspect of the invention, the polynucleotides, polypeptides, agonists and / or agonists are administered at the resection margin of a wide variety of tumors including, for example, breast, colon, brain and liver tumors. Can be done. For example, in one embodiment of the invention, the anti-angiogenic compound is present at the site of the neurological tumor after resection.
It can be administered so that the formation of new blood vessels at the site is inhibited.

The polynucleotides, polypeptides, agonists and / or agonists of the invention can also be administered with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasive factors, retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2, Plasminogen activator inhibitor-1, plasminogen activator inhibitor-2 and the lighter "group d" transition metals of various forms.

Lighter "d-group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium and tantalum species. Such transition metal species are
A transition metal complex can be formed. Suitable complexes of the above transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include, for example, metavanadate complexes and orthovanadate complexes such as 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 monohydrate and vanadyl sulfate trihydrate.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxotungsten complexes include tungstate complexes 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).
An oxide is mentioned. Suitable oxomolybdenum complexes include molybdate,
Included are 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,
Included are molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, glycerol, tartaric acid and sugar derived hydroxo derivatives.

A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include: Platelet Factor 4; Protamine Sulfate; Sulfated Chitin Derivatives (prepared from queen crab shells) (Murata et al. Cancer Res. 51: 22-26, 1991)
Sulphated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids (eg, estrogen) and tamoxifen citrate); staurosporine; regulators of substrate metabolism (eg, (Including proline analogs, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, α, α-dipyridyl, aminopropionitrile fumarate)
4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone; methotrexate; mitoxantrone; heparin; interferon; 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 tetradeca sulfate; eponomycin; camptothecin; fumagillin (
Ingber et al., Nature 348: 555-557, 1990); Sodium gold thiomalate ("GST"; Matsubara and Ziff, J.C.
lin. Invest. 79: 1440-1446, 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. Chem.
． 262 (4): 1659-1664, 1987); Bisantoren (Natio).
nal Cancer Institute); lobenzarit disodium (
N- (2) -carboxyphenyl-4-chloroanthronylic acid (chloroa)
nthoronic acid) disodium, or "CCA"; Tak
euchi et al., Agents Actions 36: 312-316, 199.
2); thalidomide; Angostatic steroids; AGM-1470; carboxynaminolmidazole.
); And metalloproteinase inhibitors (eg, BB94).

Diseases at the cellular level Diseases associated with increased cell survival or inhibition of apoptosis that can be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention and / or antagonists or agonists include: Cancer (eg, follicular lymphoma, carcinoma with p53 mutations, and hormone-dependent tumors, which are: colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer,
Intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelium, osteoblastoma, giant cell tumor of the bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and (Including but not limited to ovarian cancer); autoimmune diseases, disorders and / or
Or conditions (eg, 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 (eg, herpesvirus, poxvirus and adenovirus), inflammation, graft-versus-host disease, acute graft rejection, and chronic graft rejection. In a preferred embodiment, the polynucleotides or polypeptides, and / or antagonists of the invention are used to inhibit the growth, progression, and / or metastasis of cancers, especially those listed above.

Further diseases or conditions associated with increased cell survival that can be treated, prevented or diagnosed by the polynucleotides or polypeptides of the invention, or agonists or antagonists, include malignant disease progression and / or metastasis and the following: Associated disorders such as, but not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and (Including erythroleukemia)) and chronic leukemia (eg chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg Hodgkin's and non-Hodgkin's disease), multiple Myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcoma) And carcinomas (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma)
, Lymphangiosarcoma, lymphangioendothelioma, periosteal tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma , Adenocarcinoma, sweat adenocarcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma , Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer,
Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningiom
a), melanoma, neuroblastoma, and retinoblastoma)).

Diseases associated with increased apoptosis that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention and / or agonists or antagonists include: AIDS; neurodegenerative disorders (eg, , Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related disorders; autoimmune diseases, disorders and / or conditions (eg multiple sclerosis, Sjogren's) Syndrome, Hashimoto thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis, and rheumatoid arthritis syndrome (eg, aplastic malformation). Anemia), transplantation to host Disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver). Cancer); toxic-induced liver disease (such as that caused by alcohol), septic shock,
Cachexia and loss of appetite.

Wound Healing and Epithelial Cell Proliferation In accordance with yet a further aspect of the invention, the polynucleotides or polypeptides of the invention and / or agonists or antagonists are used for therapeutic purposes, eg, for wound healing purposes. Processes are provided for utilization to stimulate epithelial cell proliferation and basal keratinocytes, as well as to stimulate hair follicle production and skin wound healing. The polynucleotides or polypeptides of the invention, and / or agonists or antagonists may be clinically useful in stimulating wound healing, including: surgical wounds, excisional wounds,
Deep wounds (including dermal and epidermal damage), ocular tissue wounds, tooth tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, fever Exposure to or chemical burns, and other abnormal wound healing conditions (eg, uremia, malnutrition, vitamin deficiency, and systemic treatment with steroids, radiotherapy and antineoplastic drugs and antimetabolites). Related complications). The polynucleotides or polypeptides of the invention and / or agonists or antagonists can be used to promote skin recovery after skin loss.

The polynucleotides or polypeptides of the invention, and / or agonists or antagonists are intended to increase attachment of skin grafts to the wound bed and to stimulate re-epithelialization from the wound bed. Can be used for. The following are types of implants in which the polynucleotides or polypeptides, agonists or antagonists of the invention can be used to increase adhesion to the wound bed: autograft, artificial skin, allograft. , Autograft, autoepidermographic graft, avascular (av
aural) graft, Blair-Brown graft, bone graft, embryonic tissue graft,
Dermal graft, delayed graft, skin graft, epidermal graft, fascia graft, full thickness skin graft, xenograft, xenograft (xe)
nograft), allograft, proliferative graft, lamellar graft, reticular graft, mucosal graft, Olie-Tielsch graft, omental graft, patch implantation. Pieces, stalk-like grafts, penetrating grafts, split-thickness skin grafts, split-thickness skin grafts. The polynucleotides or polypeptides of the invention, and / or agonists or antagonists can be used to promote skin strength and improve the appearance of aged skin.

The polynucleotides or polypeptides of the invention and / or agonists or antagonists also result in changes in hepatocyte proliferation and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and colon. it is conceivable that. The polynucleotides or polypeptides of the present invention, and / or agonists or antagonists, can be used in epithelial cells (eg, sebocytes, hair follicles, hepatocytes, alveolar epithelial cells type II (typ).
e II pneumocyte), mucin-producing goblet cells, and other epithelial cells,
And the proliferation of the skin, lungs, liver, and their ancestors contained within the gastrointestinal tract. The polynucleotides or polypeptides, agonists or antagonists of the invention can promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides of the invention and / or agonists or antagonists may also be used to reduce the toxic side effects of intestine resulting from radiation, chemotherapy treatments or viral infections. The polynucleotides or polypeptides of the invention and / or agonists or antagonists may have a cytoprotective effect on the small intestinal mucosa. The polynucleotides or polypeptides of the invention, and / or agonists or antagonists, are also mucositis (oral mucosa) resulting from chemotherapy and viral infections.
Can stimulate healing.

The polynucleotides or polypeptides of the invention, and / or agonists or antagonists, provide sufficient regeneration of skin (ie, hair follicles, sweat glands, and Regrowth of sebaceous glands),
It can further be used in the treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides of the invention, and / or agonists or antagonists, can affect epidermal bullosa, an endogenous dermis that produces frequent open and painful blisters by promoting re-epithelialization of these lesions. It can be used to treat defects in epidermal adhesions. A polynucleotide or polypeptide of the invention,
And / or agonists or antagonists may also be used to treat gastric and duodenal ulcers, and to aid healing by scarring the mucosal lining and regeneration of the more rapid glandular and duodenal mucosal lining. Inflammatory bowel disease (eg, Coulomb's disease and ulcerative colitis) are diseases that result in the disruption of the mucosal surface of the small or large intestine, respectively. Thus, the polynucleotides or polypeptides of the invention, and / or agonists or antagonists, promote the resurfacing of mucosal surfaces to aid more rapid healing and prevent the progression of inflammatory bowel disease. Can be used for: Treatment with the polynucleotides or polypeptides of the invention and / or agonists or antagonists is expected to have a significant effect on mucosal production throughout the gastrointestinal tract, and the intestinal mucosa may be ingested or surgically treated. It can be used to protect against harmful substances after surgery. The polynucleotides or polypeptides of the invention and / or agonists or antagonists can be used to treat diseases associated with the expression of the polynucleotides of the invention.

In addition, the polynucleotides or polypeptides of the invention and / or agonists or antagonists can be used to prevent and cure damage to the lung due to various pathological conditions. Polynucleotides or polypeptides of the invention, and / or agonists or antagonists can stimulate proliferation and differentiation to prevent or treat acute or chronic lung injury, and repair alveolar and bronchiola epithelium. Can be promoted. For example, emphysema (which results in progressive loss of alveoli) and inhalation injuries (ie, resulting from smoke inhalation and burns) resulting in necrosis of bronchial epithelium and aveoli of the present invention. Polynucleotides or polypeptides, and / or agonists or antagonists can be effectively treated, prevented and / or diagnosed. Also, the polynucleotides or polypeptides of the invention, and / or agonists or antagonists can be used to stimulate alveolar epithelial cell type II proliferation and differentiation, which results in hyaline membrane disease in premature infants (eg, , Infantile respiratory distress syndrome and bronchopulmonary dysplasia) can help treat or prevent diseases.

Polynucleotides or polypeptides of the invention, and / or agonists or antagonists may stimulate proliferation and differentiation of hepatocytes and, thus, liver diseases and conditions, such as fulminant liver failure caused by cirrhosis, Hepatitis virus and toxic substances (ie acetaminophen, carbon tetrachloride (carbo)
n tetraholloide) and other liver damages known in the art)).

In addition, the polynucleotides or polypeptides of the invention and / or agonists or antagonists can be used to treat or prevent the onset of diabetes mellitus. In patients newly diagnosed with type I diabetes and type II diabetes, if some islet cell function remains, the polynucleotide or polypeptide of the invention, and / or agonist or antagonist, may affect the persistence of the disease. Can be used to maintain its islet function so as to mitigate, delay or prevent the expression of. The polynucleotides or polypeptides of the invention and / or agonists or antagonists may also be used as an aid in islet cell transplantation to improve or promote islet cell function.

Neurological Disorders Nervous system diseases, disorders and / or that can be treated, prevented and / or diagnosed with the compositions (eg, polypeptides, polynucleotides and / or agonists or antagonists) of the invention. Conditions include, but are not limited to, nervous system damage and diseases, disorders and / or conditions that cause either axotomy, neuronal loss or degeneration, or demyelination. Nervous system lesions that may be treated in patients (including human and non-human mammal patients) according to the present invention include lesions of either the following, or of the central nervous system (including spinal cord, brain) or peripheral nervous system. (1) ischemic lesions (where oxygen deprivation in a part of the nervous system results in neuronal damage or death, including cerebral infarction or ischemia, or spinal infarction or Ischemia); (2) traumatic lesions (lesions caused by physical injury or associated with surgery,
For example, a lesion that cuts a part of the nervous system, or includes a compression injury); Is destroyed or damaged by malignant tissue which is); (4)
Infectious lesions, where parts of the nervous system are destroyed or damaged, for example as a result of infection by an abscess, or associated with infection by human immunodeficiency virus, herpes zoster or herpes simplex virus, Lyme disease , Associated with tuberculosis, syphilis; Degeneration associated with atrial sclerosis (ALS), including but not limited to; (6) lesions associated with trophic diseases, disorders and / or conditions, wherein a portion of the nervous system is Destroyed or damaged by sexual or metabolic disorders, which include vitamin B12 deficiency, folate deficiency, Wernicke's disease, tobacco Call amblyopia, Marchiafava Afar Valentino - Binyami disease including but and alcohol cerebellar degeneration (primary degeneration of the corpus callosum), but not limited to)
(7) Neurological lesions associated with systemic diseases (diabetes (diabetic neuropathy,
Bell's palsy), systemic lupus erythematosus, cancer or sarcoidosis); (8) lesions caused by toxic substances (including alcohol, lead or certain neurotoxins); and (9) prolapse. Myelinated lesions, in which a part of the nervous system is destroyed or damaged by demyelinating detention, which includes multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive (Including but not limited to multifocal leukoencephalopathy and central pontine myelinolysis))
.

In a preferred embodiment, the polypeptides, polynucleotides or agonists or antagonists of the invention are used to protect neurons from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat, prevent and / or diagnose neuronal cell damage 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 neuronal cell damage associated with cerebral ischemia. In another aspect of this embodiment, the polypeptide, polynucleotide, or agonist or antagonist of the invention treats neuronal cell damage associated with cerebral infarction,
Used for prophylaxis and / or diagnosis or prophylaxis. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neuronal cell damage associated with stroke. In a further aspect of this embodiment, the polypeptide of the invention,
The polynucleotides, or agonists or antagonists, are used to treat or prevent nerve cell damage associated with heart attack.

Compositions of the invention useful for treating or preventing nervous system disorders can be selected by testing for biological activity in promoting neuronal survival or differentiation. For example, and not by way of limitation, compositions of the invention that induce any of the following effects may be useful according to the invention: (1) increased survival time of neurons in the medium; (2) medium. Or increased sprouting of neurons in vivo; (3) neuron-related molecules in media or in vivo (eg,
With respect to motor neurons, increased production of choline acetyltransferase or acetylcholinesterase; or (4) diminished symptoms of neuronal dysfunction in vivo. Such effects can be measured by any method known in the art. Preferably, in a non-limiting embodiment, the increased survival time of neurons is determined by the methods described herein or otherwise known in the art (eg, Arakawa et al. (J. Neurosci. 10: 3
507-3515 (1990))); increased sprouting of neurons can be measured by methods known in the art (eg, Pest).
ronk et al. (Exp. Neurol. 70: 65-82 (1980)) or B
row et al. (Ann. Rev. Neurosci. 4: 17-42 (1981).
Increased production of neuron-related molecules is based on the molecule to be measured, using techniques known in the art, bioassays, enzyme assays, antibody binding, Northern blots. Can be measured, such as by an assay; and motor neuron dysfunction can be measured by assessing the physical expression of motor neuron impairment (eg, weakness, motor neuron conduction velocity, or dysfunction).

In certain embodiments, a motor neuron disease, disorder and / or condition that can be treated, prevented and / or diagnosed in accordance with the present invention includes a disease, disorder that can affect motor neurons and other components of the nervous system. And / or conditions (eg, infarction, infection, toxin exposure, trauma, surgical injury, degenerative disease, or malignancy), and diseases, disorders and / or conditions that selectively affect neurons (eg,
Amyotrophic lateral sclerosis), including but not limited to:
Progressive spinal muscular atrophy, progressive bulbar bulbar palsy, primary lateral sclerosis, pediatric muscular and juvenile muscular atrophy, childhood progressive bulbar palsy (Fatio-Ronde disease), polio and post-polio symptoms, As well as hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease).

Infectious Diseases The polynucleotides or polypeptides, and / or agonists or antagonists of the invention can be used to treat, prevent and / or diagnose infectious agents. For example, infectious diseases can be treated, prevented, and / or diagnosed by raising an immune response, particularly by increasing B cell and / or T cell proliferation and differentiation. The immune response can be elevated by either raising an existing immune response or initiating a new immune response. Alternatively, the polynucleotides or polypeptides of the invention and / or agonists or antagonists also do not necessarily elicit an immune response,
Infectious agents can be directly inhibited.

A virus is an example of an infectious agent that can cause a disease or condition that can be treated, prevented and / or diagnosed with a polynucleotide or polypeptide of the invention, and / or an agonist or antagonist. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and viridae: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Calciviridae,
Sarcoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (eg cytomegalovirus, herpes simplex, herpes zoster), mononegavirus (Mononegavirus) (eg, Paramyxoviridae, measles virus, rhabdoviridae), Orthomyxoviridae (eg, influenza A, influenza B, and parainfluenza), papillomavirus, papovaviridae, parvoviridae, picornavirus. Family,
Poxviridae (eg, smallpox or vaccinia), Reoviridae (eg, rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (eg, Rubivirus). Viruses that fall within these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, ocular infections (eg, conjunctivitis). , Keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic activity, Delta), Japanese encephalitis B, Argentine hemorrhagic fever, Chingnia, Rift Valley fever, yellow fever, meninges flame,
Opportunistic infections (eg AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, colds, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg , Capoge, warts), and viremia.
Any of these conditions or diseases can be treated, prevented and / or diagnosed with the polypeptides or polynucleotides of the invention, or agonists or antagonists. In certain embodiments, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used for the treatment of: Meningitis, Dengue, EBV, and / or Hepatitis (eg Hepatitis B). In a further specific embodiment, the polynucleotides, polypeptides of the invention,
Or agonists or antagonists are used to treat patients who are non-responsive to one or more other commercially available hepatitis vaccines. In a more specific embodiment, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.

Similarly, bacterial or fungal agents which can cause a disease or condition and which can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention and / or agonists or antagonists are: Includes, but is not limited to, Gram-negative and Gram-positive bacteria and bacteria and fungi: Actinomycetales (eg, Corynebac
terium, Mycobacterium, Norcardia), Cryp
tococcus neoformans, Aspergillosis, Ba
cillaceae (eg, Anthrax, Clostridium), B
acteroidaceae, Blastomycosis, Bordetel
la, Borrelia (eg, Borrelia burgdorferi
), Brucellosis, Candidiasis, Campylobacter
ter, Coccidioidomycosis, Cryptococcosi
S., Dermatocycleses, E.I. coli (for example, Enteroto
xigenic E. E. coli and Enterohemorrhagic E
． E. coli), Enterobacteriaceae (Klebsiella)
, Salmonella (eg, Salmonella typhi, and Salmonella paratyphi), Serratia, Yersi.
nia), Erysipelothrix, Helicobacter, Leg
ionellosis, Leptospirosis, Listeria, My
coplasmatales, Mycobacterium leprae, V
ibrio cholerae, Neisseriaceae (eg Aci
netobacter, Gonorrhea, Menigococcal), M
eisseria meningitidis, Pasteurellacea
Infectious diseases (for example, Actinobacillus, Hemophilus (
For example, Hemophilus influenza B), Pasteurell
a), Pseudomonas, Rickettsiaceae, Chlamy
diaceae, Syphilis, Shigella spp. , Staph
ylococcal, Meningiococcal, Pneumococca
l and Streptococcal (eg, Streptococcus
Pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause diseases or conditions including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunism. Infections (eg, AIDS-related infections), periungual inflammation, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg, pertussis or pyometra), sepsis, Lyme disease, cat scratch disease, dysentery, Paratyphoid fever, Food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (eg meningitis type A and B), chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulinum poisoning, gangrene, tetanus, impetigo , Rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatomycosis (de)
rmatocycles)), toxemia, urinary tract infection, wound infection. The polypeptides or polynucleotides, agonists or antagonists of the invention may be used to treat, prevent and / or diagnose any of these conditions or diseases. In a specific embodiment, the polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent and / or diagnose the following: tetanus, diphtheria, botulinum, and / or type B meninges. flame.

Furthermore, diseases or conditions caused by parasite factors that can be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention and / or agonists or antagonists include the following families or classes: Are not limited to these: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dinuclear amebiosis (Dientamoebi)
as), copulation, ectoparasite, giardiasis, helminthosis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomiasis, and Trichomonas disease, and sporozoan disease (Sporozoan).
(For example, Plasmodium virax, Plasmodium fal
ciparium, Plasmodium malariae and Plasm
odium ovale). These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, scrub typhus, ocular infections, intestinal diseases (eg, dysentery, giardia giardiasis), liver diseases, lungs. Diseases, opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may be used to treat, prevent and / or diagnose any of these conditions or diseases. In a particular embodiment, the polynucleotide of the invention,
The polypeptide, or agonist or antagonist, is used to treat malaria.

Preferably, treatment or prophylaxis using a polynucleotide or polypeptide of the invention and / or an agonist or antagonist involves administering to the patient an effective amount of the polypeptide or removing cells from the patient Either by supplying the polynucleotide of the invention to the cells and returning the engineered cells to the patient (ex vivo treatment). Moreover, the polypeptides or polynucleotides of the invention can be used as antigens in vaccines to elicit an immune response against infectious diseases.

Regeneration Polynucleotides or polypeptides of the invention and / or agonists or antagonists can be used to differentiate, proliferate and attract cells to guide tissue regeneration (Science 276: 59-). 87 (1997)). Tissue regeneration can be used to give birth defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis).
tissue, periodontal disease, liver failure), surgery including cosmetic surgery, fibrosis, reperfusion injury, or tissue damaged by systemic cytokine damage may be repaired, replaced, or protected.

Tissues that can be regenerated using the present invention include the following: Organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, or myocardium),
Tissues of the vascular system (including blood vessels and lymph vessels), nerves, hematopoiesis, and skeleton (bones, cartilage, tendons, and ligaments). Preferably, regeneration occurs without or with reduced scarring. Regeneration can also include angiogenesis.

Furthermore, the polynucleotides or polypeptides of the invention and / or agonists or antagonists may increase the regeneration of tissues that are difficult to heal. For example, increasing tendon / ligament regeneration speeds recovery time after injury. The polynucleotides or polypeptides of the invention and / or agonists or antagonists may also be used prophylactically in an attempt to avoid injury. Specific diseases that can be treated, prevented and / or diagnosed include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Further examples of tissue regeneration of non-healing wounds include ulcers associated with decubitus ulcers, vascular insufficiency, surgical wounds, and traumatic wounds.

Similarly, neural and brain tissue can also be regenerated by using the polynucleotides or polypeptides of the invention and / or agonists or antagonists to proliferate and differentiate neural cells. Treatment using this method,
Diseases that can be prevented and / or diagnosed include central and peripheral nervous system diseases, neuropathy, or mechanical and traumatic disorders (eg, myelopathy, head trauma, cerebrovascular disease, and stroke). ) Is mentioned. In particular, diseases associated with peripheral nerve injury, peripheral neuropathy (eg resulting from chemotherapy or other medical therapies), localized neuropathy, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington). Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention and / or agonists or antagonists.

Chemotaxis The polynucleotides or polypeptides and / or agonists or antagonists of the invention may have chemotactic activity. Chemotactic molecules allow cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to move to specific sites in the body (eg, Site of inflammation, infection, or hyperproliferation). The recruited cells may then repel and / or heal the particular trauma or abnormality.

The polynucleotides or polypeptides and / or agonists or antagonists of the invention may increase the chemotactic activity of particular cells. These chemotactic molecules are then used to increase the number of cells targeted to specific locations in the body, resulting in inflammation, infection, hyperproliferative diseases, disorders and / or conditions,
Or any immune system disorder may be treated, prevented and / or diagnosed. For example, chemotactic molecules can be used to treat, prevent, and / or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. The chemotactic molecules of the invention may also attract fibroblasts, which may be used to treat, prevent and / or diagnose wounds.

It is also contemplated that the polynucleotides or polypeptides and / or agonists or antagonists of the invention may inhibit chemotactic activity. These molecules can also be used to treat, prevent and / or diagnose disorders.
Thus, the polynucleotides or polypeptides and / or agonists or antagonists of the invention can be used as chemotaxis inhibitors.

Binding Activity The polypeptides of the invention can be used to screen for molecules that bind to this polypeptide, or for molecules to which this polypeptide binds. The binding of the polypeptide to the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of the bound polypeptide or molecule.
Examples of such molecules include antibodies, oligonucleotides, proteins (eg receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand (eg, fragment of the ligand) of the polypeptide or natural substrate, ligand, structural mimetic, or functional mimic (Coligan et al. , Current Prot
ocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, the molecule may be intimately associated with the natural receptor to which the polypeptide binds, or at least a fragment of the receptor (eg, the active site) that may be bound by the polypeptide. In any case, the molecule can be rationally designed using known techniques.

Preferably, screening for these molecules involves producing appropriate cells that express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include mammals, yeast, Drosoph
ila, or E.I. cells derived from E. coli. The cells expressing the polypeptide (or cell membranes containing the expressed polypeptide) are then preferably observed for binding, stimulating, or inhibiting activity of either the polypeptide or the molecule. Are contacted with a test compound potentially containing the molecule of

The assay may simply test binding of a candidate compound to the polypeptide, where binding is detected by the label or in an assay for competition with a labeled competitor. In addition, the assay can test whether a candidate compound produces a signal generated by binding to this polypeptide.

Alternatively, the assay can be performed with cell-free preparations, polypeptides / molecules attached to a solid support, chemical libraries, or a mixture of natural products. The assay also simplifies the steps of mixing the candidate compound with a solution containing the polypeptide, measuring the activity or binding of the polypeptide / molecule, and comparing the activity or binding of the polypeptide / molecule to a standard. May be included.

Preferably, the ELISA assay may measure the level or activity of a polypeptide in a sample (eg, biological sample) using monoclonal or polyclonal antibodies. Antibodies may be bound to the polypeptide either directly or indirectly, or by competition with the polypeptide for a substrate,
The level or activity of the polypeptide can be measured.

Furthermore, the receptors to which the polypeptides of the invention bind can be obtained by a number of methods known to those skilled in the art, such as ligand panning and FACS sorting (Coliga).
n et al., Current Protocols in Immun. , 1 (2),
Chapter 5 (1991)). For example, expression cloning is used when polyadenylated RNA is prepared from cells responsive to this polypeptide, eg, NIH3T3 cells, which are known to contain multiple receptors for FGF family proteins, And SC-3 cells, and a cDNA library made from this RNA are pooled and used to transfect COS cells or other cells that are not responsive to the polypeptide. Transfected cells growing on glass slides are exposed to the polypeptides of the invention after labeling them. The polypeptide may be labeled by various means, including iodination or encapsulation of recognition sites for site-specific protein kinases.

After immobilization and incubation, slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an iterative subpooling and rescreening process to ultimately yield a single clone encoding the putative receptor.

As an alternative approach for receptor identification, labeled polypeptides can be photoaffinity-linked or extracted with a preparation that expresses the receptor molecule. The crosslinked material is separated by PAGE analysis and exposed to X-ray film. A labeled complex containing the receptor for the polypeptide can be excised,
It can be separated into peptide fragments and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

Furthermore, the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”) can be used to modulate the activity of the polypeptides of the invention, thereby. Effectively produces agonists and antagonists of the polypeptides of the invention. Generally, US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,83.
7,458, and Patten, P .; A. Curr. Opinion
Biotechnol. 8: 724-33 (1997); Harayama,
S. Trends Biotechnol. 16 (2): 76-82 (1998)
); Hansson, L .; O. Et al., J. Mol. Biol. 287: 265-7
6 (1999); and Lorenzo, M .; M. And Blasco, R .;
See Biotechniques 24 (2): 308-13 (1998), each of which patents and publications are incorporated herein by reference. In one embodiment, alteration of the polynucleotides of the invention and corresponding polypeptides can be accomplished by DNA shuffling. DNA shuffling is the process of homologous recombination or site-specific recombination of two or more DNA fragments.
Including the construction of the segment into the desired polynucleotide sequence of the molecule of the present invention. In another embodiment, the polynucleotides of the invention and the corresponding polypeptides are subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Can be changed. In another embodiment, one or more components of the polypeptide of the invention,
A motif, segment, portion, domain, fragment, etc. can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules. In a preferred embodiment, the heterologous molecule is a member of the family. In a further preferred embodiment, the heterologous molecule is, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF) -α, epidermal growth factor (EGF), fibroblast. Cell growth factor (FGF), TGF-β, bone morphogenetic protein (BMP) -2, BMP-4, BM
P-5, BMP-6, BMP-7, activin A and activin B, decapentaplegic (dpp), 60A, OP-
2, dosalin, growth differentiation factor (GDF), nodule (nod)
al), MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3.
, TGF-β5, and growth factors such as glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. A biologically active fragment is a fragment that exhibits an activity similar to, but not necessarily identical to, the activity of this polypeptide. The biological activity of this fragment may include an improved desired activity, or a decreased undesired activity.

The present invention further provides methods of screening compounds to identify those that modulate the action of the polypeptides of the present invention. An example of such an assay involves combining mammalian fibroblasts, a polypeptide of the invention, the compound to be screened and ³ [H] thymidine under cell culture conditions in which the fibroblasts normally grow. . Control assays are performed in the absence of the compound to be screened to obtain, and compared to the amount of fibroblast proliferation in the presence of the compound, ³ in each case [H] thymidine incorporation of The determination can determine whether the compound stimulates proliferation. The amount of fibroblast proliferation is ³ [H
] Measured by liquid scintillation chromatography, which measures thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention is incubated with a labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured. Alternatively, the response and receptor of a known second messenger system according to the interaction of the compound to be screened is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to Determine if it is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays treat, prevent, and / or diagnose a disease by activating or inhibiting a polypeptide / molecule, or have a particular outcome in a patient (eg, blood vessel growth). Can be used to bring. In addition, the assay can discover factors that can inhibit or enhance production of the polypeptides of the invention from appropriately engineered cells or tissues. Accordingly, the invention includes a method of identifying a compound that binds to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) binding. A step of determining whether or not has occurred. Further, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying biological activity, And (b) determining whether the biological activity of the polypeptide has been modified.

Also, the β-pleated sheet region contained in the polypeptide sequence of the protein can be used to experimentally identify molecules that bind the polypeptide of the invention. Accordingly, certain embodiments of the invention include the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences, or alternatively, the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences. Consisting of a polynucleotide encoding a polypeptide. A further embodiment of the invention encodes a polypeptide comprising any combination or all contained in a polypeptide sequence of the invention, or consisting of any combination or all contained in a polypeptide sequence of the invention. To a polynucleotide. A further preferred embodiment of the invention comprises the amino acid sequence of each of the β pleated sheet regions in one of the polypeptide sequences of the invention, or β in one of the polypeptide sequences of the invention.
It relates to a polypeptide consisting of the amino acid sequence of each of the pleated sheet regions. Further embodiments of the invention include any combination or all of the β-pleated sheet regions in one of the polypeptide sequences of the invention, or any of the β-pleated sheet regions in one of the polypeptide sequences of the invention. It relates to polypeptides which consist of combinations or all.

Targeted Delivery In another embodiment, the invention provides a composition comprising a targeted cell expressing a receptor for a polypeptide of the invention, or a cell-bound form of a polypeptide of the invention. Methods of delivering to expressing cells are provided.

As discussed herein, a polypeptide or antibody of the invention is a hydrophobic, hydrophilic, ionic and / or covalently linked heterologous polypeptide, heterologous nucleic acid, toxin or prodrug. May be associated through interaction. In one embodiment, the invention provides a polypeptide of the invention ((a) associated with a heterologous polypeptide or nucleic acid (
A method for specific delivery of a composition of the invention to cells by administering (including an antibody) is provided. In one example, the invention provides a method for delivering therapeutic proteins into targeted cells. In another example, the invention can be a single-stranded nucleic acid (eg, an antisense or ribozyme) or a double-stranded nucleic acid (eg, integrated into the genome of a cell, or can be episomally replicating, and transcribed). , DNA) for delivery to targeted cells.

In another embodiment, the invention provides for cell specificity by administering a polypeptide of the invention (eg, a polypeptide of the invention or an antibody of the invention) associated with a toxin or a cytotoxic prodrug. Methods for selective destruction (eg, destruction of tumor cells).

A “toxin” is an endogenous cytotoxic effector system, radioisotope, holotoxin, modified toxin, catalytic subunit of a toxin, or a cell that causes cell death under defined conditions. Alternatively, it means a compound that binds and activates any molecule or enzyme not normally present on the cell surface. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, compounds that bind an intrinsic or induced endogenous cytotoxic effector system. (For example, antibody (or complement fixation including a part thereof), thymidine kinase, endonuclease, RNAse, α-toxin, ricin,
Abrin, Pseudomonas endotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "
By "cytotoxic prodrug" is meant a non-toxic compound that is converted into a cytotoxic compound by the enzymes normally present in cells. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. However, the present invention is not limited to these.

Drug Screening Further contemplated is the use of the polypeptides of the invention, or the polynucleotides encoding these polypeptides, to screen for molecules that modify the activity of the polypeptides of the invention. Such methods include the steps of contacting the polypeptides of the invention with selected compounds suspected of having antagonistic or agonistic activity, and assaying the activity of these polypeptides following binding. To do.

The present invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment used in such a test can be immobilized on a solid support, expressed on the cell surface, free in solution, or localized intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the polypeptide of the invention can be measured.

Accordingly, the invention provides methods of screening for drugs or any other agents that affect the activity mediated by the polypeptides of the invention. These methods involve contacting such agents with a polypeptide of the invention or fragment thereof by methods well known in the art, and the presence of a complex between the agent and the polypeptide or fragment thereof. The step of assaying is included. In such competitive binding assays, the drug to be screened is typically labeled. After incubation, free drug is separated from drug present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular drug to bind a polypeptide of the invention. .

Another technique for drug screening provides high throughput screening for compounds with appropriate binding affinity for the polypeptides of the invention, and European Patent Application 84/03564 (published September 13, 1984). ), Which is incorporated herein by reference, in greater detail. Simply put,
Large numbers of different small peptide test compounds are synthesized on a solid substrate (eg, plastic pins or some other surface). Peptide test compounds are reacted with the polypeptides of the invention and washed. Bound polypeptide is then detected by methods well known in the art. The purified polypeptide is coded directly on the plate for use in the drug screening techniques described above. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

The present invention also contemplates the use of competitive drug screening assays, wherein a neutralizing antibody capable of binding a polypeptide of the invention specifically competes with a test compound for binding to the polypeptide or fragment thereof. To do. In this style,
Antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with a polypeptide of the invention.

Antisense and Ribozymes (Antagonists) In certain embodiments, an antagonist according to the invention comprises a nucleic acid corresponding to the sequence contained in SEQ ID NO: X or its complementary strand and / or the nucleotide sequence contained in the disclosed clones. Is a nucleic acid corresponding to. In one embodiment, the antisense sequences are produced internally by the organism, and in another embodiment the antisense sequences are administered separately (eg, O'Connor, Neurochem.
56: 560 (1991)). Oligodeoxynucleo
tides as Antisense Inhibitors of Gen
e Expression, CRC Press, Boca Raton, FL
(1988). Antisense DNA or RN using antisense technology
Gene expression may be regulated through A or through the formation of triple helices. Antisense technology is described, for example, in Okano, Neurochem. 56: 560 (1
991); Oligodeoxynucleotides as Antise
ns Inhibitors of Gene Expression, CR
C Press, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Research.
ch 6: 3073 (1979); Cooney et al., Science, 241:
456 (1988); and Dervan et al., Science, 251: 130.
0 (1991). These methods use complementary DNA or R
Based on the binding of the polynucleotide to NA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines has been previously described (Wickstrom et al. 1988); Anfossi et al.
9)). These experiments were performed in vitro by incubating cells with oligoribonucleotides. A similar procedure for in vivo use is
It is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is generated as follows: a sequence complementary to the first 15 bases of the open reading frame, an EcoRI site at the 5'end and a HindIII at the 3'end. Adjacent to the site. The oligonucleotide pairs are then heated at 90 ° C. for 1 min, then 2 × ligation buffer (20 mM TR
Annealed in IS HCl pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated into the EcoR1 / HindIII sites of the retroviral vector PMV7 (WO91 / 15580).

For example, the 5'coding portion of the polynucleotide encoding the mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby inhibiting transcription and production of receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a receptor polypeptide.

In one embodiment, the antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, the vector or a portion thereof is transcribed to produce the antisense nucleic acid (RNA) of the invention. Such a vector contains a sequence encoding the antisense nucleic acid of the present invention. Such a vector can be episomal or chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. The vector can be a plasmid, virus, etc. known in the art used for replication and expression in vertebrate cells. Expression of sequences encoding the polypeptides of the present invention or fragments thereof may be obtained by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernist and Chambon, Nature, 29: 304-3.
10 (1981)), a promoter contained in the 3'-long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-797 (1980)),
Herpes thymidine promoter (Wagner et al., Proc. Natl. Aca
d. Sci. U. S. A. 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296: 39).
-42 (1982)) and the like, but are not limited thereto.

The antisense nucleic acid of the present invention contains a sequence complementary to at least a part of RNA transcript of the gene of interest. However, perfect complementarity, although preferred, is not necessary. A sequence "complementary to at least a portion of RNA" as referred to herein means a sequence that has sufficient complementarity to hybridize with RNA and forms a stable duplex; Thus, in the case of the double-stranded antisense nucleic acids of the invention, single strands of double-stranded DNA can be tested or triplex formation can be assayed.
The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, longer hybridizing nucleic acids may contain more base mismatches with the RNA sequences of the invention, which may include stable duplexes (or possibly triplexes), And yet it can be formed. One of skill in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting temperature of the hybridizing complex.

Oligonucleotides that are complementary to the 5'end of the message (eg, 5'untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. is there. However, sequences complementary to the 3'untranslated sequences of mRNAs have likewise been shown to be effective in inhibiting translation of mRNAs. Generally, Wagner, R .; , Nature 372: 333-335 (199).
See 4). Therefore, 5'- or 3'of the polynucleotide sequence of the present invention
Oligonucleotides complementary to any of the untranslated non-coding regions of
It can be used in an antisense approach that inhibits translation of RNA. 5 of mRNA
The oligonucleotide complementary to the'untranslated region should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region, although less efficient inhibitors of translation, can be used in accordance with the present invention. Whether designed to hybridize to the 5'region, 3'region, or coding region of the mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably 6 to about 50. An oligonucleotide that spans the length of a nucleotide. In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides.
Nucleotides or at least 50 nucleotides.

The polynucleotides of the present invention may be DNA, or RNA, or chimeric mixtures, or derivative or modified versions thereof, single-stranded, or double-stranded. The oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone to improve, eg, molecular stability, hybridization, and the like.
The oligonucleotide may be attached to other additional groups such as peptides (eg, for targeting host cell receptors in vivo) or factors that facilitate transport across cell membranes (eg, Letsinger et al., Proc. Natl. Acad.
． Sci. U. S. A. 86: 6553-6556 (1989); Lemait.
Re et al., Proc. Natl. Acad. Sci. 84: 648-652 (19
87); PCT Publication No. WO88 / 09810 (Published December 15, 1988)
, Or the blood-brain barrier (eg PCT Publication No. WO89 / 101).
34 (published April 25, 1988)), hybridization-triggered cleavag.
e.g.) (eg Kroll et al., BioTechniques, 6: 9).
58-976 (1988)), or intercalating agents (see, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide is a different molecule (for example,
Peptides, hybridization-triggered cross-linking agents, transport agents, hybridization-triggered cleavage agents, etc.).

Antisense oligonucleotides may include at least one modified base moiety, which is selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5 -Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosyl cuocosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2
-Methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v
), Wybutoxosine, pseudouracil, queocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy. Acetic acid (v), 5-methyl-2-thiouracil,
3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)
w, and 2,6-diaminopurine.

Antisense oligonucleotides may also include at least one modified sugar moiety selected from the group including, but not limited to: arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to: phosphorothioate, phosphorodithioate, phosphoro. Amidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof.

[0583] In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. The a-anomeric oligonucleotide forms a specific double-stranded hybrid with complementary RNA, which makes the strands parallel to each other as opposed to the normal b-unit (Gautier et al., Nucl. Acids Re.
s. 15: 6625-6641 (1987)). This oligonucleotide is
2-0-methyl ribonucleotide (Inoue et al., Nucl. Acid)
s Res. , 15: 6131-6148 (1987)), or chimeric RNA.
-A DNA analogue (Inoue et al., FEBS Lett. 215: 327).
-330 (1987)).

Polynucleotides of the invention may be prepared using standard methods known in the art (eg, automated D
By NA synthesizer (such equipment is Biosearch, Applied B
(commercially available from iosystems, etc.)). For example, phosphorothioate oligonucleotides are described by Stein et al. (Nuc
l. Acids Res. 16: 3209 (1988)),
Methylphosphonate oligonucleotides are used as controlled pore glass (con
polymerized polymer support (Sarin et al., Pr)
oc. Natl. Acad. Sci. U. S. A. , 85: 7448-7451
(1988)) and the like.

On the other hand, antisense nucleotides complementary to the coding region sequences of the invention may be used, with antisense nucleotides complementary to the transcribed, untranslated region being most preferred.

Potential antagonists according to the present invention also include catalytic RNA, ie ribozymes (eg PCT International Publication WO 90/11364, October 4, 1990).
Published: Sarver et al., Science, 247: 1222-1225 (19).
90)). On the other hand, a ribozyme that cleaves an mRNA at a site-specific recognition sequence can be used to destroy the mRNA corresponding to the polynucleotide of the present invention, but it is preferable to use a hammerhead ribozyme. Hammerhead ribozymes cleave mRNAs at positions determined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 '. Construction and production of hammerhead ribozymes is well known in the art, and Haseloff and Ge
Rlach, Nature, 334: 585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the Sequence Listing. Preferably, the ribozyme is such that a cleavage recognition site is located near the 5'end of the mRNA corresponding to the polynucleotide of the invention; ie, it increases efficiency and promotes intracellular accumulation of non-functional mRNA transcripts. Manipulated to minimize.

In the case of the antisense approach, the ribozymes of the invention may be composed of modified oligonucleotides (improved for stability, targeting, etc.) and express the polynucleotides of the invention in vivo. Should be delivered to cells. The DNA construct encoding the ribozyme can be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery is to "encode" the ribozyme under the control of a strong constitutive promoter (such as the pol III or pl II promoter).
Including the use of NA constructs, so that the transfected cells disrupt the endogenous message and produce a sufficient amount of ribozyme to inhibit translation. Ribozymes, unlike antisense molecules, are catalytic, so lower intracellular concentrations are required for efficiency.

Antagonist / agonist compounds are utilized to grow and proliferate the polypeptides of the invention to neoplastic cells and tissues.
feration) effect. That is, stimulating a tumor agonist,
It slows or prevents aberrant cell growth and proliferation (eg, in tumorigenesis or proliferation).

Antagonists / agonists may also be utilized to inhibit hypervascular disease and prevent proliferation of epithelial lens cells following extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the invention may also be required in cases such as restenosis after balloon angioplasty.

Antagonists / agonists may also be utilized to prevent the growth of scar tissue during wound healing.

Antagonists / agonists may also be utilized to treat the diseases described herein.

Accordingly, the present invention includes diseases, disorders and / or conditions (including those diseases, disorders and / or conditions listed throughout this application which are associated with overexpression of a polynucleotide of the invention). (But not limited thereto) by treating (a) an antisense molecule directed to the polynucleotide of the present invention and / or (b) a ribozyme directed to the polynucleotide of the present invention to a patient. Provide a way to do.

Other Activities The polypeptides of the invention are responsible for various disease states (eg, thrombosis, arteriosclerosis, and other cardiovascular conditions) as a result of their ability to stimulate vascular endothelial cell proliferation. Can be used in a procedure for stimulating the revascularization of ischemic tissue. These polypeptides may also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

The polypeptide may also be utilized in the treatment of wounds due to injury, burns, post-operative tissue repair, and scarring. Because they are mitogenic for a variety of cells of different origin, such as fibroblasts and skeletal muscle cells, and therefore facilitate repair or replacement of damaged or diseased tissue.

The polypeptides of the invention also stimulate neuronal growth and treat neuronal damage that occurs in certain neuronal disorders or neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complexes. , For prevention and / or diagnosis. The polypeptides of the present invention may have the ability to stimulate chondrocyte proliferation and thus enhance bone and periodontal remodeling and may be utilized for assistance in tissue grafts or bone grafts. .

The polypeptides of the present invention may also be utilized to prevent skin aging due to sunburn by stimulating keratinocyte proliferation.

The polypeptides of the present invention may also be utilized to prevent hair loss. This is because members of the FGF family activate hair-forming cells and promote melanocyte proliferation. Along the same line, the polypeptides of the invention can be utilized to stimulate hematopoietic and myeloid cell proliferation and differentiation when used in combination with other cytokines.

The polypeptides of the present invention may also be utilized to maintain organs prior to transplantation, or may be used to support cell culture of primordial tissue.

The polypeptides of the present invention may also be utilized to induce tissue of mesoderm origin for differentiation in early embryos.

The polypeptides or polynucleotides and / or agonists or antagonists of the invention may also increase or decrease the differentiation or proliferation of embryonic stem cells in addition to hematopoietic lineages as discussed above.

The polypeptides or polynucleotides and / or agonists or antagonists of the invention may also have mammalian characteristics (eg height, weight, hair color,
It can be used to regulate eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (eg, cosmetic surgery). Similarly, the polypeptides or polynucleotides and / or agonists or antagonists of the invention can be used to regulate mammalian metabolism that affects catabolism, anabolic activity, processing, utilization, and energy storage.

Polypeptides or polynucleotides and / or agonists or antagonists of the invention may be used in biorhythms, caricadic
) Rhythm, depression (including depressive diseases, disorders and / or conditions), tendency to violence, resistance to pain, fertility (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite , Can be used to alter a mammal's mental or physical condition by affecting libido, memory, stress, or other cognitive qualities.

The polypeptides or polynucleotides and / or agonists or antagonists of the invention may also increase, for example, storage capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional components or It can be used as a food additive or preservative to reduce.

Other Preferred Embodiments Another preferred embodiment of the present invention comprises a nucleotide sequence that is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X, Included is an isolated nucleic acid molecule, where X is any integer as defined in Table 1.

The sequence of contiguous nucleotides begins at the nucleotide approximately 5 ′ nucleotide position of the clone sequence, as defined for SEQ ID NO: X in Table 1,
Also preferred is a nucleic acid molecule comprised in the nucleotide sequence of SEQ ID NO: X, in the range of positions ending with nucleotides at approximately the 3'nucleotide position of the clone sequence.

The sequence of contiguous nucleotides begins at the nucleotide approximately 5 ′ nucleotide position of the start codon, as defined for SEQ ID NO: X in Table 1, and approximately 3 ′ nucleotide position of the clone sequence. Also preferred are nucleic acid molecules comprised in the nucleotide sequence of SEQ ID NO: X, in the range of positions ending in nucleotides.

The sequence of contiguous nucleotides begins approximately at the 5'nucleotide of the first amino acid of the signal peptide, as defined for SEQ ID NO: X in Table 1, and approximately 3 of the cloned sequence. Also preferred are nucleic acid molecules comprised in the nucleotide sequence of SEQ ID NO: X, in the range of positions ending with the nucleotide at the nucleotide position.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

A further preferred embodiment is as defined for SEQ ID NO: X in Table 1,
Includes a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: X that begins approximately at the 5'nucleotide position of the first amino acid of the signal peptide and ends approximately at the 3'nucleotide position of the clone sequence. It is a nucleic acid molecule.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence of SEQ ID NO: X.

Also preferred is an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule under stringent hybridization conditions, wherein the hybridizing nucleic acid molecule above is an A residue under stringent hybridization conditions. Alone or does not hybridize to a nucleic acid molecule having a nucleotide sequence consisting of only T residues.

Also preferred is a composition of matter comprising a DNA molecule comprising a human cDNA clone identified by the cDNA clone ID in Table 1, said DNA molecule being comprised in the material deposited with the American Type Culture Collection, and The ATCC registration number shown in Table 1 is given for the above cDNA clone ID.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of the human cDNA clone identified by the cDNA clone ID in Table 1. ,
This DNA molecule is contained in the deposit given the ATCC registration number shown in Table 1.

Also preferred is an isolated nucleic acid molecule wherein the sequence of at least 50 contiguous nucleotides described above is contained within the nucleotide sequence of the complete open reading frame sequence encoded by the human cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by the human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by the above human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence encoded by the above human cDNA clone.

A further preferred embodiment detects in a biological sample a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: Is a nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1; and identified by the cDNA clone ID in Table 1 and in Table 1. A nucleotide sequence encoded by a human cDNA clone contained in a deposit having the ATCC accession number indicated for said cDNA clone; said method comprising a sequence selected from the group above and at least one of the above samples Comparing the nucleotide sequence of the nucleic acid molecule, and the sample Determining whether the sequence of the nucleic acid molecule in the sequence is at least 95% identical to the selected sequence.

Comparing the sequences described above comprises determining the degree of nucleic acid hybridization between the nucleic acid molecule in the sample and a nucleic acid molecule comprising the sequence selected from the group above. The above method is also preferable. Similarly, the step of comparing the sequences described above, with a nucleotide sequence determined from the nucleic acid molecules in the sample,
Also preferred is the above method carried out by the step of comparing with a sequence selected from the above group. Nucleic acid molecules can include DNA or RNA molecules.

[0621] A further preferred embodiment is a method for identifying the species, tissue, or cell type of a biological sample, the method comprising at least 50 contiguous sequences in a sequence selected from the group consisting of: Detecting a nucleic acid molecule (if any) in the sample that contains a nucleotide sequence that is at least 95% identical to the sequence of the nucleotides selected: SEQ ID NO: X, wherein X is Table 1 Any integer as defined in Table 1; and A identified by the cDNA clone ID in Table 1 and shown in Table 1 for the cDNA clone above.
The nucleotide sequence encoded by the human cDNA clone contained in the deposit with the TCC accession number.

The method for identifying the species, tissue, or cell type of a biological sample can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein At least one sequence in the panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the sequence selected from the group above.

Also preferred is a method for diagnosing in a subject a pathological condition associated with aberrant structure or expression of a gene encoding a secreted protein identified in Table 1, which method comprises the group consisting of: Detecting a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from, in a biological sample obtained from the subject (if any). A nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1; and a cDNA clone identified by the cDNA clone ID in Table 1 and described above in Table 1. Encoded by the human cDNA clone contained in the deposit having the ATCC accession number shown for Nucleotide sequence.

The method for diagnosing a pathological condition can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in the panel. Is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the above group.

Also preferred is a composition of matter comprising an isolated nucleic acid molecule, wherein the nucleotide sequence of said nucleic acid molecule comprises a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is , At least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1. And the nucleotide sequence encoded by the human cDNA clone contained in the deposit identified by the cDNA clone ID in Table 1 and having the ATCC accession number shown for the above cDNA clone in Table 1. Nucleic acid molecules can include DNA or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least about 10 consecutive amino acids in the amino acid sequence of SEQ ID NO: Y, where Y is in Table 1 It is an arbitrary integer as specified.

The sequence of contiguous amino acids begins at the residue approximately at the first amino acid position in the secretory portion, as shown for SEQ ID NO: Y in Table 1, and at approximately the last amino acid in the open reading frame. Within the range of positions ending with a residue, SEQ ID NO: Y
Also preferred is a polypeptide comprised in the amino acid sequence of

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 30 consecutive amino acids in the amino acid sequence of SEQ ID NO: Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 100 consecutive amino acids in the amino acid sequence of SEQ ID NO: Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the complete amino acid sequence of SEQ ID NO: Y.

The cDNA clones identified by the cDNA clone ID in Table 1 and above in Table 1
Human c contained in the deposit with the ATCC accession number shown for the A clone
Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a DNA clone.

The sequence of contiguous amino acids above is identified by the cDNA clone ID in Table 1 and encoded by the human cDNA clone contained in the deposit with the ATCC accession number shown for the above cDNA clone in Table 1. Also preferred is a polypeptide comprised in the amino acid sequence of the secretory portion of a secreted protein.

This cDNA identified in Table 1 by the cDNA clone ID and in Table 1
Amino acids that are at least 95% identical to a sequence of at least about 30 consecutive amino acids in the amino acid sequence of the secretory portion of the protein encoded by the human cDNA clone contained in the deposit having the ATCC accession number shown for the A clone. Also preferred are isolated polypeptides that include sequences.

This cDNA identified in Table 1 by the cDNA clone ID and in Table 1
Amino acids that are at least 95% identical to a sequence of at least about 100 consecutive amino acids in the amino acid sequence of the secretory portion of the protein encoded by the human cDNA clone contained in the deposit having the ATCC accession number shown for the A clone. An isolated polypeptide comprising the sequence is also preferred.

This cDNA identified in Table 1 by the cDNA clone ID and in Table 1
Human c contained in the deposit with the ATCC accession number shown for the A clone
Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the secretory portion of the protein encoded by the DNA clone.

An isolated that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: The antibody is further preferred: the amino acid sequence of SEQ ID NO: Y, where Y is any integer defined in Table 1; and identified by the cDNA clone ID in Table 1 and for this cDNA clone in Table 1. The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit with the indicated ATCC accession number.

A method of detecting in a biological sample a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Preferred: amino acid sequence of SEQ ID NO: Y, where Y is any integer defined in Table 1; and the ATCC entry identified by the cDNA clone ID in Table 1 and shown for this cDNA clone in Table 1. Complete amino acid sequence of the protein encoded by the human cDNA clone contained in the numbered deposit; the method compares the amino acid sequence of at least one polypeptide molecule in this sample to a sequence selected from this group. The process and distribution of this polypeptide molecule in this sample. A row contains at least 9 sequences of this sequence of at least 10 consecutive amino acids.
Includes determining if 0% identity.

This step of comparing the amino acid sequence of at least one polypeptide molecule in this sample with a sequence selected from this group includes Determining the degree of specific binding to an antibody that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids. The method is also preferred: the amino acid sequence of SEQ ID NO: Y, where Y is any integer defined in Table 1; and the cDNA clone ID in Table 1.
The complete amino acid sequence of the protein encoded by the human cDNA clone identified in Table 1 and contained in the deposit with the ATCC accession number shown for this cDNA clone in Table 1.

Also preferred is the above method, wherein the step of comparing the sequences is carried out by comparing the amino acid sequence determined from the polypeptide molecules in the sample with this sequence selected from this group.

Also preferred is a method of identifying a species, tissue or cell type of a biological sample, wherein the method comprises a polypeptide molecule in the sample (where the polypeptide, if present, comprises: At least 10 in sequences selected from the group
Including an amino acid sequence that is at least 90% identical to a sequence of contiguous amino acids)
Detecting the amino acid sequence of SEQ ID NO: Y, where Y is any integer defined in Table 1; and for this cDNA clone identified in Table 1 by the cDNA clone ID and in Table 1. The complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit with the indicated ATCC accession number.

Also preferred is the above method of identifying a species, tissue or cell type of a biological sample, wherein the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences. , Wherein at least one sequence in this panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group above.

Also preferred is a method of diagnosing a pathological condition associated with aberrant structure or expression of a gene encoding a secreted protein identified in Table 1 in a subject.
Here, the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences in a biological sample obtained from the subject, wherein at least one of the panels in the panel. One sequence is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: Y, where Y is any of those defined in Table 1. And the cDNA clone ID in Table 1
The complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit identified by and having the ATCC accession number shown for this cDNA clone in Table 1.

In any of these methods, the step of detecting the polypeptide molecule comprises the steps of:
The step of using an antibody is included.

At least 95% identical to a nucleotide sequence that encodes a polypeptide that includes an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence: the amino acid sequence of SEQ ID NO: (wherein
Y is any integer defined in Table 1); and the AT identified by the cDNA clone ID in Table 1 and shown for this cDNA clone in Table 1.
The complete amino acid sequence of the secretory protein encoded by the human cDNA clone contained in the deposit with CC accession number.

Also preferred is an isolated nucleic acid molecule in which the nucleotide sequence encoding the polypeptide is optimized for expression of the polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule in which the polypeptide comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: Y, where Y is any of those defined in Table 1. Is an integer); and the complete amino acid sequence of the secretory protein encoded by the human cDNA clone identified in the cDNA clone ID in Table 1 and included in the deposit with the ATCC accession number shown for this cDNA clone in Table 1.

Further preferred is a method of making a recombinant vector comprising the step of inserting any of the above isolated nucleic acid molecules into a vector. The recombinant vector produced by this method is also preferred. Also preferred is a method of making a recombinant host cell, which comprises the step of introducing the vector into a host cell, as well as a recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide, comprising culturing the recombinant host cell under conditions such that the polypeptide is expressed, and recovering the polypeptide. . The recombinant host cell is a eukaryotic cell and the polypeptide produces an isolated polypeptide that is a secretory portion of a human secretory protein that comprises an amino acid sequence selected from the group consisting of: This method is also preferred: the amino acid sequence of SEQ ID NO: beginning with the residue at the first amino acid position of the secretory portion of SEQ ID NO: Y, where Y is an integer listed in Table 1 and SEQ ID NO: This position of the first amino acid of the secretory portion of Y is defined in Table 1); and the deposit with the ATCC accession number identified by the cDNA clone identifier (ID) in Table 1 and shown for this cDNA clone in Table 1. The amino acid sequence of the secretory portion of the protein encoded by the human cDNA clone contained in the product. Also preferred are isolated polypeptides produced by this method.

Also preferred are methods of treating an individual in need of increased levels of secreted protein activity. Here, the method comprises administering to such an individual a fixed amount of an isolated polypeptide, polynucleotide, or antibody of the invention effective to increase the level of activity of this protein in the individual. Administering the topical composition.

The applications cited above have been used in a wide variety of hosts. Such hosts include humans, mice, rabbits, goats, guinea pigs, camels, horses,
Mice, rats, hamsters, pigs, micro pigs, chickens, goats, cows, sheep, dogs, cats, non-human primates, and humans are included, but are not limited to. In a particular embodiment, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In the most preferred embodiment, the host is human.

In a particular embodiment of the present invention, for each “Contig ID” listed in the fourth column of Table 2, preferably the nucleotide sequence referenced in the fifth column of Table 2 and the general formula one or more of the nucleotide sequences described by ab, wherein a and b are independently determined for the corresponding SEQ ID NO: X referenced in column 3 of Table 2; Polynucleotides are excluded. A more particular embodiment is directed to polynucleotide sequences that exclude 1, 2, 3, 4, or more of the particular polynucleotide sequences referenced in the fifth column of Table 2. This table is in no way meant to include all of the sequences that can be excluded by the general formula, which is merely an illustrative example. All documents available through these registries are hereby incorporated by reference in their entireties.

[0652]

[Table 2] Although the invention has been generally described, the invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting.

Examples Example 1: Isolation of Selected cDNA Clones from Deposited Samples Each cDNA clone in the referenced ATCC deposit is contained in a plasmid vector. Table 1 identifies the vector used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a plasmid excised phage vector. The table immediately below correlates the relevant plasmids for each phage vector used in constructing the cDNA library. For example, if a particular clone is identified in Table 1 as being isolated in the vector "Lambda Zap", the corresponding deposited clone is "pBluescript".

[0654]

[Table 3] Vector Lambda Zap (US Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (US Pat. No. 5,128,25).
6 and 5,286,636), Zap Express (US Pat. Nos. 5,128,256 and 5,286,636), pBluescri.
pt (pBS) (Short, JM et al., Nucleic Acids Re).
s. 16: 7583-7600 (1988); Alting-Mees, M .; A
． And Short, J. et al. M. , Nucleic Acids Res. 17
: 9494 (1989)) and pBK (Alting-Mees, MA).
Et al., Strategies 5: 58-61 (1992)) by Stratag.
ene Cloning Systems, Inc. , 11011 N.I. Tor
commercially available from rey Pines Road, La Jolla, CA, 92037. pBS contains the ampicillin resistance gene and pBK contains the neomycin resistance gene. Both are E. E. coli strain XL-1 Blue, which is also available from Stratagene. pB
S arrives in four forms: SK +, SK-, KS + and KS. S and K are polylinker regions (“S” is SacI and “K” is KpnI).
Which is the first site at each end of each of the linkers)
Refers to the orientation of the polylinker relative to the T7 and T3 primer sequences flanking.
"+" Or "-" means the orientation of the f1 origin of replication ("ori") such that single-stranded rescue initiated from f1 ori in one direction produces sense strand DNA, and antisense in the other. Say.

Vectors pSport1, pCMVSport 2.0 and pCMVSpo
rt3.0 from Life Technologies, Inc. , P .; O. Box
6009, Gaithersburg, MD 20897. All Sport vectors contain the ampicillin resistance gene, and E. coli. coli strain D
H10B, which is also available from Life Technologies, can be transformed. (For example, Gruber, CE et al., Focus)
15:59 (1993)). Vector lafmid BA (Ben
to Soares, Columbia University, NY) contains the ampicillin resistance gene, and E. E. coli strain XL-1 Blue. Vector pCR® 2.1 (this is Invitroge
n, 1600 Faraway Avenue, Carlsbad, CA 92
(Available from E. 008) contains an ampicillin resistance gene and c
oli strain DH10B (available from Life Technologies) (eg, Clark, JM, Nuc. Acids).
Res. 16: 9677-9686 (1988) and Mead, D .; Et al, Bi
o / Technology 9: (1991)). Preferably, the polynucleotides of the invention do not include the phage vector sequences identified for the particular clones in Table 1, as well as the corresponding plasmid vector sequences shown above.

The deposited material in the sample given the ATCC accession number for any given cDNA clone, referred to in Table 1, also contains one or more additional plasmids, each of which is referred to as its given clone. Including different cDNA clones)
Can be included. Thus, deposits sharing the same ATCC accession number include at least the plasmid for each cDNA clone identified in Table 1. Typically, the sample of each ATCC deposit referenced in Table 1 contains a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; , Such deposit samples have more or less than 50 c
Plasmids for DNA clones (up to about 500 cDNA clones) can be included.

Two approaches can be used to isolate a clone from the deposited sample of plasmid DNA cited for that particular clone in Table 1. First, the plasmid is isolated directly by screening the clones with the polynucleotide probe corresponding to SEQ ID NO: X.

In particular, a particular polynucleotide having 30-40 nucleotides is synthesized according to the reported sequence using an Applied Biosystems DNA synthesizer. The oligonucleotide is labeled with, for example, ³² P-γ-ATP, T
Labeled with 4 polynucleotide kinase and purified according to conventional methods. (For example, Maniatis et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harb
or Press, Cold Spring, NY (1982)). Techniques known to those of skill in the art, such as those provided by vector suppliers or those provided in the relevant publications or patents cited above, are available for plasmid mixtures.
Using a suitable host as described above (eg XL-1 Blue (Strat.
a))). Transformants are plated on 1.5% agar plates (containing a suitable selection agent such as ampicillin) at a density of about 150 transformants (colony) per plate. These plates were subjected to conventional methods for bacterial colony screening (eg Sambrook et al., M.
olecular Cloning: A Laboratory Manual
, Second Edition, (1989), Cold Spring Harbor Labor.
Screening using nylon membranes according to the Atory Press, pages 1.93-1.104) or other techniques known to those of skill in the art.

Alternatively, both ends of SEQ ID NO: X (ie, the 5'N of the clone defined in Table 1)
17-2 from within the region of SEQ ID NO: X surrounded by T and 3′NT)
Two primers of 0 nucleotides are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under conventional conditions, for example in 25 μl of a reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl ₂ , 0.01% (w / v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 units of Taq polymerase. . 35 cycle PC
R (denaturation at 94 ° C. for 1 min; annealing at 55 ° C. for 1 min; extension at 72 ° C. for 1 min) is performed using a Perkin-Elmer Cetus automated thermal cycler. The amplification product is analyzed by agarose gel electrophoresis and the DNA band of the expected molecular weight is excised and purified. PCR product, D
Confirm the selected sequence by subcloning and sequencing the NA product.

Several methods are available for the identification of the 5'non-coding or 3'non-coding portions of genes that may not be present in the deposited clones. These methods include, but are not limited to: filter probe probing, clonal enrichment using specific probes, and 5'and 3 well known in the art.
'A protocol similar or identical to the "RACE" protocol. For example, 5 '
A method similar to RACE is available to generate the missing 5'end of the desired full length transcript (Frommont-Racine et al., Nucleic A.
cids Res. 21 (7): 1683-1684 (1993)).

Briefly, specific RNA oligonucleotides are ligated to the 5'end of a population of RNAs that probably contains full-length gene RNA transcripts. PCR of the 5'portion of the desired full-length gene is performed using a primer set containing a primer specific to the linked RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
Amplify. The amplified product can then be sequenced and used to generate the full length gene.

This above method starts with total RNA isolated from the desired source, but may also use poly A + RNA. The RNA preparation is then treated with phosphatase, if necessary, to degrade or damage the RN, which may interfere with subsequent RNA ligase steps.
The 5'phosphate group of A can be eliminated. The phosphatase should then be inactivated, and the RNA should be treated with tobacco acid pyrophosphatase to remove the cap structure present at the 5'end of the messenger RNA.
This reaction leaves a 5'phosphate group at the 5'end of the cap cleaved RNA that can then be ligated to the RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first-strand cDNA synthesis using gene-specific oligonucleotides. First strand synthesis reaction
It is used as a template for PCR amplification of the desired 5'end using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest. The resulting product is then sequenced and analyzed to confirm that the 5'end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to Polynucleotides Human Genome P1 Library (Genomic Systems, Inc.)
Are screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO: X according to the method described in Example 1 (Sa
See also mbrook).

[0665]   (Example 3: Tissue distribution of polypeptide)   The tissue distribution of mRNA expression of the polynucleotides of the invention can be determined, inter alia, by Samb
Use the protocol for Northern blot analysis described by look et al.
Decide. For example, the cDNA produced by the method described in Example 1.
Probe the rediprime^TM  DNA labeling system
(Amersham Life Science) and follow the manufacturer's instructions.
That's P³²Label with. After labeling, the probe was attached to CHROMA SPIN-100. ^TM Using a column (Clontech Laboratories, Inc.)
And purify according to manufacturer's protocol number PT1200-1. Then refined
Various human tissues are tested for mRNA expression using labeled probes
.

Multiple Tissue Northern (MTN) blots (Clontech) containing various human tissues (H) or human immune system tissues (IM) were analyzed using ExpressHyb ^™ Hybridization Solution (Clontech), manufacturer's protocol number P.
Test with labeled probe according to T1190-1. After hybridization and washing, the blots are mounted and exposed to film overnight at -70 ° C and the film developed according to standard procedures.

Example 4: Chromosomal Mapping of Polynucleotides A set of oligonucleotide primers is designed according to the sequence at the 5'end of SEQ ID NO: X. This primer preferably spans about 100 nucleotides.
This primer set is then used for polymerase chain reaction under the following set of conditions: 95 ° C for 30 seconds; 56 ° C for 1 minute; 70 ° C for 1 minute. This cycle is repeated 32 times and then once at 70 ° C. for 5 minutes. Somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc)
In addition, human, mouse, and hamster DNA are used as templates. Reactions are analyzed on either an 8% polyacrylamide gel or a 3.5% agarose gel. Chromosome mapping is performed for about 100 in specific somatic cell hybrids.
Determined by the presence of the bp PCR fragment.

Example 5: Bacterial Expression of Polypeptides Polynucleotides encoding the polypeptides of the present invention, as outlined in Example 1, are PCR oligos corresponding to the 5'and 3'ends of the DNA sequence. Amplification is performed using nucleotide primers to synthesize the insert fragment. The primer used to amplify the cDNA insert should preferably contain restriction sites such as BamHI and XbaI at the 5'end of the primer for cloning the amplification product into an expression vector. For example, BamHI and XbaI are bacterial expression vectors pQE-9 (Qiagen, Inc., Chatsworth,
CA) corresponding to the restriction enzyme site. This plasmid vector
Amp ^r ), bacterial origin of replication (ori), IPTG-regulatable promoter /
It encodes an operator (P / O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and a restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated in bacterial RBS. The ligation mixture was then treated with the plasmid pR, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ).
E. coli, including multiple copies of EP4. E. coli strain M15 / rep4 (Qiagen,
Inc. ). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired construct were cloned into Amp (100 μg / ml) and Kan (100 μg / ml).
Liquid culture in LB medium supplemented with both (25 μg / ml) overnight (O / N)
Proliferate. The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells were placed at an optical density of 600 (OD) between 0.4 and 0.6.
． Grow to ⁶⁰⁰ ). Then IPTG (isopropyl-BD-thiogalactopyranoside) is added to a final concentration of 1 mM. IPTG is lacI
Induced by repressor inactivation, clears P / O obstructions and leads to increased gene expression.

Cells are grown for an additional 3-4 hours. The cells are then centrifuged (6000 x
g for 20 minutes). The cell pellet is chaotropic agent 6
Solubilize by stirring in M guanidine HCl for 3-4 hours at 4 ° C. Cellular debris was removed by centrifugation and the polypeptide-containing supernatant was loaded onto a nickel-nitrilotriacetic acid (“Ni-NTA”) affinity resin column (QI).
AGEN, Inc. (Available from above). Proteins with a 6xHis tag bind Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (see, in particular, The QIA expressionlist (1
995) QIAGEN, Inc. , See above).

Briefly, the supernatant was loaded onto a column of 6M guanidine-HCl, pH8, which column was first washed with 10 volumes of 6M guanidine-HCl, pH8,
It is then washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

The purified protein was then loaded with phosphate buffered saline (PBS) or 5
Regenerate by dialysis against 0 mM sodium acetate, pH 6 buffer and 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. Recommended conditions are as follows: Regeneration using a linear gradient of 6M to 1M urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. Regeneration should take a minimum of 1.5 hours. After playing
The protein is eluted by the addition of 250 mM imidazole. Imidazole in PBS or 50 mM sodium acetate pH 6 buffer and 200 mM
It is removed by a final dialysis step against NaCl. 4 purified proteins
Store at ℃ or frozen at -80 ℃.

In addition to the expression vectors described above, the invention further comprises an expression vector called pHE4a, which comprises a phage operator and a promoter element operably linked to the polynucleotide of the invention (ATCC Accession No. 209645).
, Deposited on February 25, 1998). This vector contains: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, and 6) lactose operon repressor gene (laqIq). The origin of replication (oriC) is pUC19 (LTI, Ga
Itersburg, MD). Promoter and operator sequences are made synthetically.

The vector was restricted with NdeI and XbaI, BamHI, XhoI, or Asp718, the restriction products were run on a gel, and the larger fragment (the stuffer fragment should be approximately 310 base pairs). DNA) can be inserted into pHEa by isolating D
The NA insert was labeled with NdeI (5 ′ primer) and XbaI, BamHI, Xh.
PCR with restriction sites for oI or Asp718 (3 'primer)
Primers are used and generated according to the PCR protocol described in Example 1.
The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

The engineered vector can be easily replaced in the above protocol to express the protein in bacterial systems.

Example 6: Purification of Polypeptides from Inclusion Bodies Another method described below is the procedure described in E. coli when the polypeptides are present in the form of inclusion bodies. co
It can be used to purify the polypeptide expressed in li. Unless otherwise specified, all the following steps are performed at 4-10 ° C.

E. After completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 ° C. and the cells are harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). Based on the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste (by weight) was added to 100 mM Tris, 50 mM EDTA, pH 7.
4. Suspend in buffer solution containing 4. The cells are dispersed in a homogenous suspension using a high shear mixer.

The cells were then placed in a microfluidizer (
Microfluidics, Corp. Or APV Gaulin, Inc
． ) Is dissolved twice by passing the solution at 4000-6000 psi.
The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 minutes. The obtained pellet was treated with 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.
Wash again using 4.

The resulting washed inclusion bodies were treated with 1.5 M guanidine hydrochloride (GuHCl) to
Solubilize for 4 hours. After centrifugation at 7000 xg for 15 minutes, the pellet is discarded, and the polypeptide-containing supernatant is incubated at 4 ° C overnight to allow additional Gu.
Allows HCl extraction.

Following high speed centrifugation (30,000 × g) to remove insoluble particles, Gu
HCl solubilized protein was extracted with GuHCl extract, 50 mM sodium, pH
Refold by rapid mixing with 20 volumes of buffer containing 4.5, 150 mM NaCl, 2 mM EDTA with vigorous stirring. The refolded diluted protein solution is kept at 4 ° C. without mixing for 12 hours before further purification steps.

To clarify the refolded polypeptide solution, a pre-prepared tangential filtration unit equipped with a 0.16 μm membrane filter with appropriate surface area, equilibrated with 40 mM sodium acetate, pH 6.0 (eg, Filtro
n) is used. The filtered sample is treated with a cation exchange resin (eg, Poros).
HS-50, Perseptive Biosystems). The column was washed with 40 mM sodium acetate, pH 6.0, and 250 mM, 500 mM, 1000 mM, and 1500 mM NaC in the same buffer.
With l, elute in a stepwise fashion. The absorbance of the eluate at 280 nm is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample was then added to a pre-prepared strong anion (Poros HQ
-50, Perseptive Biosystems exchange resin and weak anion (Poros CM-20, Perseptive Biosystems).
) Load into a set of in-line columns of exchange resin. The column is equilibrated with 40 mM sodium acetate, pH 6.0. Both columns were loaded with 40 mM sodium acetate,
Wash with 200 mM NaCl, pH 6.0. Next, the CM-20 column is replaced with 10
Linear gradient of column volume (0.2 M NaCl, 50 mM sodium acetate, pH
Elution with 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5). Fractions are collected under constant A ₂₈₀ monitoring of the eluate. Fractions containing the polypeptide (eg, found by 16% SDS-PAGE) are then pooled.

The resulting polypeptide should exhibit greater than 95% purity after the refolding and purification steps described above. When 5 μg of purified protein is loaded, no major contaminating band should be observed from Coomassie blue stained 16% SDS-PAGE gels. The purified protein is also endotoxin / LPS
It can be tested for contamination and typically the LPS content is less than 0.1 ng / ml according to the LAL assay.

Example 7: Cloning and Expression of Polypeptides in Baculovirus Expression System In this example, plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector is used for Autographa californica nuclear polyhedrosis virus (
AcMNPV) strong polyhedrin promoter followed by BamHI, Xba
I, and convenient restriction sites such as Asp718. Simian virus 40
The polyadenylation site of ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, this plasmid was engineered into E. coli under the control of the weak Drosophila promoter in the same orientation. It contains the β-galactosidase gene from E. coli, followed by the polyadenylation signal of the polyhedrin gene. The inserted gene is flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA, which produces a viable virus expressing the cloned polynucleotide.

Many other baculovirus vectors (eg pAc373, pVL941)
, And pAcIM1) are signals that the construct is properly positioned for transcription, translation, secretion, etc. (including signal peptide and in-frame AUG, if required), as will be readily appreciated by those of skill in the art. Can be used in place of the above vector as long as Such a vector is, for example, Lucco.
W et al., Virology 170: 31-39 (1989).

Specifically, using the PCR protocol described in Example 1, the cDNA sequence contained in the deposited clone containing the AUG start codon and naturally linked to the leader sequence identified in Table 1 was used. And amplify. The pA2 vector does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Alternatively, Summers et al., “A Manual o
f Methods for Baculovirus Vectors an
d Insect Cell Culture Procedures ", Te
xas Agricultural Experimental Status
n Bulletin NO. : 1555 (1987), this vector may be modified to include a baculovirus leader sequence (pA2GP).

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO
101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified on a 1% agarose gel.

This plasmid can be digested with the corresponding restriction enzymes and, if desired, dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. This DNA is then added to a commercially available kit (“Geneclean” BIO 10
1 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel.

This fragment and the dephosphorylated plasmid are ligated together using T4 DNA ligase. E. coli HB101 cells or XL-1 Blu
e (Stratagene Cloning Systems, La Joll)
a, CA) other suitable E. The E. coli host is transformed with the ligation mixture and spread on culture plates. Bacteria containing this plasmid are identified by digesting the DNA from individual colonies and analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of plasmid containing this polynucleotide was isolated from Felgner et al., Pr.
oc. Natl. Acad. Sci. USA 84: 7413-7417 (19).
87), using 1.0 μg of commercially available linearized baculovirus DNA ("BaculoGold ^™ baculovi").
rus DNA ", Pharmingen, San Diego, CA). 1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid were treated with 50 μl of serum-free Grace's medium (Life Tech).
Nologies Inc. , Gaithersburg, MD) in a sterile well of a microtiter plate. Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then seeded on a 35 mm tissue culture plate containing 1 ml of Grace's medium without serum (Sf9 insect cells (A
TCC CRL 1711). The plate is then incubated at 27 ° C for 5 hours. The transfection solution is then removed from the plate and 1 ml Grace's insect medium supplemented with 10% fetal bovine serum is added. The culture is then continued for 4 days at 27 ° C.

Supernatants are collected after 4 days and plaque assay performed as described by Summers and Smith (supra). "Blue Gal" (Life
Technologies Inc. , Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones (resulting in blue-stained plaques). (A detailed description of this type of "plaque assay" can also be found in Life Technologies Inc.
, Gaithersburg, pages 9-10, which can be found in the user guide for insect cell culture and baculovirology). After appropriate incubation, blue-stained plaques are picked up with a micropipette tip (eg Eppendorf). The agar containing the recombinant virus was then resuspended in a microcentrifuge tube containing 200 μl Grace's medium and the suspension containing the recombinant baculovirus was used to seed Sf9 cells seeded in a 35 mm dish. Infect. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C.

[0693]   In order to confirm the expression of the polypeptide, Glein supplemented with 10% heat-inactivated FBS was used.
Sf9 cells are grown in sucrose medium. The cells were infected with an infection efficiency of about 2 (“MO
I ") with a recombinant baculovirus containing this polynucleotide.
If radiolabeled protein is desired, remove the medium after 6 hours and
Methionine- and cysteine-free SF900 II medium (Life T
technologies Inc. Available from Rockville, MD
Noh). 42 hours later 5 μCi³⁵Of S-methionine and 5 μCi ³⁵ Add S-cysteine (available from Amersham). Cells further
Incubate for 16 hours, then harvest by centrifugation. Tampa in the supernatant
Cytoplasmic as well as intracellular proteins by SDS-PAGE followed by
Analyze by geography (if radiolabeled).

[0694] Microsequencing of the amino terminal amino acid sequence of purified proteins can be used to determine the amino terminal sequence of the produced protein.

Example 8 Expression of Polypeptides in Mammalian Cells The polypeptides of the present invention can be expressed in mammalian cells. A typical mammalian expression vector contains promoter elements that mediate the initiation of transcription of mRNA, protein coding sequences, and signals necessary for termination of transcription and polyadenylation of the transcript. Further elements are enhancers, Kozak (Koz
ak) sequences and intervening sequences flanking the donor and acceptor sites for RNA splicing. Very efficient transcription is achieved by the SV40-derived early and late promoters, retroviruses (eg RSV, HTLVI, H
IVI) -derived long terminal repeat (LTR), and cytomegalovirus (CMV)
Is achieved using the early promoter of. However, intracellular elements (eg,
The human actin promoter) can also be used.

Expression vectors suitable for use in practicing the present invention are, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVc.
at (ATCC 37152), pSV2dhfr (ATCC 37146),
Includes vectors such as pBC12MI (ATCC 67109), pCMVSport2.0, as well as pCMVSport3.0. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurka.
t cells, mouse NIH3T3 cells and C127 cells, Cos 1 cells, Co
s 7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide comprises a polynucleotide integrated into a chromosome,
It can be expressed in stable cell lines. Co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is
Useful for developing cell lines that carry hundreds or even thousands of copies of the gene of interest (eg, Alt, FW et al., J. Biol. Chem. 253: 1357).
-1370 (1978); Hamlin, J .; L. And Ma, C.I. , Bioc
hem. et Biophys. Acta, 1097: 107-143 (199
0); Page, M .; J. And Sydenham, M .; A. Et al, Biotec
hnology, 9: 64-68 (1991)). Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Bi.
ochem J. 227: 277-279 (1991); Bebbington.
Et al., Bio / Technology 10: 169-175 (1992)). These markers are used to grow mammalian cells in selective medium and select the cells with the highest resistance. These cell lines contain the amplified gene integrated into the chromosome. Chinese Hamster Ovary (CHO) and NS
O cells are often used for protein production.

Expression Vectors pC4 (ATCC Accession No. 209646) and pC6 (AT
CC Accession No. 209647) is a Rous sarcoma virus (Cullen et al., Mol.
electrical and Cellular Biology, 438-447 (
March 1985)), a strong promoter (LTR), and a fragment of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites with BamHI, XbaI, and Asp718 restriction enzyme cleavage sites facilitate cloning of the gene of interest. This vector also contains a 3'intron, polyadenylation and termination signals of the rat preproinsulin gene, and S
Contains the mouse DHFR gene under the control of the V40 early promoter.

Specifically, for example, the plasmid pC6 is digested with an appropriate restriction enzyme,
Calf intestinal phosphatase (phosph) is then prepared by procedures known in the art.
ate) to dephosphorylate. The vector is then isolated from a 1% agarose gel.

Polynucleotides of the invention are amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used for the production of secreted protein, the vector need not include a second signal peptide. Alternatively,
If the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO96 / 34891).

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO 10).
1 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. Then E. E. coli HB101 cells or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC6 are identified using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of expression plasmids pC6 and pc4 are cotransfected with 0.5 μg of plasmid pSVneo using lipofectin (Felgner et al., Supra). The plasmid pSV2-neo contains the neo gene from Tn5 which encodes an enzyme that confers resistance to a group of antibiotics including G418, which is a dominant and selectable marker. This cell
Seed in α-minus MEM supplemented with 1 mg / ml G418. Two days later, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with 10, 25, or 50 ng / ml methotrexate and 1 mg / ml G418. After about 10-14 days, single clones were trypsinized and then at different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM.
nM) is used to seed 6-well Petri dishes or 10 ml flasks.
Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product can be achieved, for example, by SDS.
-Analyze by PAGE and Western blot or by reverse phase HPLC analysis.

Example 9 Protein Fusions The polypeptides of the invention are preferably fused to other proteins. These fusion proteins can be used in various applications. For example, fusion of a polypeptide of the invention to a His tag, HA tag, protein A, IgG domain, and maltose binding protein facilitates purification (see Example 5; EP.
See also A 394,827; Traunecker et al., Nature.
e, 331: 84-86 (1988)). Similarly, fusions to IgG-1, IgG-3, and albumin increase half-life in vivo. Nuclear localization signals fused to the polypeptides of the invention can target proteins to specific subcellular localizations. On the other hand, covalent heterodimers or homodimers may increase or decrease the activity of the fusion protein. Fusion proteins can also create chimeric molecules that have more than one function. Finally, fusion proteins may increase the solubility and / or stability of fusion proteins as compared to non-fusion proteins. All forms of the above fusion proteins can be made by modifying the protocol below, which outlines the fusion of polypeptides to IgG molecules, or the protocol described in Example 5.

Briefly, the human Fc portion of an IgG molecule can be PCR amplified using primers spanning the 5'and 3'ends of the sequences described below. These primers should also have convenient restriction enzyme sites which facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, if pC4 (accession number 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3'BamHI site should be destroyed. Then, the vector containing the human Fc portion is transformed into B
The polynucleotide of the invention, which was again restricted with amHI, linearized the vector and isolated by the PCR protocol described in Example 1,
It is linked to the amHI site. Note that the polynucleotide is cloned without a stop codon, otherwise no fusion protein will be produced.

PC4 does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891).

[0709]

[Chemical 8] (Example 10: Production of antibody from polypeptide) The antibody of the present invention can be prepared by various methods. (Current Pr
otocols, Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of secreted protein is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

In the most preferred method, the antibodies of the invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies are prepared using hybridoma technology (Kohler et al., Nature 25.
6: 495 (1975); Kohler et al., Eur. J. Immunol. 6:
511 (1976); Kohler et al., Eur. J. Immunol. 6:29
2 (1976); Hammerling et al .: Monoclonal Antib.
ands and T-Cell Hybridomas, Elsevier
, N .; Y. , Pp. 563-681 (1981)). Generally, such a procedure involves immunizing an animal, preferably a mouse, with the polypeptide, or more preferably with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, they are preferably supplemented with 10% fetal bovine serum (inactivated at about 56 ° C.), and about 10 g / l of non-essential amino acids, About 1,
It is cultured in Earle's modified Eagle medium supplemented with 000 U / ml penicillin and about 100 μg / ml streptomycin.

The splenocytes of such mice are extracted and fused with the appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, AT
It is preferred to use the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
Cloning by limiting dilution as described by 1981)). The hybridoma cells obtained through such selection are then assayed to identify clones secreting antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to this polypeptide may be generated in a two step procedure using anti-idiotypic antibodies. Such a method takes advantage of the fact that the antibody itself is the antigen and therefore it is possible to obtain an antibody that binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify antibody-producing clones whose ability of the antibody to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies include anti-idiotypic antibodies to protein-specific antibodies and are used to immunize animals to induce the formation of additional protein-specific antibodies.

It is clear that Fab and F (ab ′) 2 and other fragments of the antibodies of the invention can be used according to the methods described herein. Such fragments are typically generated by proteolytic cleavage using an enzyme such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments). To be done. Alternatively, secreted protein-binding fragments may be produced by the application of recombinant DNA technology or synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced by using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (For a review, see Morrison, Science 229: 1202.
(1985); Oi et al., BioTechniques 4: 214 (1986).
Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neube.
rger et al., WO 8601533; Robinson et al., WO 870267.
1; Boulianne et al., Nature 312: 643 (1984); Ne.
Uberger et al., Nature 314: 268 (1985)).
.

Example 11: Production of Secreted Proteins for High Throughput Screening Assays The following protocol produces a supernatant containing the polypeptide to be tested. This supernatant can then be used in the screening assay described in Examples 13-20.

First, poly-D-lysine (644 587 Boehringer-Man).
nheim) stock solution (1 mg / ml in PBS) in PBS (17-516F Biowhittaker without calcium or magnesium).
Dilute to 20 to make working solution 50 μg / ml. 200 μl of this solution
Add to each well (24-well plate) and incubate for 20 minutes at room temperature. Make sure to distribute the solution over each well (Note: a 12 channel pipettor can be used with tips on every other channel).
Aspirate the poly-D-lysine solution and rinse with 1 ml PBS (phosphate buffered saline). PBS should remain in the wells just prior to plating the cells, and the plates can be pre-coated with polylysine by 2 weeks.

293T cells (passing P + 20 and carrying no cells) were treated with 2 × 10 ⁵ cells / well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium) (4.
5 G / L glucose and L-glutamine (12-604F Biowhit
10% heat-inactivated FBS (14-503F Biowhi)
ttaker) / 1 × Penstrep (17-602E Biohitter)
plate). Allow cells to grow overnight.

Next day, in a sterile solution basin, 300 μl lipofectamine (1
8324-012 Gibco / BRL) and 5 ml Optimem I (
31985070 Gibco / BRL) / 96 well plate are mixed together. Using a small volume of multi-channel pipettor, approximately 2 μg of the expression vector containing the polynucleotide insert produced by the method described in Example 8 or 9 was placed in an appropriately labeled 96-well round bottom plate. Aliquot.
Using a multichannel pipettor, 50 μl of Lipofectamine / Opt
Add the imem I mixture to each well. Mix gently by pipetting up and down gently. Incubate for 15-45 minutes at room temperature. After about 20 minutes,
Add 150 μl Optimem I to each well using a multi-channel pipettor. As a control, one plate of vector DNA lacking the insert should be transfected with each set of transfections.

[0719] Preferably, the transfection is carried out in a tag team (
tag-teaming). By forming a tag team,
The time effort is halved, and the cells do not spend too much time on PBS. First, Person A aspirates the medium from the four 24-well plates of cells, and then Person B rinses each well with 0.5-1 ml PBS. Mr. A then aspirates the PBS rinse and Mr. B uses 200 μl of DNA using a 12-channel pipettor with tips on every other channel.
/ Lipofectamine / Optimem I complex is added to each row on a 24-well plate, first to odd wells, then to even wells. Incubate at 37 ° C for 6 hours.

[0720]   While incubating the cells, add 1 × penstrep
1% BSA in DMEM with or 2 mM glutamine and 1x pens
CHO-5 medium containing rep (116.6 mg / L CaCl 2₂(Anhydrous); 0
． 00130mg / L CuSO_Four-5H₂O; 0.050 mg / L Fe (NO ₃ )₃-9H₂O; 0.417 mg / L FeSO_Four-7H₂O; 311.80 mg
/ L KCl; 28.64 mg / L MgCl₂48.84 mg / L MgS
O_Four6995.50 mg / L NaCl; 2400.0 mg / L NaHCO₃ 62.50 mg / L NaH₂PO_Four-H₂O; 71.02 mg / L Na₂HP
O_Four0.4320 mg / L ZnSO_Four-7H₂O; 0.002 mg / L ara
Chidonic acid; 1.022 mg / L cholesterol; 0.070 mg / L DL-
α-tocopherol acetate; 0.0520 mg / L linoleic acid; 0.010 mg
/ L linolenic acid; 0.010 mg / L myristic acid; 0.010 mg / L
Oleic acid; 0.010 mg / L of palmitooleic acid (palmitric
acid); 0.010 mg / L palmitic acid; 100 mg / L Pluro
nic F-68; 0.010 mg / L stearic acid; 2.20 mg / L T
ween 80; 4551 mg / L D-glucose; 130.85 mg / ml
L-alanine; 147.50 mg / ml L-arginine-HCl; 7.50 m
g / ml L-asparagine-H₂O; 6.65 mg / ml L-asparagine
Acid; 29.56 mg / ml L-cystine 2HCl-H₂O; 31.29 mg /
ml L-cystine-2HCl; 7.35 mg / ml L-glutamic acid; 36
5.0 mg / ml L-glutamine; 18.75 mg / ml glycine;
48 mg / ml L-histidine-HCl-H₂O; 106.97 mg / ml
L-isoleucine; 111.45 mg / ml L-leucine; 163.75 mg
/ Ml L-lysine HCl; 32.34 mg / ml L-methionine; 68.4
8 mg / ml L-phenylalanine; 40.0 mg / ml L-proline; 2
6.25 mg / ml L-serine; 101.05 mg / ml L-threonine;
19.22 mg / ml L-tryptophan; 91.79 mg / ml L-tyro.
Shinrosine-2Na-2H₂O; 99.65 mg / ml L-
Valine; 0.0035 mg / L biotin; 3.24 mg / L D-Ca panto
Tenic acid; 11.78 mg / L choline chloride; 4.65 mg / L folic acid; 15.6
0 mg / L i-inositol; 3.02 mg / L niacinamide; 3.0
0 mg / L pyridoxal HCl; 0.031 mg / L pyridoxine HCl
0.319 mg / L riboflavin; 3.17 mg / L thiamine HCl; 0
． 365 mg / L thymidine; and 0.680 mg / L vitamin B₁₂25
mM HEPES buffer; 2.39 mg / L Na hypoxanthine; 0.105
mg / L lipoic acid; 0.081 mg / L putrescine sodium-2HCl; 5
5.0 mg / L sodium pyruvate; 0.0067 mg / L sodium selenite
Thorium; 20 μM ethanolamine; 0.122 mg / L ferric citrate
Methyl-B-cyclodext complexed with 41.70 mg / L linoleic acid
Phosphorus; Methyl-B-cyclodoxy complexed with 33.33 mg / L oleic acid
Methyl-B-sik complexed with string; and 10 mg / L retinal
Any suitable medium (rodextrin) is prepared. (10% BSA stock
For solution, BSA (81-068-3 Bayer) 1 in 1 L DMEM
00g dissolved). The medium is filtered and 50 μl for endotoxin assay
Collect in 15 ml polystyrene conical.

The transfection reaction is terminated at the end of the incubation time, preferably in tag teams. Person A aspirates off the transfection medium, while person B adds 1.5 ml of the appropriate medium to each well. Incubate at 37 ° C for 45 or 72 hours depending on the medium used:
45% for 1% BSA or 72 hours for CHO-5.

On day 4, 600 μl using a 300 μl multichannel pipettor
Aliquot into 1 ml deep well plate and 2 ml of remaining supernatant
Aliquot into deep wells of. The supernatant from each well was then added to Examples 13-2.
0 may be used in the assay described in 0.

If activity is obtained in any of the assays described below using the supernatant, the activity may be obtained directly from the polypeptide (eg as a secreted protein) or
It is specifically understood that either derived from, or by a polypeptide that induces the expression of other proteins, which are then secreted into the supernatant.
Thus, the invention further provides methods for identifying proteins in supernatants characterized by activity in a particular assay.

Example 12: Construction of GAS Reporter Constructs One signaling pathway involved in cell differentiation and proliferation is Jaks-ST.
Called the AT route. Proteins activated in the Jaks-STAT pathway bind to the gamma activation site "GAS" element or interferon-sensitive response element ("ISRE") located in the promoters of many genes.
Binding of proteins to these elements alters the expression of related genes.

GAS and ISRE elements are recognized by signal transducers and activators of transcription, a class of transcription factors called “STATs”. There are 6 members in the STAT family. S
tat1 and Stat3, as well as Stat2 (as the response to IFNα is widespread) are present in many cell types. Stat4 is more restricted and is not present in many cell types, but is found in T helper class I cells after treatment with IL-12. Stat5, originally called breast growth factor, is found in higher concentrations in other cells, including bone marrow cells. It can be activated in tissue culture cells by many cytokines.

STATs are activated and translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a unique family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3.
These kinases show significant sequence similarity and are generally catalytically inactive in resting cells.

Jaks are activated by a wide range of receptors as summarized by the table below. (Schidler and Darnell, Ann. Rev. Bi
ochem. 64: 621-51 (1995). Jaks
The cytokine receptor family that is capable of activating sac can be divided into two groups:
(A) Class 1 includes IL-2, IL-3, IL-4, IL-6, IL-7, IL
-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CS
F, GM-CSF, LIF, CNTF, and receptors for thrombopoietin; and (b) class 2 includes IFN-a, IFN-g, and IL-.
Including 10. Class 1 receptors contain a conserved cysteine motif (a set of 4 conserved cysteines and 1 tryptophan), and WSXWS.
Shares the motif (membrane adjacent region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO: 2)).

Thus, upon binding of the ligand to the receptor, Jaks is activated, which in turn activates STAT, which then translocates and binds to the GAS element. This entire process is involved in the Jaks-STAT signaling pathway.

Therefore, the Jak reflected by the binding of GAS or ISRE elements
Activation of the s-STAT pathway can be used to indicate proteins involved in cell growth and differentiation. For example, growth factors and cytokines are described in Jaks-S
It is known to activate the TAT pathway. (See table below). Therefore, by using a GAS element linked to a reporter molecule, Jak
Activators of the s-STAT pathway can be identified.

[0730]

[Table 4] To construct synthetic GAS containing promoter elements used in the biological assays described in Examples 13-14, a PCR-based strategy is used to generate the GAS-SV40 promoter sequence. The 5'primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind to STAT upon induction with a range of cytokines (Rothman et al. , Immunity 1: 457-
468 (1994)), other GAS or ISRE elements may be used instead. The 5'primer also contains an 18 bp sequence complementary to the SV40 early promoter sequence and flanks the XhoI site. The sequence of the 5'primer is:

[0731]

[Chemical 9] The downstream primer is complementary to the SV40 promoter and is Hin
Adjacent to dIII site: 5 ': GCGGCAAGCTTTTTGCAAAG
CCTAGGC: 3 '(SEQ ID NO: 4).

PCR amplifications are performed with the SV40 promoter template present in the B-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI / HindIII and subcloned into BLSK2- (Stratagene). Sequencing with forward and reverse primers confirms that the insert contains the following sequences:

[0733]

[Chemical 10] When this GAS promoter element is bound to the SV40 promoter,
The GAS: SEAP2 reporter construct is then engineered. Here, the reporter molecule is secretory alkaline phosphatase or "SEAP". However, clearly any reporter molecule can substitute for SEAP in either this or other examples. Well-known reporter molecules that can be used in place of SEAP include chloramphenicol acetyl transferase (C
AT), luciferase, alkaline phosphatase, β-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

The synthetic GAS-SV40 promoter element identified by the above sequence was added to HindIII and XhoI to generate a GAS-SEAP vector.
Is used to effectively replace the SV40 promoter with the amplified GAS: SV40 promoter element and subcloned into the pSEAP-promoter vector obtained from Clontech. However, this vector does not contain the neomycin resistance gene and is therefore not preferred for mammalian expression systems.

Therefore, in order to generate a stable mammalian cell line expressing the GAS-SEAP reporter, the GAS-SEAP cassette was removed from the GAS-SEAP vector with SalI and NotI and GAS-SEAP / Neo. To create a vector, these restriction sites in the multiple cloning site were used to create a backbone vector containing the neomycin resistance gene (eg pGFP).
-1 (Clontech)). Once the vector is transfected into mammalian cells, the vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.

Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, the construction of a reporter molecule containing NFK-B and EGR promoter sequences is described in Examples 15 and 16. However, many other promoters can be replaced using the protocols described in these examples. For example, the SRE, IL-2, NFAT, or osteocalcin promoters alone or in combination (eg, GAS / NF-KB / EGR, G
AS / NF-KB, Il-2 / NFAT, or NF-KB / GAS). Similarly, other cell lines (eg, HELA (epithelial cells), HUVEC (endothelial cells), Reh (B cells), Saos-2 (osteoblasts), HUVAC (aortic cells), or cardiomyocytes) can be used as reporters. It can be used to test the activity of the construct.

Example 13: High Throughput Screening Assay for Activity of T Cells The following protocol was used to identify the factors and the supernatant containing the polypeptides of the present invention expanded T cells. And / or assess T cell activity by determining whether to differentiate. GA of T cell activity produced in Example 12
Assess with the S / SEAP / Neo construct. Thus, factors that increase SEAP activity show the ability to activate the Jaks-STATS signaling pathway.
The T cells used in this assay were Jurkat T cells (ATCC accession number T
IB-152), but Molt-3 cells (ATCC accession number CRL-155).
2) and Molt-4 cells (ATCC accession number CRL-1582) cells may also be used.

Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. Approximately 2 million Jurkat cells were DMRed to generate stable cell lines.
GAS-SEAP using IE-C (Life Technologies)
/ Neo vector (transfection procedure described below). Transfected cells are seeded at a density of approximately 20,000 cells / well, and transfectants that are resistant to 1 mg / ml of geneticin are selected. Growing resistant colonies, then
Test for their response to increasing concentrations of interferon-γ. The dose response of selected clones is shown.

In detail, the following protocol yields sufficient cells for 75 wells containing 200 μl of cells. Therefore, in order to produce sufficient cells for multiple 96-well plates, this is either scaled up or performed in multiples. Jurkat cells in RPMI + 10% serum and 1% Pen
-Keep in Strep. 2.5 ml OPTI-MEM in a T25 flask
(Life Technologies) and 10 μg of plasmid DNA. Add 2.5 ml OPTI-MEM containing 50 μl DMRIE-C and incubate at room temperature for 15-45 minutes.

During the incubation time, the cell concentration was counted, the required number of cells (10 ⁷ per transfection) was spun down, and the final concentration was 10 ⁷ cells / m 2.
Resuspend to 1 in OPTI-MEM. Then 1 ml of 1 × 10 ⁷ cells in OPTI-MEM is added to the T25 flask and incubated at 37 ° C. for 6 hours. After incubation, 10 ml RPMI + 15% serum is added.

Jurkat: GAS-SEAP stable reporter strain was added to RPMI + 10% serum,
Maintain in 1 mg / ml Geneticin, and 1% Pen-Strep.
These cells are treated with a supernatant containing a polypeptide of the invention and / or are induced with a polypeptide of the invention to be produced by the protocol described in Example 11.

The cells should be washed on the day of treatment with the supernatant and 500,00 ml / ml
It should be resuspended in fresh RPMI + 10% serum to a density of 0 cells. The exact number of cells required depends on the number of supernatants screened.
Approximately 10 million cells are needed per 96-well plate (100 million cells per 10 plates).

Transfer cells to triangular reservoir boats to dispense cells into 96-well dishes using a 12-channel pipette. Transfer 200 μl of cells to each well using a 12-channel pipette (thus 1 per well)
Add 0,000 cells).

After seeding all plates, 50 μl of supernatant is transferred directly from the 96-well plate containing the supernatant to each well using a 12-channel pipette. further,
Exogenous interferon gamma doses (0.1, 1.0, 10 ng) were added to well H
Add to 9, well H10, and well H11 to serve as an additional positive control for the assay.

96-well dishes containing Jurkat cells treated with supernatant are placed in an incubator for 48 hours (note: this time can be varied between 48 and 72 hours).
. Then 35 μl of sample from each well is transferred to an opaque 96-well plate using a 12-channel pipette. The opaque plate should be covered (using a cover of cellophane) and stored at -20 ° C until the SEAP assay described in Example 17 is performed. The plate containing the remaining treated cells is placed at 4 ° C. and serves as a source of material for repeating the assay on specific wells, if desired.

As a positive control, 100 units / ml of interferon-γ can be used, which is known to activate Jurkat T cells. Typically,> 30-fold induction is observed in the positive control wells.

The protocol described above can be used to generate both transient and stable transfected cells, which will be apparent to those of skill in the art.

Example 14: High Throughput Screening Assay to Identify Myeloid Activity The following protocol is used to determine whether a polypeptide of the invention proliferates and / or differentiates myeloid cells. To assess myeloid activity. The activity of myeloid cells is assessed using the GAS / SEAP / Neo construct produced in Example 12. Therefore, a factor that increases SEAP activity is Jaks-S.
Shows the ability to activate the TATS signaling pathway. The myeloid cells used in this assay are U937 (pre-monocyte cell line), but TF-1, HL60, or KG1 may also be used.

To transiently transfect U937 cells with the GAS / SEAP / Neo construct produced in Example 12, the DEAE-Dextran method (
Kharbanda et al., 1994, Cell Growth & Differ.
Orientation, 5: 259-265). First, 2 × 10e ⁷ U937 cells are harvested and washed with PBS. U937 cells are usually
Grow in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 00 units / ml penicillin and 100 mg / ml streptomycin.

Next, 0.5 mg / ml DEAE-Dextran, 8 μg of GAS-SE.
AP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM
_{Na 2 HPO 4 · 7H 2 O} , 1mM MgCl 2, and 675μM CaCl ₂
The cells are suspended in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing Incubate at 37 ° C for 45 minutes.

Cells were washed with RPMI 1640 medium containing 10% FBS, then 10
Resuspend in ml complete medium and incubate at 37 ° C for 36 hours.

GAS-SEAP / U937 stable cells are obtained by growing the cells in 400 μg / ml G418. G418-free medium is used for routine growth, but every 1-2 months the cells are 400 μg / ml G418 for two passages.
Should be regrown in.

These cells are tested by harvesting 1 × 10 ⁸ cells, which is sufficient for 10 96-well plate assays, and washing with PBS. The cells are suspended in the above 200 ml of growth medium at a final density of 5 × 10 ⁵ cells / ml. Plate 200 μl cells per well (ie 1 × 10 ⁵ cells / well) in a 96-well plate.

Add 50 μl of the supernatant prepared according to the protocol described in Example 11. Incubate at 37 ° C for 48-72 hours. 1 as a positive control
00 units / ml of interferon gamma can be used, which is known to activate U937 cells. Typically,> 30-fold induction is observed in positive control wells. Supernatant supernatants according to the protocol described in Example 17
EAP assay.

Example 15: High Throughput Screening Assay to Identify Neuronal Activity When a cell undergoes differentiation and proliferation, a panel of genes is activated via many different signaling pathways. One of these genes, EGR1 (Early Growth Response Gene 1), is induced in various tissues and cell types upon activation. EG
The R1 promoter is responsible for such induction. EGR bound to a reporter molecule
One promoter can be used to assess cell activation.

In detail, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (rat pheochromocytoma
ocytoma) cells proliferate and / or differentiate by activation with many mitogens such as TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor), and EGF (epithelial growth factor). Is known. EGR1 gene expression is activated during this treatment. Therefore, PC12 cell activation can be assessed by stably transfecting PC12 cells with a construct containing an EGR promoter linked to a SEAP reporter.

The EGR / SEAP reporter construct can be assembled by the following protocol.
EGR-1 promoter sequence (-633 to +1) (Sakamoto K et al., O.
ncogene 6: 867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers: 5'GCCGCTCGAGGGATGACAGCGATAGAACCCCGG-.
3 '(SEQ ID NO: 6) 5'GCGAAGCTTCCGCGACTCCCCGGATCCGCCCTC-3
'(SEQ ID NO: 7).

The GAS: SEAP / Neo vector generated in Example 12 can then be used to insert the EGR1 amplification product into this vector. Restriction enzyme XhoI / H
Linearize the GAS: SEAP / Neo vector using indIII
Remove the S / SV40 stuffer. EGR1 amplification products are restricted with these same enzymes. Connect the vector and the EGR1 promoter.

To prepare 96-well plates for cell culture, coating solution (
Collagen type I (Upstate Biotech Inc. Catalog No. 08
-115) diluted with 30% ethanol (sterile filtration) 1:30) into one 10c.
Add 2 ml per m-plate, or 50 ml per well in a 96-well plate and allow to air dry for 2 hours.

PC12 cells were plated on a 10 cm tissue culture dish pre-coated with 10% horse serum (JRH BIOSCIENCES, catalog # 124) supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml).
49-78P), RPMI-164 containing 5% heat-inactivated fetal bovine serum (FBS)
Routinely grow in 0 medium (Bio Whittaker). Divide from 1 to 4 every 3-4 days. The cells are removed from the plate by scraping and resuspended by pipetting up and down more than 15 times.

The EGR / SEAP / Neo construct was prepared using the Lipofecta described in Example 11.
Transfect PC12 using the mine protocol. EGR-S
EAP / PC12 stable cells are obtained by growing the cells in 300 μg / ml G418. G418-free medium is used for routine growth, but every 1-2 months cells should be regrown in 300 μg / ml G418 for 2 passages.

To assay neuronal activity, 10 cm plates with cells at approximately 70-80% confluence are screened by removing old media. The cells are washed once with PBS (phosphate buffered saline). The cells were then treated with low serum medium (1% horse serum and 0.5% FBS with antibiotics).
Starve overnight in RPMI-1640).

The next morning, remove the medium and wash the cells with PBS. The cells are scraped from the plate and the cells are suspended in 2 ml of low serum medium. Cell numbers are counted and lower serum medium is added to reach a final cell density of 5 × 10 ⁵ cells / ml.

200 μl of cell suspension is added to each well of a 96-well plate (1 × 1
0 ⁵ cells / well). 50 μl of the supernatant produced according to Example 11
Add at 37 ° C for 48-72 hours. As a positive control, P via EGR
Growth factors known to activate C12 cells can be used, such as 50 ng / μl nerve growth factor (NGF). Typically,> 50-fold SEAP induction is found in positive control wells. The supernatant is treated with SE according to Example 17.
AP assay.

Example 16: High Throughput Screening Assay for T Cell Activity NF-KB (Nuclear Factor KB) is used in a wide variety of agents (inflammatory cytokines IL-1 and TNF, CD30 and CD40). , Lymphotoxin-α and lymphotoxin-β), by exposure to LPS or thrombin, and by expression of specific viral gene products. As a transcription factor, NF-KB expresses genes involved in activation of immune cells, controls apoptosis (NF-KB seems to protect cells from apoptosis),
It regulates B and T cell development, antiviral and antibacterial responses, and multiple stress responses.

In unstimulated conditions, NF-KB is retained in the cytoplasm with I-KB (inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1.
MHC is mentioned.

Due to its central role and ability to respond to a range of stimuli, NF-
A reporter construct utilizing the KB promoter element is used to screen the supernatant produced in Example 11. Activators or inhibitors of NF-KB are useful in treating disease. For example, inhibitors of NF-KB can be used to treat diseases associated with acute or chronic NF-KB activation, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, PCR
Use a strategy based on. The upstream primer has an NF-KB binding site (
GGGGACTTTCCC) (SEQ ID NO: 8), containing an 18 bp sequence complementary to the 5'end of the SV40 early promoter sequence and flanked by XhoI sites:
GGGGACTTTCCGGGAACTTTCCATCCTGGCCATCTCAA
TTAG: 3 '(SEQ ID NO: 9).

The downstream primer is complementary to the 3'end of the SV40 promoter,
And adjacent to the HindIII site: 5 ': GCGGCAAGCTTTTTGCAAAAGCCATAGGC: 3' (SEQ ID NO: 4).

PCR amplification is performed using the SV40 promoter template present in the pB-gal: promoter plasmid obtained from Clontech.
The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2- (Stratagene). T7 and T
Confirm that the insert contains the following sequences by sequencing with 3 primers:

[0771]

[Chemical 11] XhoI and HindIII are then used to replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB / SV40 fragment. However, this vector does not contain the neomycin resistance gene and is therefore not preferred for mammalian expression systems.

To generate a stable mammalian cell line, the NF-KB / SV40 / SEAP cassette was constructed using the restriction enzymes SalI and NotI as described above for NF-KB / SEA.
Remove from P vector and insert into vector containing neomycin resistance.
Specifically, after restriction treatment of pGFP-1 with SalI and NotI, NF-
The KB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech) to replace the GFP gene.

Once the NF-KB / SV40 / SEAP / Neo vector was prepared, stable Jurkat T cells were prepared according to the protocol described in Example 13.
And maintain. Similarly, a method for assaying supernatants containing these stable Jurkat T cells is also described in Example 13. As a positive control, exogenous TNFα (0.1, 1, 10 ng) was added to wells H9, H10, and H11, and typically 5-10 fold activation is observed.

Example 17: Assay for SEAP activity SE as a reporter molecule for the assays described in Examples 13-16.
AP activity was measured by Tropix Phospho-li according to the following general procedure.
Assay using the ght Kit (catalog number BP-400). Troop
The ix Phospho-light Kit is a dilution buffer used below,
Supply assay buffer and reaction buffer.

Fill the dispenser with 2.5 × Dilution Buffer and dispense 15 μl of 2.5 × Dilution Buffer into an Optiplate containing 35 μl of supernatant. Seal the plate with a plastic sealer and incubate at 65 ° C for 30 minutes. Separate Optiplates to avoid uneven heating.

Cool the sample to room temperature for 15 minutes. Empty the dispenser and fill with assay buffer. Add 50 ml assay buffer and incubate at room temperature for 5 minutes. Empty the dispenser and fill with reaction buffer (see table below). Add 50 μl reaction buffer and incubate for 20 minutes at room temperature. The intensity of the chemiluminescent signal is time-dependent, and it takes about 10 minutes to read 5 plates on the luminometer, so 5 plates are processed at a time and the second set is started 10 minutes later. Should do.

Read the relative light unit in the luminometer. Set H12 as blank and print the result. An increase in chemiluminescence indicates reporter activity.

[0778]

[Table 5] Example 18: High Throughput Screening Assay Identifies Changes in Small Molecule Concentration and Membrane Permeability Binding of a ligand to a receptor alters intracellular levels of small molecules such as calcium, potassium, sodium and pH. , And changing the membrane potential is known. These changes can be measured in assays that identify the supernatant that binds to the receptor on a particular cell. The following protocol describes an assay for calcium, but this protocol is easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe. You can

The following assay measures changes in fluorescent molecules (Molecular Probes) that bind small molecules using a fluorimetric imaging plate reader ("FLIPR"). Apparently, any fluorescent molecule that detects small molecules is used herein, the calcium fluorescent molecule fluo-4 (Molecular Probes,
Inc. ; Catalog No. F-14202).

For adherent cells, seed cells at 10,000-20,000 cells / well in Co-star black 96-well plates with clear bottoms. CO _{2 the} plate
Incubate in incubator for 20 hours. The adherent cells were treated with 200 μl of HBSS (Hank's Balanced Salt) in a Biotek washer.
Wash twice with Solution, leaving 100 μl of buffer after the last wash.

A stock solution of 1 mg / ml fluo-4 was added to 10% pluronic acid (pluro acid).
nic acid) Made of DMSO. To load the cells with fluo-4,
12 μg / ml fluo-4 (50 μl) is added to each well. The plate is incubated for 60 minutes at 37 ° C. in a CO ₂ incubator. The plate is washed 4 times with HBSS in the Biotek washer, leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from the culture medium. 50 cells
Resuspend to 2-5 × 10 ⁶ cells / ml with HBSS in a ml conical tube. 4 μl of 1 mg / ml fluo-4 10% pluronic acid DMSO solution is added per 1 ml of cell suspension. Then, place the tube in a 37 ° C water bath for 30-6
Leave for 0 minutes. The cells were washed twice with HBSS and resuspended at 1 × 10 ⁶ cells / ml,
Then, dispense 100 μl / well to the microplate. Plate 100
Centrifuge at 0 rpm for 5 minutes. Next, plate the Denley CellWa
After washing once with 200 μl in sh, the final volume is brought to 100 μl by an aspiration step.

For non-cell based assays, each well contains a fluorescent molecule such as fluo-4. The supernatant is added to the wells and the change in fluorescence is detected.

[0784]   In order to measure the fluorescence of intracellular calcium, FLIPR was set to the following parameters.
Set: (1) System gain is 300-800 mW; (2)
Exposure time is 0.4 seconds; (3) camera F / stop is F / 2;
(4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 μl. The increase in luminescence at 530 nm is due to intracellular Ca ⁺⁺ Figure 7 shows extracellular signaling events that result in increasing concentrations.

Example 19: High Throughput Screening Assay to Identify Tyrosine Kinase Activity Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytosolic kinases. Within the receptor protein tyrosine kinase (RPTK) family, there is a range of receptors for mitogenic and metabolic growth factors, including PDGF, FGF, EGF, NGF, HGF and the insulin receptor subfamily. In addition, there is a large RPTK family whose corresponding ligands are unknown. RPTK ligands include primarily secreted low molecular weight proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTK by a ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of the receptor subunit and activation of cytoplasmic tyrosine kinase. As cytoplasmic tyrosine kinase, src family (
Examples include receptor-associated tyrosine kinases of src, yes, lck, lyn, fyn), and non-receptor bound and cytosolic protein tyrosine kinases such as the Jak family. These members include cytokine superfamily (eg, interleukins, interferons, GM
-CSF and leptin).

Because of the wide variety of known factors that can stimulate tyrosine kinase activity, the identification of these novel human secreted proteins that can activate the tyrosine kinase signaling pathway is of interest. Therefore, the following protocol is designed to identify novel human secreted proteins that can activate the tyrosine kinase signaling pathway.

Target cells (eg, primary keratinocytes) were purchased from Nalge Nunc (Naperville, IL) at a density of approximately 25,000 cells / well 96.
Seed wells Loprodyne Silent Screen Plate. The plates are sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsed with water and then dried overnight. Several plates were loaded with 100 ml of cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (
50 mg / ml) (all of these are Sigma Chemicals (St. L.
ouis, MO) or at Becton Dickinso
n (Bedford, MA) 10% Matrigel or calf serum for 2 hours, rinsed with PBS and stored at 4 ° C. Growth medium was seeded at 5,000 cells / well and manufactured by the manufacturer Alamar Biosie.
nces, Inc. (Sacramento, CA), as described by ala
Cell proliferation is assayed on these plates by indirect quantification of cell number after 48 hours using marBlue. Becton Dickinson
(Bedford, MA) using Falcon plate cover # 3071
Cover the Loprodyne Silent Screen Plate. Fal
The con Microtest III cell culture plate may also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded (20,000 / 200 ml / well) on nylon membranes of Loprodyne plates and cultured overnight in complete medium. The cells are incubated in serum-free basal medium for 24 hours to make them rest. 5-2 (50 ng of EGF (60 ng / ml) or the supernatant produced in Example 11)
After treatment for 0 minutes, the medium was removed and 100 ml of extraction buffer (20 mM HEP
ES pH 7.5, 0.15M NaCl, 1% Triton X-100, 0.
1% SDS, 2 mM Na ₃ VO ₄ , 2 mM Na ₄ P ₂ O ₇ and Boeheri
nger Mannheim (Indianapolis, IN) mixture of protease inhibitors (# 1836170)) is added to each well, and the plate is shaken on a rotary shaker for 5 minutes at 4 ° C. The plate is then placed in a vacuum transfer manifold (vacuum transfer manifold).
ld) and place the extract at the bottom of each well using house vacuum.
． Filter through a 45 mm membrane. The extracts are collected in a 96 well capture / assay plate at the bottom of the vacuum manifold and immediately placed on ice. To obtain a clarified extract by centrifugation, solubilize with detergent for 5 minutes, then remove the contents of each well and centrifuge at 16,000 xg for 15 minutes at 4 ° C.

The filtered extract is tested for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one of the methods is described herein.

[0971] Supernatant tyrosine kinase activity is generally assessed by determining the ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (cell division kinase cd
and amino acids 6-20 of c2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a series of tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, after adding 10 μl of 5 μM biotinylated peptide, ATP / Mg ²⁺ (
5 mM ATP / 50 mM MgCl ₂ 10 μl, 5 × assay buffer (40
mM imidazole hydrochloride, pH 7.3, 40 mM β-glycerophosphate, 1 mM
EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg / ml
BSA) 10 μl, sodium vanadate (1 mM) 5 μl and finally water 5 μl. The components are mixed gently and the reaction mixture is preincubated for 2 minutes at 30 ° C. The reaction is started by adding 10 μl of control enzyme or filtered supernatant.

The tyrosine kinase assay reaction is then stopped by adding 10 μl of 120 mM EDTA and the reaction is placed on ice.

Tyrosine kinase activity is measured by transferring a 50 μl aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. As a result, streptavidin (streptava)
din) Coat 96-well plate with biotinylated peptide.
Wash MTP module 4 times with 300 μl / well PBS. Next, anti-phosphotyrosine conjugated to horseradish peroxidase (phospotyro).
Sine) antibody (anti-P-Tyr-POD (0.5 u / ml)) (75 μl) is added to each well and then incubated at 37 ° C. for 1 hour. Wash wells as above.

Next, a peroxidase substrate solution (Boehringer Mannheim) was used.
) Add 100 μl and incubate at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the sample at 405 nm is measured using an ELISA reader. The level of bound peroxidase activity was quantified using an ELISA reader, which reflects the level of tyrosine kinase activity.

Example 20: High Throughput Screening Assay to Identify Phosphorylation Activity Primary cells as a potential alternative and / or compliment of the protein tyrosine kinase activity assay described in Example 19. Assays that detect activation (phosphorylation) of internal signaling intermediates may also be used.
For example, as described below, one particular assay is Erk-1 and Erk-2.
Tyrosine phosphorylation of the kinase can be detected. However, Raf, JNK, p38M
Phosphorylation of other molecules such as AP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec and Janus, as well as any other phosphoserine, phosphotyrosine or phosphothreonine molecule. , Erk-1 or Er in these molecules in the following assay
It can be detected by replacing k-2.

Specifically, the wells of a 96-well ELISA plate were loaded with protein G (1 μg /
ml) 0.1 ml to coat at room temperature (RT) for 2 hours to make an assay plate. The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Next, the protein G plate was subjected to Erk-1 and E.
Two commercially available monoclonal antibodies against rk-2 (100 ng / well) (S
Ant Cruz Biotechnology) (RT for 1 hour). (This step can be easily modified by replacing the monoclonal antibody that detects any of the molecules described above to detect other molecules). After rinsing 3-5 times with PBS, plates are stored at 4 ° C until use.

A431 cells are seeded at 20,000 / well in 96 well Loprodyne filter plates and cultured overnight in growth medium. Next, the cells are treated with basal medium (D
EGF (6 ng / well) or Example 1 after starvation in MEM) for 48 hours.
Treat with 50 μl of the supernatant obtained in 1 for 5 to 20 minutes. The cells are then solubilized and the extract filtered and placed directly into the assay plate.

After incubating with the extract for 1 hour at RT, the wells are rinsed again. Commercially available MAP kinase preparation (10 ng / well) as a positive control
In place of the A431 extract. The plates are then loaded with Erk-1 and E.
Treatment with a commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes a phosphorylated epitope of rk-2 kinase (1 hour at RT). The antibody is biotinylated by standard procedures. Next, the bound polyclonal antibody was combined with Europium streptavidin and Europium fluorescence enhancer and Wallac DELF.
Quantification by continuous incubation (time-resolved fluorescence) in an IA instrument. An increase in fluorescent signal above background indicates phosphorylation.

Example 21: Method for Determining Changes in Genes Corresponding to Polynucleotides RNA isolated from whole families or individual patients presenting with the phenotype of interest (eg, disease) is isolated. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook).
This cDNA is then used as a template for PCR with primers that surround the region of interest in SEQ ID NO: X. Suggested PCR conditions are Sid
ranky, D.A. Et al., Science 252: 706 (1991), consisting of 35 cycles of 95 ° C for 30 seconds; 52-58 ° C for 60-120 seconds; and 70 ° C for 60-120 seconds.

Next, the PCR product was subjected to SequiTherm Polymerase (Epi).
Center Technologies) and sequenced using primers labeled with T4 polynucleotide kinase at the 5'end. The intron-exon boundaries of the selected exons are also determined and the genomic PCR products are analyzed to confirm the results. The PCR product with the suspected mutation is then cloned and sequenced to confirm the direct sequencing results.

The PCR product was transformed into Holton, T .; A. And Graham, M .; W. , Nu
Cleic Acids Research, 19: 1156 (1991) and cloned into a T-tail vector and labeled with T7 polymerase (United).
Sequencing at the States Biochemical). Affected individuals are identified by mutations that are not present in unaffected individuals.

Genomic rearrangements are also observed as a way to determine alterations in the gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 were transformed with digoxigenin deoxy-uridine 5'-triphosphate (Boehringer Manh).
nick translation using eim), and Johnson, Cg
． Et al., Methods Cell Biol. 35: 73-99 (1991)
FISH is performed as described in (). For specific hybridization to the corresponding genomic locus, a large excess of human cot-1 DNA is used for hybridization with the labeled probe.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C and R bands. Alignment images for accurate mapping can be obtained by triple band filter set (Chroma T
technology, Brattleboro, VT) and cooled charge coupled device cameras (Photometrics, Tucson, AZ) and tunable excitation wavelength filters (Johnson, Cv. et al., Genet. Anal. Tech. App.
l. , 8:75 (1991)). ISee Gra
physical Program System (Invision Cor
poration, Durham, NC) for image collection, analysis and chromosome segment length measurement. Chromosomal changes in the genomic region to which the probe hybridizes are identified as insertions, deletions and translocations. These changes are used as diagnostic markers for related diseases.

Example 22: Method for Detecting Abnormal Levels of Polypeptides in Biological Samples Polypeptides of the invention can be detected in biological samples, and elevated or decreased polypeptide levels are detected. If so, the polypeptide is a marker of a particular phenotype. It is understood that there are numerous detection methods, and therefore one of skill in the art can modify the following assays to suit their particular needs.

For example, an antibody sandwich ELISA is used to detect a polypeptide in a sample, preferably a biological sample. The wells of a microtiter plate are coated with specific antibody at a final concentration of 0.2-10 μg / ml. This antibody is either monoclonal or polyclonal and is produced by the method described in Example 10. The well is blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated with the polypeptide-containing sample at RT for> 2 hours. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed three times with deionized or distilled water to remove unbound polypeptide.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was added to 25-400 n.
Add at a concentration of g and incubate at room temperature for 2 hours. Plates are washed again three times with deionized or distilled water to remove unbound conjugate.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate at room temperature for 1 hour. Reactions are measured by microtiter plate reader. A standard curve is constructed using serial dilutions of control samples and the polypeptide concentration is plotted on the X-axis (log scale) and fluorescence or absorbance on the Y-axis (linear scale). A standard curve is used to interpolate the concentration of polypeptide in the sample.

Example 23: Formulation The present invention also provides a disease or disorder (eg, any one or more of the diseases disclosed herein, etc.) by administering to a subject an effective amount of a therapeutic agent. ) Are treated and / or prevented. The therapeutic agent is a pharmaceutically acceptable carrier type (
For example, it means a polynucleotide or polypeptide of the present invention (including fragments and variants), an agonist or antagonist thereof, and / or an antibody thereto, in combination with a sterile carrier.

Therapeutic agents will take into account the clinical condition of the individual patient (particularly the side effects of treatment with the secreted polypeptide alone), the site of delivery, the mode of administration, the dosing regimen and other factors known to those of skill in the art, and the practice of medical practice. Formulated and dosed in a manner that adheres to the (good medical practice). Therefore, the "effective amount" intended in the present specification is
It is decided by taking such a consideration into consideration.

As a general proposition, the total pharmaceutically effective amount of the therapeutic agent administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient body weight,
This is at therapeutic discretion, as described above. More preferably, for the hormone, the dose is at least 0.01 mg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. In the case of continuous administration, the therapeutic agent is typically about 1 μg / kg / hour to about 50 μg / kg.
Administered by either 1 to 4 injections per day or by continuous subcutaneous infusion (eg using a minipump) at a dosing rate of / hour. Intravenous bag solutions may also be used. The duration of treatment required to observe changes and the post-treatment interval at which a response occurs is:
It seems to change depending on the desired effect.

The therapeutic agent may be administered orally, rectally, parenterally, intracistemally.
y), vaginal, intraperitoneal, topical (such as by powder, ointment, gel, drops, or transdermal patch), buccal or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the invention are also suitably administered by a sustained release system. The therapeutic agent is
Oral, rectal, parenteral, intracisternally, vaginal,
Intraperitoneal, topical (such as by powder, ointment, gel, drops, or transdermal patch),
It is given by mouth, oral, or as a nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are suitable polymeric substances (eg semipermeable polymer matrices in the form of shaped articles (eg films or microcapsules)), suitable hydrophobic substances (eg in acceptable quality oils). As an emulsion) or an ion exchange resin, and a poorly soluble derivative (eg, a poorly soluble salt).

Sustained-release matrices include polylactide (US Pat. No. 3,773,919).
No., EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-5.
56 (1983)), poly (2-hydroxyethyl methacrylate) (Lang)
er et al. Biomed. Mater. Res. 15: 167-277 (19
81), and Langer, Chem. Tech. 12: 98-105 (19
82)), ethylene vinyl acetate (Langer et al., Ibid.) Or poly-D.
-(-)-3-hydroxybutyric acid (EP133,988) is mentioned.

Sustained-release therapeutic agents also include liposome-encapsulated compositions of the invention (generally Langer, Science 249: 1527-1533 (1990).
); Treat et al., Liposomes in the Therapy of
Infectious Disease and Center, Lopez
-Berstein and Fidler (ed.), Liss, New Yo
rk, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se:
DE 3,218,121; Epstein et al., Proc. Natl. Acad.
Sci. USA 82: 3688-3692 (1985); Hwang et al., Pr.
oc. Natl. Acad. Sci. USA 77: 4030-4034 (19).
80); EP52,322; EP36,676; EP88,046; EP143.
, 949; EP 142, 641; Japanese Patent Application No. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545 and EP 1
02,324. Usually, liposomes are small (about 200-800 Å) unilamellar form, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for the optimal therapeutic agent. It

In yet a further embodiment, the therapeutic agents of the invention are delivered by pump (Langer, supra; Sefton, CRC Crit. Ref. Biome.
d. Eng. 14: 201 (1987); Buchwald et al., Surgery.
88: 507 (1980); Saudek et al., N. et al. Engl. J. Med. Three
21: 574 (1989)).

Another controlled release system is the Langer (Science 249: 1527-15).
33 (1990)).

For parenteral administration, in one embodiment, the therapeutic agent will generally be administered to the recipient in the desired degree of purity and pharmaceutically acceptable carrier, ie, the dosage and concentration employed. Formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with a non-toxic and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be deleterious to therapeutic agents.

[0821] Generally, the formulations are prepared by contacting the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, in a uniform and intimate contact. The product is then shaped, if desired, into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipients at the dosages and concentrations used, and include such substances as phosphates, citrates, succinates, acetic acid and other organic acids or their salts. Buffers such as; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone. Hydrophilic polymers such as; glycine, glutamic acid, aspartic acid or amino acids such as arginine; cellulose or its derivatives, mono-, di-, and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA ; Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

The therapeutic agent is typically about 0.1 mg / ml to 100 mg / ml, preferably 1
It is formulated in such a vehicle at a concentration of -10 mg / ml and a pH of about 3-8. It is understood that the use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.

Any therapeutic agent used for therapeutic administration can be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (eg 0.2 micron membrane). Generally, the therapeutic agent is placed in a container having a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

Therapeutic agents are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
As an example of a lyophilized formulation, sterile filtered 1% (w / v) into 10 ml vials.
) Fill 5 ml of aqueous polypeptide solution and freeze-dry the resulting mixture.
The lyophilized polypeptide is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the therapeutic agents of the invention. A notice in the form of a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products may accompany such containers, which notice may include government notices regarding manufacture, use, or sale for human administration. Indicates approval by the institution. In addition, therapeutic agents may be used in combination with other therapeutic compounds.

The therapeutic agents of the present invention can be administered alone or in combination with an adjuvant. Adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to, alum, alum and deoxycholic acid (ImmunoAg), MTP-PE (Biocine Corp.), QS2.
1 (Genentech, Inc.), BCG, and MPL. In certain embodiments, therapeutic agents of the invention are administered in combination with alum. In another specific embodiment, the Therapeutic Agents of the invention are administered in combination with QS21. Additional adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to, monophosphoryl lipid immunomodulators, Adj.
uVax 100a, QS-21, QS-18, CRL1005, aluminum salt, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the therapeutic agents of the present invention include, but are not limited to, MMR (measles, mumps, rubella), polio, chickenpox, tetanus / diphtheria, A. Hepatitis B, hepatitis B, influenza B, whooping cough (w
Vaccines directed against protection against hoofing cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, polimyelitis, rabies, typhoid fever, and pertussis. Combinations can be administered either concomitantly (eg, as a mixture, separately but simultaneously or in parallel); or sequentially. This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and the combined agents are administered separately but simultaneously (eg, when administered to the same individual via separate intravenous lines). It also includes procedures. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second.

Therapeutic agents of the invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered with the therapeutic agents of the present invention include, but are not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory agents. , Conventional immunotherapeutic agents, cytokines and / or growth factors. The combination can be administered either, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and also in procedures where the combined agents are administered separately but simultaneously (eg, to the same individual via separate intravenous lines). Also includes. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second. 1
In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF molecules, TNF that can be administered with the therapeutic agents of the invention
Related or TNF-like molecules include, but are not limited to, soluble forms of TNF-α, lymphotoxin-α, (LT-α, TNF-.
also known as β), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1.
BBL, DcR3, OX40L, TNF-γ (International Publication No. WO96 / 1432
8), AIM-I (International Publication No. WO97 / 33899), endokine (en)
dokine) -α (International Publication No. WO98 / 07880), TR6 (International Publication No. WO98 / 30694), OPG, and Neutrokine (neutrokine).
kine) -α (International Publication No. WO98 / 18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, soluble forms of CD30, soluble forms of CD27, soluble forms of CD40 and soluble forms of 4-IBB. , T
R2 (International Publication Number WO96 / 34095), DR3 (International Publication Number WO97 /
33904), DR4 (International Publication Number WO98 / 32856), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO98 / 30694).
, TR7 (International Publication Number WO98 / 41629), TRANS, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number WO98 / 54202).
) 312C2 (International Publication No. WO98 / 06842), and TR12, and soluble forms of CD154, soluble forms of CD70, and soluble forms of CD.
153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and / or protease inhibitors. Examples of nucleoside reverse transcriptase inhibitors that can be administered in combination with the therapeutic agent of the present invention include the following:
Not limited to: RETROVIR ^™ (Zidovudine / AZT), VIDE
Z ^™ (Didanocin / ddI), HIVID ^™ (Zalcitabine / ddC), ZER
IT ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC), and COMBIVIR ^™ (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to, VIRAMUNE ^™ (nevirapine), RESCRI.
PTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirenz). Protease inhibitors that can be administered in combination with the therapeutic agents of the present invention include:
Including but not limited to: CRIXIVAN ^TM (indinavir), NORVIR ^TM (ritonavir), INVIRASE ^TM (saquinavir))
, And VIRACCEPT ^™ (Nelfinavir). In certain embodiments, an antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is for treating AIDS and / or for preventing or treating HIV infection. , And can be used in any combination with the therapeutic agents of the invention.

In other embodiments, Therapeutics of the invention can be administered in combination with anti-opportunistic infection agents. Anti-opportunistic infection agents that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to, TRIMETHOP.
RIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTA
MIDINE ^™ , ATOVAQONE ^™ , ISONIAZID ^™ , RIFAM
PIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ , RIFAB
UTIN ^™ , CLARITHROMYCIN ^™ , AZITHROMYCIN ^™ ,
GANCICLOVIR ^™ , FOSCARNET ^™ , CIDOFOVIR ^™ , F
LUCONAZOLE ^™ , ITRACONAZOLE ^™ , KETOCONAZO
LE ^™ , ACYCLOVIR ^™ , FAMCICOLVIR ^™ , PYRIMETH
AMINE ^™ , LEUCOVORIN ^™ , NEUPOGEN ^™ (filgrastim / G-CSF), and LEUKINE ^™ (sargramostin (sargra
mostim) / GM-CSF). In certain embodiments, the Therapeutic Agents of the present invention are used to treat or prevent opportunistic Pneumocystis carinii pneumonia infection prophylactically.
OLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , and / or AT
Used in any combination with OVAQUONE ^™ . In another specific embodiment, the therapeutic agents of the invention are ISONIAZID ^™ , RIFA for prophylactically treating or preventing opportunistic Mycobacterium avium combined infections.
MPIN ^™ , PYRAZINAMIDE ^™ , and / or ETHAMBUTO
Used in any combination with L ^™ . In another particular embodiment, the therapeutic agents of the invention are RIFABUTIN ^™ , CLARIT for the prophylactic treatment or prevention of opportunistic Mycobacterium tuberculosis infections.
Used in combination with HRMYCIN ^™ , and / or AZITHROMYCIN ^™ . In another particular embodiment, the therapeutic agents of the invention are administered to GANCI to prophylactically treat or prevent opportunistic cytomegalovirus infections.
Used in any combination with CLOVIR ^™ , FOSCARNET ^™ , and / or CIDOFOVIR ^™ . In another particular embodiment, the therapeutic agents of the invention are FLUCONA for the prophylactic treatment or prevention of opportunistic fungal infections.
ZOLE ^™ , ITRACONAZOLE ^™ , and / or KETOCONAZ
Used in any combination with OLE ^™ . In another particular embodiment, the therapeutic agents of the invention are ACYCLOVIR ^™ and / or FA for the prophylactic treatment or prevention of opportunistic herpes simplex virus type I and / or type II infections.
Used in any combination with MCICOLVIR ^™ . In another particular embodiment, the therapeutic agents of the invention are PYRIMETHAMINE ^™ and / or for the prophylactic treatment or prevention of opportunistic Toxoplasma gondii infection.
Or used in any combination with LEUCOVORIN ^™ . In another particular embodiment, the Therapeutics of the invention are used in any combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to prophylactically treat or prevent opportunistic bacterial infections.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with antiviral agents. Antiviral agents that may be administered with the therapeutic agents of the present invention include acyclovir, ribavirin, amantadine, and remantidine (remantidine).
Tidine), but is not limited thereto.

In a further embodiment, the Therapeutics of the invention are administered with an antibiotic. Antibiotics that can be administered with the therapeutic agent of the present invention include amoxicillin, β
-Lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,
Macrolide antibiotics, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamides, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin are included without limitation.

Conventional non-specific immunosuppressive agents that can be administered with the therapeutic agents of the present invention include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxysperm. Guarin (15-deoxyspergualin), and response T
Other immunosuppressive agents that act by suppressing the function of cells include, but are not limited to.

In certain embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressant preparations that can be administered with the therapeutic agents of the invention include ORT
HOCLONE ^TM (OKT3), SANDIMMUNE ^TM / NEORAL ^TM / S
ANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CE
Examples include, but are not limited to, LLCCEPT ^™ (mycophenolate), azathioprine, glucocorticosteroids, and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents may be used to prevent rejection of organ or bone marrow transplants.

In a further embodiment, the Therapeutics of the invention are administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that may be administered in combination with the therapeutic agents of the present invention include GAMMAR ^™ , IV
Examples include, but are not limited to, EEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™ and GAMIMUNE ^™ . In certain embodiments, therapeutic agents of the invention are administered in combination with an intravenous immunoglobulin preparation in transplantation therapy (eg, bone marrow transplant).

In a further embodiment, the Therapeutic Agents of the invention are administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that can be administered with the therapeutic agent of the present invention include glucocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, Pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrin
e), bendazac, benzydamine, bucolome, diphenpyramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaseprol, paranyline, perisoxal,
Examples include pifoxime, procazone, proxazole, and tenidap,
It is not limited to these.

In another embodiment, the compositions of this invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the therapeutic agents of the invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5). -FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin
, Mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin). , And vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and test lactone); Nitrogen mustard derivatives (eg mephalen, chlorambucil, mechlorethamine Roger emissions mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium); and others (
Examples include, but are not limited to, dicarbazine, asparaginase, mitoten, vincristine sulfate, vinblastine sulfate, and etoposide.

In certain embodiments, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or in any combination of the components of CHOP. In another embodiment, the Therapeutic Agents of the invention are administered in combination with Rituximab. In a further embodiment, the therapeutic agents of the invention are Rituxmab and CHOP.
Or with any combination of Rituxmab and CHOP components.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with cytokines. ILs include cytokines that can be administered with the therapeutic agents of the invention.
2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13
, IL15, anti-CD40, CD40L, IFN-γ and TNF-α, but are not limited thereto. In another embodiment, the therapeutic agent of the present invention comprises any interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4.
, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1
1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-1
7, IL-18, IL-19, IL-20 and IL-21).

In a further embodiment, Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that can be administered with the therapeutic agents of the invention include glioma-derived growth factor (GDGF) as disclosed in European Patent No. EP-399816; platelet-derived as disclosed in European Patent No. EP-682110. Growth factor A (PDGF-A); Platelet-derived growth factor B (PDGF-B) as disclosed in European Patent No. EP-228317; International Publication No. WO92 / 0.
Placental Growth Factor (PIGF) as disclosed in 6194; Hauser et al., G.
placental growth factor 2 (PIGF-2) as disclosed in orwt Factors, 4: 259-268 (1993); vascular endothelial growth factor (VEGF) as disclosed in International Publication No. WO90 / 13649; European Patent No. EP-5064
Vascular Endothelial Growth Factor A (VEGF-A) as disclosed in 77; International Publication Number W
Vascular Endothelial Growth Factor 2 (VEGF-2) as disclosed in O96 / 39515
Vascular endothelial growth factor B (VEGF-3); vascular endothelial growth factor B-186 (VEGF-B186) as disclosed in International Publication No. WO 96/26736; as disclosed in International Publication No. WO 98/02543. Vascular endothelial growth factor D (VE
GF-D); vascular endothelial growth factor D (VEGF-D) as disclosed in WO98 / 07832; and vascular endothelial growth factor E (VEGF-E) as disclosed in German Patent No. DE19639601. ), But is not limited thereto. The above references are incorporated herein by reference.

In a further embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that can be administered with the therapeutic agents of the present invention include LEU
KINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FIL
GRASTIM ^™ ), but is not limited thereto.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered together with the therapeutic agent of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5 and FG.
F-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11,
FGF-12, FGF-13, FGF-14, and FGF-15 include, but are not limited to.

In a further embodiment, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation treatment).

Example 24: Method for Treating Decreased Levels of Polypeptides The present invention relates to a method for treating an individual in need of increasing levels of a polypeptide of the invention in the body, the method comprising: Administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including a polypeptide of the invention). Furthermore, it is understood that conditions caused by reduced normal or normal expression levels of secreted proteins in an individual can be treated by administering the polypeptides of the invention, preferably in secreted form. Accordingly, the invention also provides a method of treating an individual in need of increased levels of this polypeptide. The method involves administering to such an individual a therapeutic agent comprising an amount of the polypeptide that increases the activity level of the polypeptide in such individual.

For example, a patient with reduced levels of a polypeptide will receive the polypeptide at a daily dose of 0.1-100 μg / kg for 6 consecutive days. Preferably the polypeptide is in secreted form. The exact details of the dosing and formulation-based regimen are:
Provided in Example 23.

Example 25: Method of Treating Elevated Levels of Polypeptides The present invention also relates to a method for treating an individual in need of decreasing levels of a polypeptide of the invention in the body, the method comprising: , Administering a composition comprising a therapeutically effective amount of an antagonist of the invention (including the polypeptides and antibodies of the invention) to such an individual.

In one example, antisense technology is used to inhibit production of a polypeptide of the invention. This technique is one example of how to reduce the levels of polypeptides, preferably secreted forms of the polypeptides, that result from various etiologies such as cancer. For example, in patients diagnosed with abnormally elevated levels of polypeptide, 0.5, 1.0, 1.5, 2.0 and 3.0 mg of antisense polynucleotide may be administered.
/ Kg / day for 21 days. If well tolerated, the procedure is repeated after a 7 day washout period. The formulation of this antisense polynucleotide is provided in Example 23.

Example 26 Treatment Method Using Gene Therapy-Ex Vivo One method of gene therapy involves transplanting fibroblasts capable of expressing a polypeptide into a patient. Fibroblasts are generally obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. A blob of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted and tightly closed, then left overnight at room temperature. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask, and fresh medium (eg,
Ham's F12 containing 10% FBS, penicillin and streptomycin
Medium) is added. The flask is then incubated at 37 ° C for approximately 1 week.

At this point, fresh medium is added and then replaced every few days. After culturing for another two weeks, a monolayer of fibroblasts appears. Trypsinizing this monolayer,
Scale up to a larger flask.

PMV-7 (Kirsc) flanked by long terminal repeats of Moloney murine sarcoma virus.
hmeier, P.M. T. Et al., DNA, 7: 219-25 (1988)).
After digestion with RI and HindIII, it is treated with calf intestinal phosphatase.
The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNAs encoding the polypeptides of the present invention were prepared as described in Example 1 using primers and with appropriate restriction sites and start / stop codons, respectively, the 5'and 3'terminal sequences, respectively. Can be amplified using PCR primers corresponding to Preferably, the 5'primer contains an EcoRI site and the 3'primer contains a HindIII site. Equal amounts of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under suitable conditions for ligating the two fragments. The ligation mixture is then used to transform bacterial HB101. It is then plated on agar containing kanamycin to confirm that the vector has the gene of interest inserted correctly.

Amphotropic pA317 or GP + am12 packaging cells were
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle Medium (DMEM) with 0% Calf Serum (CS), Penicillin and Streptomycin. The MSV vector containing the gene of interest is then added to the medium and the packaging cells are transduced with this vector. At this time, the packaging cells produce infectious viral particles containing the gene (herein, the packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells and this medium is then harvested from 10 cm plates of confluent producer cells. The spent medium containing infectious viral particles is filtered through a Millipore filter to remove detached producer cells and then used to infect fibroblasts. Media is removed from the subconfluent plates of fibroblasts and quickly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts will be infected and no selection is required. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker such as neo or his. Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if the protein is being produced.

The engineered fibroblasts are then transplanted into the host either alone or after being grown to confluence on Cytodex 3 microcarrier beads.

Example 27: Gene Therapy Using an Endogenous Gene Corresponding to a Polynucleotide of the Invention Another method of gene therapy according to the present invention is the use of endogenous polynucleotide sequences of the invention, eg, in US Pat. No. 5,641,670 (issued June 24, 1997);
International Publication Number WO 96/29411 (Published September 26, 1996); International Publication Number WO 94/12650 (Published August 4, 1994); Koller et al., P.
roc. Natl. Acad. Sci. USA, 86: 8932-8935 (1
989); and Zijlstra et al., Nature, 342: 435-438.
Operably linked to the promoter via homologous recombination, as described in (1989). The method involves activation of a gene that is present in the target cell but is not expressed in the cell or is expressed at a lower level than desired.

A polynucleotide construct containing a promoter and targeting sequence is made. This target sequence is homologous to the 5'non-coding sequence of the endogenous polynucleotide sequence, which flanks the promoter. The targeting sequence is 5'of the polynucleotide sequence.
Close enough to the ends so that the promoter is operably linked to the endogenous sequence upon homologous recombination. PCR of this promoter and targeting sequence
Can be used to amplify. Preferably, the amplified promoter contains different restriction enzyme sites on the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence is the 3'end of the amplified promoter. Contains the same restriction sites as.

The amplified promoter and amplified targeting sequence are digested with the appropriate restriction enzymes, followed by treatment with calf intestinal phosphatase. The digested promoter and digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent such as liposomes, viral sequences, viral particles, precipitating agents and the like. Such methods of delivery are known in the art.

Once the cells have been transfected, homologous recombination will occur and the promoter will be operably linked to the endogenous polynucleotide sequence. This results in expression of the polynucleotide corresponding to the polynucleotide in the cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the surface of the plastic with nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is mixed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM K).
Cl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. This final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to a polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, NY) was constructed.
) Is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site at the 5'end and a BamHI site at the 3'end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5'end and an XbaI site at the 3'end; the other non-coding sequence (fragment). 2) is amplified with a BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promoter and fragments thereof (1 and 2) were cloned into the appropriate enzymes (CMV promoter-XbaI and BamHI; fragment 1-XbaI; fragment 2-B.
amHI) and digested together. The resulting ligation product was HindII
I digested and ligated with HindIII digested pUC18 plasmid.

Plasmid DNA was loaded onto a sterile cuvette (B
io-Rad). Final DNA concentration is generally at least 120μ
g / ml. Then 0.5 ml of the cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably integrate the introduced DNA into their genome. Given these parameters, the pulse time is about 14-2
0 mSec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells were placed in a 10 cm dish containing pre-warmed nutrient medium (D containing 15% bovine serum).
MEM) 10 ml directly and incubate at 37 ° C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours.

The engineered fibroblasts are then injected into the host, either alone or after being grown to confluence on cytodex 3 microcarrier beads. To do. here,
The fibroblasts produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28: Method of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. This method of gene therapy involves the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease expression of the polypeptide. The polynucleotide of the invention may be operably linked to a promoter, or any other genetic element required for expression of the polypeptide by the target tissue. Techniques and methods for such gene therapy and delivery are known in the art, eg, WO 90/11.
092, WO98 / 11779; U.S. Pat. Nos. 5,693,622, 5,705,51.
51, ibid. 5580859; Tabata et al., Cardiovasc. Re
s. 35 (3): 470-479 (1997); Chao et al., Pharmaco.
l. Res. 35 (6): 517-522 (1997); Wolff, Neur.
omuscul. Disord. 7 (5): 314-318 (1997), Sc.
hwartz et al., Gene Ther. 3 (5): 405-411 (1996)
); Tsurumi et al., Circulation 94 (12): 3281-3.
290 (1996), incorporated herein by reference.

The polynucleotide construct is delivered by any method that delivers an injectable substance to cells of an animal, such as injection into the interstitial space of a tissue (heart, muscle, skin, lung, liver, intestine, etc.). obtain. The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term "naked" polynucleotide, DNA or RNA refers to any delivery vehicle (viral sequence, viral particle, liposome formulation, lipofectin, or (Including a precipitating agent) is not included. However, the polynucleotides of the invention can also be prepared in liposome formulations (eg, Felgner) that can be prepared by methods well known to those of skill in the art.
P. L. (1995) Ann. NY Acad. Sci. 772: 126-1
39 and Abdallah B. (1995) Biol. Cell 85 (
1): 1-7)).

The polynucleotide vector construct used in this gene therapy method comprises:
Preferred are constructs that do not integrate into the host genome and do not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for the production of desired polypeptides for periods of up to 6 months.

This polynucleotide construct is used to determine the tissue (animal, skin, brain, lung, liver,
(Including spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue) It can be delivered to the space. The interstitial spaces of this tissue are intercellular fluids, mucopolysaccharide substrates (between reticulum fibers of organ tissue, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue), or connective tissue sheaths. Includes the same matrix within natural muscle cells or in the hiatus of bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space is preferred for the reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression can be effected on undifferentiated cells or cells that are not fully differentiated (eg, blood stem cells). Or skin fibroblasts). In vivo, muscle cells are particularly competent for their ability to take up and express polynucleotides.

For naked polynucleotide injections, an effective dosage of DNA or RNA is in the range of about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily 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,
This includes, for example, inhalation of aerosol formulations, especially for delivery to the mucosa of the lungs or bronchial tissues, throat, or nose. In addition, the naked polynucleotide construct can be delivered to the artery during the angioplasty procedure by the catheter used in this procedure.

The dose response effect of injected polynucleotide in muscle in vivo is determined as follows. Appropriate template DNA for the production of mRNA encoding the polypeptide of the present invention is prepared according to standard recombinant DNA methodologies. Mold DN
Either A, which can be either circular or linear, is used as naked DNA or is complexed with liposomes. The mouse quadriceps is then injected with varying amounts of template DNA.

To 5 and 6 week old female and male Balb / C mice, 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. Template DNA is placed in a 0.1 ml carrier and injected with a 1 cc syringe through a 27 gauge needle for 1 minute into the knee about 0.5 cm from the distal insertion site of the muscle at a depth of about 0.2 cm. . Sutures are made over the injection site for future localization and the skin is closed with stainless steel clips.

After a suitable incubation time (eg 7 days), muscle extracts are prepared by cutting out whole quads. Every 5th 15 μm section of an individual quadriceps is histochemically stained for protein expression. The time course for protein expression can be done in a similar fashion except that four animals from different mice are harvested at different times. Persistence of DNA in muscle after injection can be determined by Southern blot analysis after preparation of total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice are shown as naked D
NA can be used to extrapolate appropriate dosages and other treatment parameters in humans and other animals.

Example 29: Transgenic Animals The polypeptides of the present invention can also be expressed in transgenic animals. Mouse, rat, rabbit, hamster, guinea pig, pig, miniature pig, goat,
Animals of any species, including but not limited to sheep, cows and non-human primates such as baboons, monkeys and chimpanzees, can be used to produce transgenic animals. In certain embodiments, techniques described herein or otherwise known in the art are used for expression of a polypeptide of the invention in humans as part of a gene therapy protocol.

Any technique known in the art can be used to introduce a transgene (ie, a polynucleotide of the invention) into an animal to establish the foo of the transgenic animal.
nder line) can be generated. Such techniques are described in Pronuclear Microinjection (Patterson et al., Appl. Microbiol. Biotec.
hnol. 40: 691-698 (1994); Carver et al., Biotec.
hology (NY) 11: 1263-1270 (1993); Wright
Et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); Germline retrovirus-mediated gene transfer (Van der Putten et al., Pr.
oc. Natl. Acad. Sci. USA 82: 6148-6152 (19).
85)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompso)
n et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 180).
3-1814 (1983)); introduction of the polynucleotide of the present invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993)); to embryonic pluripotent stem cells. Introduction of the nucleic acid construct and transfer of this stem cell into the blastocyst; and sperm-mediated gene transfer (La
Vitrano et al., Cell 57: 717-723 (1989)); and the like, but are not limited thereto. For a review of such techniques, see Gordon, "Transgenic An," which is incorporated herein by reference in its entirety.
imals ", Intl. Rev. Cytol. 115: 171-229 (19
89).

Any technique known in the art can be used to generate transgenic clones containing a polynucleotide of the invention (eg, cultured, quiescently induced embryonic cells, fetal cells or adult cells). Nuclear Transfer of Cell-Derived Enucleated Oocytes to the Nucleus (Campell et al., Nature 380: 64-66 (1996); Wil
mut et al., Nature 385: 810-813 (1997))).

The present invention provides transgenic animals that carry the transgene in all their cells, as well as animals that carry the transgene in some (but not all) of the cells (ie, mosaic animals or chimeras). To do. The transgene may be as a single transgene or in a concatemer (eg head-to-head tandem or head-to-head).
Multiple copies, such as tail tandem type). This transgene is also described, for example, in the teaching of Lasko et al. (Lasko et al., Proc. Natl. Ac.
ad. Sci. USA 89: 6232-6236 (1992)) and can be selectively introduced into and activated by specific cell types. The regulatory sequences required for such cell type-specific activation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. Targeting of the gene is preferred when it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene. Briefly, if such a technique is utilized, a vector containing several nucleotide sequences homologous to the endogenous gene may be used to bind the nucleotide sequence of the endogenous gene via homologous recombination with the chromosomal sequence. And is designed for the purpose of destroying the function of the nucleotide sequence. Transgenes can also be selectively introduced into specific cell types, and thus, for example, Gu et al. (Gu et al. Science 26
5: 103-106 (1994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type of interest, and they will be apparent to those of skill in the art.

Once a transgenic animal is produced, expression of its recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to confirm that transgene integration has occurred by analysis of animal tissue. The level of transgene mRNA expression in the tissues of transgenic animals was also determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P of tissue samples obtained from the animals.
It can be assessed using techniques including, but not limited to CR (rt-PCR). Samples of transgenic gene expressing tissues can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once founder animals are generated, they can be bred, inbred, outbred or crossed to generate colonies of particular animals. Examples of such mating strategies include, but are not limited to: Founder outcrosses with more than one integration site to establish another lineage; additive expression of each transgene. By effect, inbreeding of separate strains to produce composite transgenics expressing higher levels of the transgene; a given routine for both enhancing expression and eliminating the need for screening animals by DNA analysis. Hybridization of heterozygous transgenic animals that produce animals homozygous for the site of integration; crossing of separate homozygous strains to generate complex heterozygous or homozygous lines; and to the desired experimental model Mating to place the transgene on a suitable different background.

The transgenic animals of the invention may be used to describe the biological function of the polypeptides of the invention, to study diseases, disorders and / or conditions associated with aberrant expression, and to study such diseases, disorders and / or conditions. Alternatively, it has applications including, but not limited to, animal model systems useful in screening for compounds that are effective in ameliorating the condition.

Example 30: Knockout Animals Endogenous gene expression is also achieved using targeted homologous recombination to gene and / or
Alternatively, it can be reduced by inactivating or “knocking out” its promoter (eg, Smithies et al., Nature 317: 230-2).
34 (1985); Thomas and Capecchi, Cell 51: 5.
03-512 (1987); Thompson et al., Cell 5: 313-32.
1 (1989); each of which is incorporated herein by reference in its entirety). For example, a variant, non-functional polynucleotide (or completely unrelated DNA) of the invention flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of this gene). Sequence) can be used with or without a selectable marker and / or a negative selectable marker to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to make knockouts in cells that contain the gene of interest but do not express it. Insertion of this DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such an approach is particularly suited to the research and agricultural fields where modifications to embryonic stem cells may be used to produce animal progeny that have inactive targeted genes (eg, Thomas and Capecchi 1987 and Thompson 1989). , See above). However, this approach is conventional for use in humans when the recombinant DNA construct is directly administered or targeted to the required site in vivo using appropriate viral vectors, which will be apparent to those skilled in the art. Can be adapted to.

In a further embodiment of the invention, a cell genetically engineered to express a polypeptide of the invention, or a cell genetically engineered not to express a polypeptide of the invention (eg, a knockout), Administer to patients in vivo. Such cells can be obtained from patients (ie, animals, including humans) or MHC compatible donors, and can be fibroblasts, bone marrow cells, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Can be included, but is not limited to. The cells can be transformed, for example, by transduction (using a viral vector and preferably a vector that integrates the transgene into the cell genome) or transfection procedures (plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Endogenous regulation to introduce a coding sequence of a polypeptide of the invention into a cell or to bind to this coding sequence and / or a polypeptide of the invention. To destroy the array
Genetically engineered in vitro using recombinant DNA technology. The coding sequence of the polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the polypeptide of the invention. Engineered cells that express and preferably secrete a polypeptide of the invention can be introduced systemically into the patient, eg, in circulation or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and transplanted into the body (
For example, genetically engineered fibroblasts can be transplanted as part of a skin graft); genetically engineered endothelial cells can be transplanted as part of a lymphoid or vascular graft (eg Anderson et al., US Patent) No. 5,399,349 and Mu
See Lligan and Wilson, US Pat. No. 5,460,959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-self or non-MHC compatible cells, they may be administered using well known techniques that interfere with the development of a host immune response against the introduced cells. For example, the cells can be introduced in a capsular form, which does not allow the introduced cells to be recognized by the host immune system, while permitting exchange of components with the immediate extracellular environment.

The transgenic and “knockout” animals of the present invention may be used to describe the biological function of the polypeptides of the present invention, to study diseases, disorders and / or conditions associated with aberrant expression, as well as to such diseases. , Including, but not limited to, animal model systems useful in screening compounds effective in ameliorating disorders and / or conditions.

Example 31: Production of Antibodies a. Hybridoma Technology The antibodies of the invention can be prepared by a variety of methods. (Current Pr
otocols, Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of the polypeptide of the invention is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

Monoclonal antibodies specific for the polypeptides of the invention are prepared using hybridoma technology (Kohler et al., Nature 256: 495).
(1975); Kohler et al., Eur. J. Immunol. 6: 511 (1
976); Kohler et al., Eur. J. Immunol. 6: 292 (197)
6); Hammerling et al .: Monoclonal Antibodies.
and T-Cell Hybridomas, Elsevier, N .; Y.
, Pp. 563-681 (1981)). Generally, an animal, preferably a mouse, is immunized with a polypeptide of the invention, or more preferably with secretory polypeptide expressing cells. Such polypeptide-expressing cells are preferably supplemented with 10% fetal bovine serum (inactivated at about 56 ° C.) in any suitable tissue culture medium and contain about 10 g / l of non-essential amino acids, about 1. Cultured in Earle's modified Eagle's medium supplemented with 1,000 U / ml penicillin and about 100 μg / ml streptomycin.

The splenocytes of such mice are extracted and fused with the appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, AT
It is preferred to use the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
Cloning by limiting dilution as described by 1981)). The hybridoma cells obtained by such selection are then assayed to identify clones secreting antibodies capable of binding the polypeptides of the invention.

Alternatively, additional antibodies capable of binding the polypeptides of the present invention may be generated in a two step procedure using anti-idiotypic antibodies. Such a method
Taking advantage of the fact that the antibody itself is the antigen and it is therefore possible to obtain an antibody which binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones producing antibodies whose ability to bind protein-specific antibodies can be blocked by the polypeptides of the invention. Such antibodies include anti-idiotypic antibodies to protein-specific antibodies and are used to immunize animals to induce the formation of additional protein-specific antibodies.

For use in vivo use of the antibody in humans, the antibody is "humanized." Such antibodies can be produced by using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (for a review, Morrison, Science 229: 120).
2 (1985); Oi et al., BioTechniques 4: 214 (1986).
); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi
Et al., EP 171496; Morrison et al., EP 173494; Neub.
erger et al., WO 8601533; Robinson et al., WO 87026.
71; Boulianne et al., Nature 312: 643 (1984); N.
See Euberger et al., Nature 314: 268 (1985)).

(B) Isolation of Antibody Fragments Against Polypeptides Derived from a Library of scFvs) [0891] A naturally-occurring V gene isolated from human PBLs of the present invention, to which a donor may or may not be exposed. It is constructed into a library of antibody fragments that include reactivity to the polypeptide (see, eg, US Pat. No. 5,885,793, which is incorporated herein by reference in its entirety).

Library rescue. A library of scFvs is constructed from RNA of human PBLs as described in PCT Publication WO 92/01047. To rescue the phage displaying the antibody fragment, about 10 ⁹ E. coli carrying the phagemid were rescued. 50 ml 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) was inoculated with E. coli and 0.8 OD with shaking. D. Grow to. 5 of this culture
50 ml of 2 × TY-AMP-GLU was inoculated with 2 ml of 2 × 10 8 TU Δ gene 3 helper (M13Δ gene III, PCT publication WO92 / 01047).
) Is added and the culture is incubated at 37 ° C. for 45 minutes without shaking, then at 37 ° C. for 45 minutes with shaking. This culture was incubated for 10 minutes at 4000 rpm. p. m. Centrifuge at rt and resuspend pellet in 2 liters of 2xTY containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and grow overnight. PCT published WO
Prepare as described in 92/01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking and then for another hour at 37 ° C with shaking. Centrifuge the cells (IEC-Centra 8,400r
． p. m. / Min for 10 minutes) and 100 μg / ml ampicillin and 25 μm
2 × TY broth containing 2 g / ml of kanamycin (2 × TY-AMP-KAN
) Resuspended in 300 ml and grown overnight at 37 ° C. with shaking. Phage particles were purified and concentrated from the culture medium by two rounds of PEG precipitation (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius), about 1 μm.
A final concentration of 0 ¹³ transducing units / ml (ampicillin resistant clones) is obtained.

Panning the library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg / ml or 10 μg / ml of the polypeptides of the invention. Tubes are 3% with 2% Marvel-PBS
Block for 2 hours at 7 ° C, then wash 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to the tubes and incubated for 30 minutes at room temperature with tumbling up and down on a turntable, then left to stand for a further 1.5 hours. The tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.
The phages were eluted by adding 1 ml of 100 mM triethylamine and spinning up and down for 15 minutes on a turntable, then 0.5 ml of 1.
Immediately neutralize with 0 M Tris-HCl, pH 7.4. The eluted phage were then incubated with the bacteria for 30 minutes at 37 ° C. to allow 10 ml of mid-log phase E. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then
Rescue with Δ gene 3 helper phage as described above to prepare phage for subsequent rounds of selection. This process was then repeated for all 4 rounds of affinity purification, with 3rd and 4th tube washings with PBS, 0.1% Tw.
Increase 20-fold with een-20 and 20-fold with PBS.

Characterization of binders. Phage eluted from the third and fourth round of selection were used to transform E. coli. E. coli HB2151 and soluble scFv are generated from a single colony for the assay (Marks et al., 1991). The ELISA is performed using microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in 50 mM bicarbonate, pH 9.6. ELISA
PCR positive printing (for example, PCT publication WO
92/01047), and then further characterized by sequencing. These ELISA positive clones can also be cloned using techniques known in the art (eg, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody / antigen binding, and competitive agonist activity. Or competitive antagonist activity, etc.).

Example 32: Assay for Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation Generation of a functional humoral immune response can be achieved between B lineage cells and their microenvironment.
It requires both soluble and cognate signaling. The signal can convey a positive stimulus (which causes cells of the B lineage to sustain programmed development) or a negative stimulus (which directs the cell to block the current-generating pathway). To date, IL-2, IL-4, IL-5, IL-6, IL-7, IL
A number of stimulatory and inhibitory signals have been found to influence B cell responsiveness, including -10, IL-13, IL-14 and IL-15. Interestingly, these signals, which are weak effectors in their own right, can be combined with various costimulatory proteins to induce activation, proliferation, differentiation, homing, resistance, and death in B cell populations.

One of the most studied classes of B cell costimulatory proteins is the TNF superfamily. Within this family, CD40, CD27 and CD30
It has been found to regulate a variety of immune responses with its respective ligands, CD154, CD70 and CD153. Assays that allow the detection and / or observation of proliferation and differentiation of these B cell populations and their progenitor cells show the effects that various proteins may have on these B cell populations in terms of proliferation and differentiation. A valuable tool in making decisions. Listed below are two assays designed to allow detection of differentiation, proliferation, or inhibition of B cell populations and their precursors.

In Vitro Assay—Ability of the purified polypeptides of the invention, or truncated forms thereof, to induce activation, proliferation, differentiation or inhibition and / or death in B cell populations and their precursors. Evaluate about. Activity of the polypeptides of the invention on purified human tonsillar B cells (qualitatively 0.1-10,000 n
(measured over a dose range of g / mL) is evaluated in a standard B lymphocyte costimulation assay. In this assay, purified tonsil B cells were used as a priming factor in formalin-fixed Staphylococcus aureus.
Culture in the presence of either Cowan I (SAC) or immobilized anti-human IgM antibody. Secondary signals such as IL-2 and IL-15 cooperate with SAC and IgM cross-linking to induce B cell proliferation as measured by tritiated thymidine incorporation. New synergists can be easily identified using this assay. This assay involves the isolation of human tonsillar B cells by magnetic bead (MACS) depletion of CD3 positive cells. The resulting cell population is CD45R
Greater than 95% of B cells when assessed by expression of (B220).

Each sample at various dilutions was placed in an individual well of a 96-well plate, to which the culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / ml).
10 ⁵ B cells in a total volume of 150 μl suspended in penicillin, RPMI 1640 containing 10 μg / ml streptomycin, and 10 ⁻⁵ dilution of SAC) are added. Proliferation or inhibition was started 72 hours after addition of the factor and 3H-
Quantify with thymidine (6.7 Ci / mM) for 20 h pulse (1 μCi / well). Positive and negative controls are IL2 and medium, respectively.

In Vivo Assay-BALB / c mice are injected twice daily (intraperitoneally) with buffer alone, or 2 mg / kg of the polypeptides of the invention, or their shortened forms. Mice were given this treatment for 4 consecutive days, at which point they were sacrificed and various tissues and sera were collected for analysis. Comparison of H & E sections from normal spleen and spleens treated with the polypeptide of the invention results in activity of this polypeptide on spleen cells, such as diffusion of periarterial lymphoid sheaths and / or red pulp regions. A significant increase in nucleated cellularity is noted, which may indicate activation of B cell population differentiation and proliferation. Anti-CD, a B cell marker
Using immunohistochemical studies with 45R (B220), any physiologic changes to spleen cells (eg spleen tissue disruption) invade the established T cell regions, a vaguely defined B cell compartment. Determine whether due to increased B cell presentation within.

Using flow cytometric analysis of spleens from mice treated with the polypeptide, the polypeptide is ThB +, CD45R (B220) dull B.
It is shown whether the ratio of cells is specifically increased over that observed in control mice.

In addition, the putative consequences of increased in vivo presentation of mature B cells are serum I
g is the relative increase in titer. Therefore, serum IgM and IgA levels are compared between buffer-treated and polypeptide-treated mice.

The tests described in this example tested the activity of the polypeptides of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy) of the polypeptides of the invention, agonists and / or antagonists of the polynucleotides or polypeptides of the invention. .

Example 33: T Cell Proliferation Assay A CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. The assay is performed as follows. 96-well plates were loaded with 100 mAbs against CD3 (HIT3a, Pharmingen).
μl / well, or isotype-matched control mAb (B33.1) (
1 μg / ml in 0.05 M bicarbonate buffer, pH 9.5) at 4 ° C. overnight,
Coat and then wash 3 times with PBS. PBMCs were isolated from human peripheral blood by F / H gradient centrifugation and coated with mAb in RPMI containing 10% FCS and P / S in the presence of various concentrations of the polypeptides of the invention. Add to ⁴ wells (5 × 10 ⁴ / well) of the plate (total volume 200 μl).
The relevant protein buffer and media alone are controls. After 48 hours of incubation at 37 ° C., the plate was spun at 1000 rpm for 2 minutes and 100 μm.
l of supernatant was removed and stored at −20 ° C. for measurement of IL-2 (or other cytokines) if effects on proliferation were observed. 0.5 μCi in well
Supplemented with 100 μl of medium containing ³ H-thymidine and incubated at 37 ° C for 18-
Incubate for 24 hours. Wells were harvested and ³ H-thymidine incorporation was used as an indicator of proliferation. Anti-CD3 alone is a positive control for proliferation. IL-2 (
100 U / ml) is also used as a growth enhancing control. A control antibody that does not induce T cell proliferation is used as a negative control for the effects of the polypeptides of the invention.

The studies described in this example tested activity of a polypeptide of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy) of the polypeptides of the invention, agonists and / or antagonists of the polynucleotides or polypeptides of the invention. .

Example 34: Effect of Polypeptides of the Invention on MHC Class II, Co-stimulatory and Adhesion Molecule Expression, and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells Produced by proliferation of proliferative progenitors found in blood: Adherent PBMC or cleared monocyte fractions were treated with GM-CSF (50 ng / ml) and I
Incubate with L-4 (20 ng / ml) for 7-10 days. These dendritic cells have characteristic phenotypes of immature cells (CD1, CD80, CD86, CD40 and M40).
HC class II antigen expression). Treatment with activators such as TNF-α causes rapid changes in surface phenotype (MHC class I and II, increased expression of costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). Occurs. These changes are associated with increased antigen presenting capacity and functional maturation of dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated with increasing concentrations of a polypeptide of the invention or LPS (positive control) for 1 to 3 days and 1% B
Wash with PBS containing SA and 0.02 mM sodium azide, then 1
: Incubate for 30 minutes at 4 ° C. with 20 dilutions of the appropriate FITC- or PE-labeled monoclonal antibody. After additional washing, labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on Cytokine Production Cytokines produced by dendritic cells, particularly IL-12, are important in the initiation of T cell dependent immune responses. IL-12
Strongly influence the development of a Th1 helper T cell immune response and induce cytotoxic T
Induces cellular and NK cell function. IL using ELISA as follows
-12 Release is measured. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of a polypeptide of the invention for 24 hours. LPS (100 ng / ml) is added to the cell culture as a positive control. The supernatant from the cell culture is then collected and a commercially available ELISA kit (eg R & D Systems (Minneap
olis, MN)) for analysis of IL-12 content. Use the standard protocol provided in the kit.

Effect on expression of MHC class II, costimulatory molecules and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptors. Modulation of the expression of MHC class II antigens and other costimulatory molecules such as B7 and ICAM-1 can result in changes in the ability of monocytes to present antigen and to induce T cell activation. Increased Fc receptor expression may be correlated with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to test surface antigens as follows. Monocytes were enriched at 1 with increasing concentrations of a polypeptide of the invention or LPS (positive control).
~ PB treated for 5 days and containing 1% BSA and 0.02 mM sodium azide
Wash with S, then incubate for 30 minutes at 4 ° C. with a 1:20 dilution of the appropriate FITC- or PE-labeled monoclonal antibody.
After further washing, labeled cells were washed with FACScan (Becton Dickin
Son) for analysis by flow cytometry.

Molecules (Increase Monocyte Activation and / or Survival) Monocytes that Activate (or Inactivate) Monocytes and / or Increase Monocyte Survival (or Decrease Monocyte Survival) Assay is known in the art and can be routinely applied to determine whether the molecules of the invention function as monocyte inhibitors or activators. The polypeptides, agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) were treated with Histop
Single donor leuko by centrifugation through an aque gradient (Sigma)
pack (American Red Cross, Baltimore, MD
). Countercurrent centrifugal elutriation (counterflo)
Isolate from PBMC by w centrifugal elution).

Monocyte Survival Assay Human peripheral blood monocytes progressively lose viability when cultured in the absence of serum or other stimuli. Their death results from an internally regulated process (apoptosis). Addition of activators such as TNFα to the culture dramatically improves cell survival and prevents DNA fragmentation. Propidium iodine (
PI) staining is used to measure apoptosis as follows. 100n for monocytes
Incubate for 48 hours in serum-free medium (positive control) in polypropylene tubes in the presence of g / ml TNF-α (negative control) and in the presence of various concentrations of the compound to be tested. Cells are suspended in PBS containing PI at a final concentration of 5 μg / ml to a concentration of 2 × 10 ⁶ / ml and then incubated for 5 minutes at room temperature before FACScan analysis. PI uptake has been shown to correlate with DNA fragmentation in this test paradigm.

Effect on Cytokine Release An important function of monocytes / macrophages is their regulatory activity on other cell populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows.
Human monocytes are incubated at a density of 5 × 10 ⁵ cells / ml with increasing concentrations of a polypeptide of the invention and in the absence of this polypeptide under the same conditions. For production of IL-12, the cells are primed with IFN (100 U / ml) overnight in the presence of the polypeptides of the invention. Then, LPS (10
ng / ml) is added. Conditioned medium is collected after 24 hours and stored frozen until use. Then, the measurement of TNF-α, IL-10, MCP-1 and IL-8 is performed using a commercially available ELISA kit (for example, R & D Systems Minne.
apolis, MN) and applying the standard protocol provided in the kit.

96. Oxidative burst purified 96 monocytes.
Plate well plates at 2-1 × 10 ⁵ cells / well. Increasing concentrations of the polypeptides of the invention were added to wells in a total volume of 0.2 ml of culture medium (RPMI 1640+
10% FCS, glutamine and antibiotics). After 3 days of incubation, the plates are centrifuged and the medium is removed from the wells. In a monolayer of macrophages, 0.2 ml of phenol red solution (140 m
M NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO).
Add with stimulant (200 nM PMA). This plate at 37 ℃ 2
Incubate for a period of time and stop the reaction by adding 20 μl of 1N NaOH per well. Read absorption at 610 nm. To calculate the amount of H ₂ O ₂ produced by macrophages, a standard curve of H ₂ O ₂ solution of known molarity is run for each experiment.

The studies described in this example tested activity of a polypeptide of the invention. However, those skilled in the art will appreciate that the activity of the polypeptide of the invention, the activity of the polynucleotide (
For example, gene studies), agonist activity, and / or antagonist activity can be readily modified to study the illustrated studies.

Example 35 Biological Effects of Polypeptides of the Present Invention Astrocyte and Neural Assays [0916] Recombinant polypeptides of the present invention expressed and purified in Escherichia coli as described above were used. , Survival of cortical neuron cells, activity promoting neurite outgrowth or phenotypic differentiation, and induction of proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for bioassays is based on the predominant expression of FGF-1 and FGF-2 in cortical structures and the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. Thymidine incorporation assays can be used, for example, to elucidate the activity of the polypeptides of the invention on these cells.

In addition, FGF to cortical or hippocampal neurons in vitro
A previous report describing the biological effects of -2 (basic FGF) demonstrated an increase in both neuronal survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promoters".
survival of dissociated hippocampal
neurons and enhancements neuroite extensi
on ”Proc. Natl. Acad. Sci. USA 83: 3012-30
16 (1986), the assay is hereby incorporated by reference in its entirety).
. However, reports from experiments carried out on PC-12 cells indicate that these two responses are not necessarily synonymous, and not only which FGF is being tested, but which receptor is expressed on the target cells. Suggest that you may depend. The ability of the polypeptides of the invention to induce neurite outgrowth can be compared to the response obtained with FGF-2 using the primary cortical neuron culture paradigm, eg, using the thymidine incorporation assay.

Fibroblast and Endothelial Cell Assay Human lung fibroblasts are obtained from Clonetics (San Diego, CA) and maintained in growth medium from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well in 96 well plate growth medium for 1 day. The cells are then incubated for 1 day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test protein for 3 days. Alamar Blue (Almar Bios
(Ciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hours. Cell viability is C
Measure by reading with a ytoFluor fluorescence reader. For the PGE ₂ assay, human lung fibroblasts are cultured in 96-well plates for 1 day at 5,000 cells / well. After converting the medium to 0.1% BSA basal medium, the cells were
Incubate with FGF-2 or a polypeptide of the invention with or without L-1α for 24 hours. The supernatant was collected and the EIA kit (
Cayman, Ann Arbor, MI) for PGE ₂ . For the IL-6 assay, human lung fibroblasts are cultured in 96-well plates for 1 day at 5,000 cells / well. After converting the medium to 0.1% BSA basal medium, cells are incubated with FGF-2 for 24 hours with or without the polypeptide IL-1α of the invention. Supernatants were collected and IL by ELISA kit (Endogen, Cambridge, MA)
Assay for -6.

Human lung fibroblasts were cultured with FGF-2 or a polypeptide of the invention in basal medium for 3 days, after which Alam was used to assess the effect on fibroblast proliferation.
Add ar Blue. FGF-2 should show a stimulus of 10 to 2500 ng / ml which could be used comparable to that with the polypeptides of the invention.

Parkinson's Model Loss of motor function in Parkinson's disease results from striatal dopamine deficiency resulting from degeneration of nigrostriatal dopaminergic projection neurons. A widely characterized animal model of Parkinson's disease is 1-methyl-4phenyl 1,2,3
, 6-tetrahydropyridine (MPTP) systemic administration. In the CNS,
MPTP is taken up by astrocytes and 1 by monoamine oxidase B
-Catalyzed and released into methyl-4-phenylpyridine (MPP ⁺ ).
Subsequently, MPP ⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter of dopamine. MPP ⁺ is then enriched in mitochondria by an electrochemical gradient and selectively inhibits nicotinamide diphosphate: ubiquinone oxidoreductase (complex I), thereby interfering with electron transfer and To generate active oxygen.

It has been demonstrated in a tissue culture paradigm that FGF-2 (basic FGF) has trophic activity on substantia nigra dopaminergic neurons (Ferrari).
Et al., Dev. Biol. 1989). Recently, Unsicker's group has demonstrated that FGF-2 administration in striatal gel implants results in near complete protection of substantia nigra dopaminergic neurons from toxicity associated with MPTP exposure ( Otto and Unsicker, J. Neuroscience,
1990).

Based on the data using FGF-2, the polypeptide of the present invention exerts an action similar to that of FGF-2 in enhancing survival of dopaminergic neurons in vitro. Polypeptides of the invention can also be evaluated in vivo to determine if they have, and also tested in vivo for protection of dopaminergic neurons in the striatum from damage associated with MPTP treatment. The potential effects of the polypeptides of the invention are first tested in vitro in the dopaminergic neuronal cell culture paradigm. 14 days pregnant Wista
Cultures are prepared by dissecting the midbrain floor plate from r rat embryos. Tissues were detached with trypsin and seeded on polyortinin-laminin coated coverslips at a density of 200,000 cells / cm ² . The cells are maintained in Dulbecco's modified Eagle medium and F12 medium containing hormone supplements (NI). After 8 days in vitro cultures are fixed with paraformamide and processed for immunohistochemical staining with tyrosine hydroxylase, a specific marker for dopaminergic neurons. Separate cell cultures are prepared from embryonic rats.
The culture medium is changed every 3 days and the factor is also added at each time point.

Since the dopaminergic neurons are isolated from animals on day 14 of gestation (this time of development has passed the stage where dopaminergic progenitor cells proliferate), an increase in the number of tyrosine hydroxylase immunopositive neurons was observed in vitro. Shows an increase in the number of surviving dopaminergic neurons. Therefore, it is suggested that if the polypeptide of the present invention acts to prolong dopaminergic survival, this polypeptide may be involved in Parkinson's disease.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy)
, The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 36: Effect of Polypeptides of the Invention on Proliferation of Vascular Endothelial Cells On day 1, human umbilical vein endothelial cells (HUVEC) were treated with 4% fetal bovine serum (FBS).
), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.).
Seed in medium at a density of 2-5 × 10 ⁴ cells per 35 mm dish. On day 2, this medium was added to M199 containing 10% FBS, 8 units / ml heparin.
Replace with. A polypeptide having the amino acid sequence of SEQ ID NO: Y and a positive control (eg VEGF and basic FGF (bFGF)) are added at various concentrations. Change medium on days 4 and 6. Day 8, Coulter C
Determine the cell number using an outer.

An increase in HUVEC cell number indicates that the polypeptides of the present invention are capable of proliferating vascular endothelial cells.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy)
, The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 37: Stimulatory Effect of Polypeptides of the Invention on Proliferation of Vascular Endothelial Cells A colorimetric assay using the electron coupling reagent PMS (phenazine methosulfate) for the evaluation of mitogenic activity of growth factors. An analytical MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay was performed (CellTiter).
96 AQ, Promega). Cells are seeded in 96-well plates (5,000 cells / well) in 0.1 mL serum-supplemented medium and allowed to attach overnight. 0
． After 12 hours serum starvation in 5% FBS, the conditions (bFGF, VEGF ₁₆₅ or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U / ml) were applied to the wells for 48 hours. Added. 20 mg MTS / PMS
The mixture (1: 0.05) is added per well and allowed to incubate at 37 ° C. for 1 hour, after which the absorbance at 490 nm is measured on an ELISA plate reader. Background absorption of control wells (medium, no cells) is subtracted, and 7 wells are run in parallel for each condition.
Leak et al., In Vitro Cell. Dev. Biol. 30A: 512
518 (1994).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy)
, The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 38: Inhibition of PDGF-induced Vascular Smooth Muscle Cell Proliferation Stimulating Effect HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent quiescent cells grown on 4-chamber slides were
Transfect with RP or FITC labeled AT2-3LP. The cells are then pulsed with 10% bovine serum and 6 mg / ml BrdUrd. 24
After hours, immunocytochemistry is performed by using the BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with biotinylated mouse anti-BrdUrd antibody for 2 hours at 4 ° C. after exposure to denaturing solution, followed by streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination and BrdUrd-positive cells are counted. BrdU
The rd coefficient is calculated as the ratio of BrdUrd-positive cells to the total cell number. Furthermore, for individual cells, simultaneous detection of BrdUrd staining (nucleus) and FITC uptake (cytoplasmic) is performed by simultaneous use of bright and dark UV fluorescence illumination. (Hayashida et al., J. Biol. Chem. 6: 271 (3
6): 21985-21992 (1996)).

The studies described in this example tested activity of a polypeptide of the invention.
However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 39: Stimulation of Endothelial Migration This example can be used to explore the potential of the polypeptides of the invention to stimulate lymphatic endothelial cell migration.

The Endothelial Cell Migration Assay is Performed Using a 48-Well Microchemotactic Chamber (Neuroprobe Inc., Cabin John, MD; Falk).
W., J. et al. Immunological Methods 1980; 33:
239-247). Polycarbonate filter without polyvinylpyrrolidone, which has a pore size of 8 μm (Nucleopore Corp. Cam.
(Bridge, MA) coated with 0.1% gelatin for at least 6 hours at room temperature,
Then it is dried under sterile air. Test substances are diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and 25 μl of the final dilution is placed in the bottom chamber of the modified Boyden device. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required for cell detachment. After placing the filter between the bottom chamber and the top chamber, 2.5 × 10 ⁵ cells suspended in 50 μl M199 containing 1% FBS are seeded in the upper compartment. Then this device
Cells were allowed to migrate by incubating at 37 ° C. for 5 hours in a chamber moistened with 5% CO 2. After the incubation period, the filter was removed and the upper side of the filter with non-migrated cells was placed on the rubber policeman (polic).
Eman). The filter was fixed with methanol and Giem
Stain with sa solution (Diff-Quick, Baxter, McGraw P
ark, IL). Migration is quantified by counting 3 random high power fields (40x) of cells in each well. Then run all groups in quadruplicate.

The studies described in this example tested activity of a polypeptide of the invention.
However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 40: Stimulation of nitric oxide production by endothelial cells Nitric oxide released by vascular endothelium is believed to be a mediator of vascular endothelial relaxation. Thus, the activity of a polypeptide of the invention can be assayed by measuring nitric oxide production by endothelial cells in response to this polypeptide.

Nitric oxide was starved for 24 hours, followed by varying levels of positive control (
For example, in a 96-well plate of confluent microvascular endothelial cells after 4 hours of exposure to VEGF-1) and polypeptides of the invention. Nitric oxide in the medium is measured by using the Griess reagent to measure the total amount of nitrite after reduction of nitric acid from nitric oxide by nitrate reductase. The effect of the polypeptides of the invention on nitric oxide release is tested in HUVEC.

Briefly, NO release from cultured HUVEC monolayers was measured using a NO meter (Iso
-NO, World Precision Instruments Inc.
) (1049) connected to a NO-specific polarographic electrode.
The calibration of the NO element is carried out according to the following formula: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ SO ₄ calibration curve with stepwise concentrations of KNO ₂ (in a calibration solution containing KI and H ₂ SO ₄ (
0, 5, 10, 25, 50, 100, 250, and 500 nmol / L). The specificity of the Iso-NO electrode for NO is previously determined by measuring NO from an authentic NO gas (105).
0). The culture medium is removed and the HUVECs are washed twice with Dulbecco phosphate buffered saline. The cells are then placed in a 6-well plate for 5 m
Immerse it in 1 filtered Krebs-Henseleit solution and place the cell plate in a slide warmer (Lab Lin) to maintain the temperature at 37 ° C.
e Instruments Inc. ) Hold. The NO sensor probe is inserted vertically into the well, keeping the tip of the electrode 2 mm below the surface of the solution before adding different conditions. S-nitrosoacetylpenicillamine (SNAP) is used as a positive control. The amount of NO released is expressed as picomolar concentration per 1 × 10 ⁶ endothelial cells. All reported values are the average of 4-6 measurements in each group (number of cell culture wells). Leak et al., Biochem. and B
iophys. Res. Comm. 217: 96-105 (1995).

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 41: Effect of Polypeptides of the Invention on Cord Formation in Angiogenesis Another step in angiogenesis is the cord (c) characterized by endothelial cell differentiation.
ord) formation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) was used as a proliferating cell (2nd passage) in Cell.
Applications Inc. Purchased from Cell Applica
cultures in Tions' CADEMC Growth Medium, and 5
Use at the passage. For in vitro angiogenesis assay, wells of a 48-well cell culture plate were placed in Cell Applications'Attachme.
Coat with nt Factor Medium (200 ml / well) for 30 minutes at 37 ° C. CADMEC coated wells at 7,500 cells / well
Seed wells and culture overnight in Growth Medium. Then, this Growth Medium was added to 300 mg of Cell Applica containing a control buffer or the polypeptide of the present invention (0.1 to 100 ng / ml).
Tions' Chord Formation Medium, and the cells are cultured for an additional 48 hours. The number and length of the capillary-like chords are calculated by Boeck.
Quantify through use of an eler VIA-170 video image analyzer. All assays are done in triplicate.

Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. β-estradiol (1 ng / ml) is used as a negative control. Appropriate buffer (no protein) is also used as a control.

The studies described in this example tested activity of a polypeptide of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 42: Angiogenic Effect on Chicken Chorioallantoic Membrane Chicken Chorioallantoic Membrane (CAM) is a well established system for testing angiogenesis. Angiogenesis in the CAM can be easily visualized and quantified. The ability of the polypeptides of the invention to stimulate angiogenesis in CAM can be tested.

Fertilized eggs of White Leghorn chicken (Gallus gallus) and Japanese quail (Coturnix coturnix) were
Incubate at 37.8 ° C and 80% humidity. 16 day old chicken differentiated CAM and 13 day old quail embryos are studied in the following manner.

On day 4 of development, a window is made in the eggshell of chicken eggs. The embryos are examined for normal development and the eggs are sealed with Cellotape®. These one more
Incubate until day 3. Thermanox cover glass (Nunc,
(Naperville, IL) are cut into discs of approximately 5 mm diameter. Sterile and salt-free growth factor is dissolved in distilled water, and about 3.3 mg / 5 ml is pipetted onto the disc. After air drying, the inverted disc is applied to the CAM. 3 days later, sample 3
Fixed in% glutaraldehyde and 2% formaldehyde, and 0.12
Rinse with M sodium cacodylate buffer. These are stereomicroscopes [Wild M8
] And photographed and embedded for semi-thin and ultra-thin sectioning as described above. Control is performed only on the carrier disc.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 43: Angiogenesis Assay Using Matrigel Implants in Mice [0947] An in vivo angiogenesis assay of the polypeptides of the invention is based on the preexisting capillary network-like, mouse extracellular matrix material (Matrigel). ) To determine the ability to form new vessels in the implanted capsules. This protein is liquid Ma at 4 ° C.
It is mixed with trigel and then the mixture is injected subcutaneously into mice, where the mixture solidifies. After 7 days, Matrigel's solid "plugs" are removed and tested for the presence of new blood vessels. Matrigel, Bect
on Dickinson Labware / Collaborative B
Purchase from iomedical Products.

Matrigel material is a liquid when thawed at 4 ° C. This Matrix
1 is mixed with a polypeptide of the invention at 150 ng / ml at 4 ° C. and placed in a cold 3 ml syringe. Female C57B1 / 6 mice, approximately 8 weeks old, are injected with a mixture of Matrigel and experimental protein at two sites on the midventral aspect of the abdomen (0.
5 ml / site). After 7 days, the mice were sacrificed by cervical dislocation and Matrige
Remove 1 plug and wash (ie remove all attached membrane and fibrous tissue). Whole duplicate plugs are fixed in neutral buffered 10% formamide, embedded in paraffin and stained with Masson's Trichrome before use to prepare sections for histological examination. Sections from three different areas of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay was bovine basic FGF (150n
g / ml). Matrigel alone is used to determine basal levels of angiogenesis.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model To study the in vivo effect of the polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model was previously described. (Takesh
ita et al., Am J. Pathol 147: 1649-1660 (1995).
), As by surgical removal of one femoral artery. Resection of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. As a result, blood flow to the ischemic limb depends on collateral vessels arising from the internal iliac artery (Takeshita et al., Am J.
． Pathol 147: 1649-1660 (1995)). 10 day intervals allow post-surgical recovery of rabbits and development of endogenous collateral vessels. After surgery 1
On day 0 (day 0), after baseline angiography, the internal iliac artery of the ischemic limb was treated with 500 mg of the naked expression plasmid containing the polynucleotide of the invention,
(Riessen et al., Hum Gene Ther. 4: 749-758 (19
93); Leclerc et al. Clin. Invest. 90: 936-94
4 (1992)) by the arterial gene transfer technique using a hydrogel-coated balloon catheter. When the polypeptide of the invention is used for treatment, a single bolus of 500 mg of the polypeptide of the invention or control is delivered through the infusion catheter for 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits: (a) BP ratio-ischemic limb systolic blood pressure to normal limb systolic blood pressure ratio; (b) blood flow and reflux-arrest FL. : Blood flow during undilated state, and maximum FL: blood flow during fully dilated state (also an indirect measurement of vascular volume), and regurgitation, maximum FL: stop F
It is reflected in the ratio of L; (c) Angiographic Score
e) -this is measured by angiography of the collateral vessels. Values are determined by the percentage of circles in the overlaid grid (using transverse opacified arteries divided by the total number m of rabbit thighs); (d) Capillary density-determined in light microscopy sections obtained from the hindlimb. Number of collateral capillaries formed.

The studies described in this example tested the activity of the polynucleotides and polypeptides of the present invention. However, one of skill in the art could easily modify the illustrated study to
The agonists and / or antagonists of the invention may be tested.

(Example 45: Effect of the polypeptide of the present invention on vasodilation) [0952] Since dilation of vascular endothelium is important for lowering blood pressure, spontaneous hypertensive rats (SHR).
In), the ability of the polynucleotide of the invention to affect blood pressure is tested. Increasing doses (0, 10, 30, 100, 300, and 900 mg / kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as mean +/- SEM. Statistical analysis is performed using a two-tailed t-test, and statistical significance is defined as p <0.05 for response to buffer alone.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 46: Rat ischemic skin flap model Evaluation parameters include cutaneous blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of the polypeptides of the invention during skin ischemia is studied using in situ hybridization.

[0955]   The work in this model is divided into three parts:   a) ischemic skin,   b) ischemic skin wounds,   c) Normal wound.

The experimental protocol includes: a) 3 × 4 cm single-stem full-thickness single pedicles.
producing a full-thickness random skin flap) (muscle flap extending to the lower back of the animal), b) making an excision wound on the ischemic skin (skin flap) (diameter 4-6 mm), c) 1 mg-100 mg Topical treatment of excisional wounds (at 0, 1, 2, 3, 4 days after wounding) with the polypeptides of the present invention in various dosage ranges of, d) histological studies , For immunohistochemical and in situ studies,
Collect wound tissue at 3, 5, 7, 10, 14, and 21 days after wounding.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 47: Peripheral Arterial Disease Model Angioplasty therapy with the polypeptides of the invention is a novel therapeutic strategy to obtain peri-ischemic blood flow restoration in the case of peripheral arterial disease. . The experimental protocol includes: a) The femoral artery on one side is ligated to create the ischemic muscle of the hind limb, while the hind limb on the other side serves as a control.

B) Polypeptides of the invention are delivered in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2-3 weeks.

C) This ischemic muscle tissue is collected at 1, 2, and 3 weeks after ligation of the femoral artery for analysis of expression of the polypeptides of the invention and histology. Biopsies are also performed on the normal muscle of the contralateral hind limb on the other side.

The studies described in this example tested activity of a polypeptide of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 48: Ischemic Myocardial Disease Model The polypeptides of the invention are evaluated as potent mitogens that can stimulate collateral vessel development and reconstitute new blood vessels after coronary artery occlusion. . Altered expression of the polypeptide is investigated in situ. The experimental protocol includes: a) The heart is exposed via a left thoracotomy in the rat. Immediately, the left coronary artery,
Occlusate with fine suture (6-0) and close the rib cage.

B) Polypeptides of the invention are delivered in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2 to 4 weeks.

C) Thirty days after surgery, the heart is removed for morphometry and cross-sectioned and analyzed in situ.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 49: Rat Corneal Wound Healing Model This animal model shows the effect of the polypeptides of the invention on neovascularization. The experimental protocol includes: a) Making an incision 1-1.5 mm long from the center of the cornea into the stroma.

[0967]   b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.

C) Creating a pocket (its base is 1-1.5 m from the edge of the eye)
m).

D) Placing pellets containing 50 ng to 5 μg of a polypeptide of the invention in the pocket.

E) Treatment with the polypeptides of the present invention may also be applied topically to the corneal wound within a dosage range of 20 mg to 500 mg (daily treatment for 5 days).

The studies described in this example tested activity of a polypeptide of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 50 Diabetic Mice and Glucocorticoid Damage Wound Healing Model A. Diabetic db + / db + Mouse Model To demonstrate that the polypeptides of the present invention promote the healing process, A genetically diabetic mouse model is used. The full thickness wound healing model in db + / db + mice is a well characterized, clinically relevant, and reproducible model of wound wound healing. Healing of diabetic wounds depends on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389 (19).
92); Greenhalgh, D .; G. Et al., Am. J. Pathol. 136
: 1235 (1990)).

This diabetic animal has many of the characteristic properties observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) obese compared to littermates. Mutant diabetes (db + / db +)
Mice carry a single autosomal recessive mutation (db +) on chromosome 4 (C
oleman et al., Proc. Natl. Acad. Sci. USA 77:28
3-293 (1982)). Animals exhibit polyphagia, polydipsia, and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, elevated or normal insulin levels, and suppressed cell-mediated immunity (Ma.
ndel et al. Immunol. 120: 1375 (1978); Debra.
y-Sachs, M .; Et al., Clin. Exp. Immunol. 51 (1): 1
-7 (1983); Leiter et al., Am. J. of Pathol. 114:
46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular injury,
Basement membrane thickening and glomerular filtration abnormalities have been described in these animals (
Norido, F .; Et al., Exp. Neurol. 83 (2): 221-232 (
1984); Robertson et al., Diabetes 29 (1): 60-6.
7 (1980); Giacomelli et al., Lab Invest. 40 (4)
: 460-473 (1979); Coleman, D .; L. , Diabetes
31 (Supplement): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia and are resistant to insulin, similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-13).
77 (1978)).

The characteristics observed in these animals indicate that the healing in this model may be similar to that observed in human diabetes (Greenhalgh et al., Am. J. of Pathol. 136: 1235-1246 ( 1990))
.

Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates are used in this study (J
acksson Laboratories). Purchased these animals at 6 weeks of age,
And these animals are 8 weeks of age at the start of the study. Animals are housed individually and provided with food and water ad libitum. All operations are performed using aseptic technique. This experiment was performed on Human Genome Sciences, Inc's Insitu
regional Animal Care and Use Committee
and the Guidelines for the Care and
Use according to the rules and guidelines of Use of Laboratory Animals.

The wound protocol was modified according to previously reported methods (Tsuboi, R. and Rif.
kin, D.M. B. J. Exp. Med. 172: 245-251 (1990)
). Briefly, on the day of wounding, animals were treated with A dissolved in deionized water.
Anesthetize with intraperitoneal injection of vertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol. The dorsal area of the animal is shaved and the skin is washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. Then, an 8 mm full-thickness wound
It is prepared using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove the spread of the wound. The wound is opened during the experiment. The application of treatment is given topically for 5 consecutive days starting from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wounds are visually inspected and photographed at a fixed distance on the day of surgery and at 2 day intervals thereafter. Wound closure is determined by daily measurements on days 1-5 and 8. Wounds are measured horizontally and vertically using calibrated Jameson calipers. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

The polypeptides of the invention are administered in the vehicle for 8 days with different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the total area of the wound. Contraction is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). Calculations are performed using the following formula: [Open area on day 8]-[Open area on day 1] / [Open area on day 1] Specimens are fixed in 10% buffered formalin and paraffin. The embedded mass is cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of wounds is used to assess whether the healing process and morphological appearance of repaired skin have been altered by treatment with the polypeptides of the invention. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization, and epidermal maturation (Green.
nhalgh, D.N. G. Et al., Am. J. Pathol. 136: 1235 (19
90)). Blinded lens micrometer with graduation
Observer).

Tissue sections are also immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control.
Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a calibrated lens micrometer.

Proliferating Cell Nuclear Antigen / Cyclin (PCNA) in Skin Specimens was Modified with ABC El
Demonstrate by using anti-PCNA antibody (1:50) with the ite detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen contains sections with omission of primary antibody and replacement with non-immune mouse IgG. The rank of these sections is based on the extent of proliferation on a scale of 0-8 (lower scale reflecting slight proliferation to higher side reflecting vigorous proliferation).

Experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

B. Steroidopathic Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids an.
d Wound healing: Anti-Inflammatory St
eroid Action: Basic and Clinical Aspe
cts. 280-302 (1989); Wahl et al. Immunol. 1
15: 476-481 (1975); Werb et al., J. Am. Exp. Med. 147
: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert et al., An. Intern. Med. 37: 7.
01-705 (1952)), fibroblast proliferation, and collagen synthesis (Bec).
k et al., Growing Factors. 5: 295-304 (1991); Hay
nes et al. Clin. Invest. 61: 703-797 (1978)).
And resulting in a transient decrease in circulating monocytes (Hay
nes et al. Clin. Invest. 61: 703-797 (1978);
Wahl, "Glucocorticoids and wound heel
ing ": Antiflammery Steroid Action
: Basic and Clinical Aspects, Academic
Wound healing is delayed by Press, New York, 280-302 (1989)). Systemic administration of steroids for impaired wound healing is a well established phenomenon in rats (Beck et al., Growth Fact).
ors. 5: 295-304 (1991); Haynes et al. Clin.
Invest. 61: 703-797 (1978); Wahl, "Gluco.
corticoids and wound healing ": Antiin
firmware Steroid Action: Basic and
Clinical Aspects, Academic Press, New
York, 280-302 (1989); Pierce et al., Proc. Nat
l. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that the polypeptides of the present invention may promote the healing process, multiple of the polypeptides of the present invention against full-thickness excised skin wounds in rats, where healing is impaired by systemic administration of methylprednisolone. Evaluate the effect of topical application of.

Young adult male Sprague Dawley rats (weight 250-300 g)
) (Charles River Laboratories) is used for this experiment. The animals are purchased at 8 weeks of age. The start of the study is 9 weeks of age. The healing response of the rat was determined by systemic administration of methylprednisolone (17 mg / k
g / rat, intramuscular). Animals are housed individually and given food and water ad libitum. All operations are performed using aseptic technique. This research, Huma
n Genome Sciences, Inc. Institutional
Animal Care and Use Committee and the
e Guidelines for the Care and Use of
Perform according to the Laboratory Animals rules and guidelines.

The wound protocol follows Section A above. On the day of wounding, the animals were given ketamine (5 m
g / kg) and xylazine (5 mg / kg) by intramuscular injection. The dorsal area of the animal is shaved and the skin is washed with 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. 8 mm full thickness wounds are created using a Keyes tissue punch. The left side of the wound is opened during the experiment. Application of the test substance is given topically once a day for 7 consecutive days, starting from the day of wounding and then methylmethylprednisolone administration. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wound is visually inspected and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure is determined by daily measurements on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a Calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

The polypeptides of the present invention are administered in the vehicle for 8 days with different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology. Tissue specimens are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, and 3) treated group. .

Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total area of the wound. Closure is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). Calculations are performed using the following formula: [Open Area on Day 8]-[Open Area on Day 1] / [Open Area on Day 1] Specimens are fixed in 10% buffered formalin and paraffin. The embedded mass is cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of wounds makes it possible to assess whether the healing process and the morphological appearance of the repaired skin have been improved by treatment with the polypeptides of the invention. A graduated lens observer is used by a blinded observer to determine the gap distance of the wound.

Experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

The studies described in this example tested polynucleotide activity of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polypeptides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 51: Lymphedema Animal Model The purpose of this experimental approach was to identify lymphangiogenesis (lymph) in the rat hindlimb.
to develop a suitable and consistent model of lymphedema for testing the therapeutic effect of the polypeptides of the invention in reestablishing the vascular and lymphatic circulation. Efficacy is measured by swelling volume of the affected limb, quantification of lymphatic vascular mass, total plasma protein mass, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema
Continues for 3-4 weeks.

Prior to beginning surgery, blood samples are drawn for protein concentration analysis.
Male rats (weighing approximately 350 g) are dosed with pentobarbital. Then, the right limb is shaved from the knee to the hip joint. Wipe the shaved area with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. After measuring the circumference and volume, mark the two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) and then inject the dye into the foot. On the endothelium of both right and left dorsal
Inject 0.05 ml of 1% Evan's Blue. Then, after the dye is injected into the paw, circumference measurement and volume measurement are performed.

Using the knee joint as a landmark, an incision in the groin of the middle limb is made in a ring so that the position of the femoral vessel can be confirmed. The flaps are dissected and separated using forceps and hemostat. After locating the femoral vessel, the lymphatic vessels that run alongside and below the vessel are located. The major lymphatic vessels in this area are then attached to the electrocoagulated suture ligature (ele).
Critically suture ligated).

Using a microscope, the muscles of the back of the limb (semitendinosus)
And around the adductor muscle). The location of the popliteal lymph nodes is then confirmed. The two proximal lymphatic vessels and two distal lymphatic vessels as well as the distal blood supply of the popliteal node are ligated with sutures. The popliteal lymph nodes and any associated adipose tissue are then removed by cutting connective tissue.

Be careful to manage any mild bleeding that occurs with this procedure. After occlusion of lymphatic vessels, flaps are placed on the liquid skin (Vetbond) (AJ Buck).
Seal by using. Separate skin edges are sealed to the underlying muscle tissue leaving a gap of about 0.5 cm around the leg. The skin may also be anchored by suturing to the underlying muscle when needed.

[1001] Animals are individually housed using a mesh (no bedding) to avoid infection. Recovered animals are checked daily through the optimal edema peak. This peak typically occurs up to 5-7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of two designated locations on each limb are measured before the procedure and daily for 7 days. The effect of plasma proteins on lymphedema is determined, and it is also investigated whether protein analysis is a useful test perimeter. The weights of both control and edematous limbs are evaluated at two points. The analysis is performed in a blind manner.

Circumference measurement: Under short-term gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a cloth tape measure. Measurements are made on the talar and dorsal paw by two different persons and then these two readings are averaged. Readings are taken from both control and edematous limbs.

[1003] Volumetric: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume measurements, animals are placed under short-term halothane anesthesia (rapid immobilization and quick recovery), both legs shaved, and the legs marked equally with water-resistant markers. The legs are first dipped in water, then dipped into the device to each marked level, then Buxco hydrops software (Chen / Victor
or)). The data is recorded by one person, while the other is soaked in the marked area of the leg.

Blood-plasma protein measurements: Blood is drawn prior to surgery, centrifuged, and serum separated, then the total protein and Ca2 + comparisons are concluded.

[1005] Limb Weight Comparison: After blood is drawn, the animal is prepared for tissue collection.
The limb is cut with a quillitine, then both the experimental and control legs are ligated and cut and weighed. A second weighing is performed by removing the tibio-cakaneal joint and weighing the foot.

[1006] Histological Preparation: The transverse muscle located behind the knee (popliteal) area was excised and placed in a metal mold, filled with freezeGel, soaked in cold methylbutane and -80EC until cut into labeled sample bags. Place it. Upon cutting, the muscle is observed under the fluorescent microscope for lymphatic vessels.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 52 Suppression of TNFα-Induced Adhesion Molecule Expression by the Polypeptides of the Present Invention The recruitment of lymphocytes to areas of inflammation and angiogenesis is associated with cell surface adhesion molecules (CAM) on lymphocytes. It relates to specific receptor-ligand interactions with the vascular endothelium. This adhesion process is associated with intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-) in both normal and pathological settings.
1), and following a multistep cascade involving endothelial leukocyte adhesion molecule-1 (E-selectin) expression in endothelial cells (EC). Expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into local tissues during the development of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Tumor necrosis factor alpha (TNF-α), a potent pro-inflammatory cytokine, is a stimulator of all three CAMs in endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in disease. Bring physical results.

[1010] The potential of the polypeptides of the invention to mediate the suppression of TNF-α induced CAM expression may be tested. A modified ELISA assay, which uses EC as the solid phase adsorbent, was used to quantify the amount of CAM expression in the TNF-α-processed protein EC when co-stimulated with members of the FGF family of proteins. taking measurement.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord harvests and growth medium (EGM-2; supplemented with 10% FCS and 1% penicillin / streptomycin). Clonetics, San Die
go, CA) in a humidified incubator at 37 ° C. with 5% CO ₂ . HUVECs are seeded in 96 well plates at a concentration of 1 × 10 ⁴ cells / well in EGM medium at 37 ° C. for 18-24 hours or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U / ml penicillin and 100 mg / ml streptomycin and at 37 ° C. for 24 hours with the indicated cytokines and / or growth factors. Processed. After incubation, the cells are then evaluated for CAM expression.

[1012] Human umbilical vein endothelial cells (HUVEC) are grown to confluence in standard 96-well plates. Growth medium is removed from the cells and replaced with 90 μl 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (10 μl volume). The plates are incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Aspirate the plate to remove the medium and 100 μl of 0.1%
Paraformaldehyde-PBS (with Ca ++ and Mg ++) is added to each well. Hold the plate for 30 minutes at 4 ° C.

The fixative was then removed from the wells and the wells were PBS (+ Ca, Mg).
Wash once with + 0.5% BSA and drain. Do not dry this well. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VACM-1-Biotin
And anti-E-selectin-Biotin at a concentration of 10 μg / ml (0.1 mg / ml).
/ Ml stock antibody 1:10 dilution). The cells are incubated for 30 minutes at 37 ° C in a humidified environment. Wells with PBS (+ Ca, Mg
) Wash 3 times with + 0.5% BSA.

[1014] Then, 20 μl of diluted ExtrAvidin-Alkaline Ph
Add osphotase (1: 5,000 dilution) to each well and at 37 ° C.
Incubate for 30 minutes. Wells with PBS (+ Ca, Mg) + 0.5
Wash 3 times with% BSA. 1 tablet of p-nitrophenol phosphate (p
NPP) is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells were transferred to ExtrAvidin-Alkaline Pho in glycine buffer.
Working dilution of spotase (1: 5
000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 ). 5 μl of each dilution was added to triplicate wells and the resulting AP content in each well was 5.
50 ng, 1.74 ng, 0.55 ng, and 0.18 ng. Then 100
μl of pNPP reagent must be added to each standard well. The plate must be incubated at 37 ° C for 4 hours. 3M NaO
Add 50 μl volume of H to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to show the concentration of AP conjugate in each standard well [5.50].
ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are shown as the amount of bound AP conjugate in each sample.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

[1016] It will be appreciated that the present invention may be practiced other than as described in detail in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the appended claims.

[1017] Full disclosure of each document cited in the Background, Detailed Description and Examples (including patents, patent applications, scholarly literature, abstracts, laboratory manuals, books or other disclosures).
Are incorporated herein by reference. Further, both the hard copy of the Sequence Listing and the corresponding computer readout form, submitted with this specification, are hereby incorporated by reference in their entirety.

[1018]

[Table 6] ATCC Registration No. 203957 (Canada) Applicant is responsible for issuing a Canadian patent based on the application, or until the application is rejected or abandoned and cannot be recovered or withdrawn until the Commissioner of Patents. ) Request that only the independent expert nominated by the Secretary grant the sample of the deposited biological material referred to in the application, and the applicant Prior to the completion of the regulatory preparations for that, the International Bureau must be notified in writing.

[1019] (Norway) Applicants are required to provide samples here until the application has been published (by the Norwegian Patent Office) or has been decided by the Norwegian Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

[1020] (Australia) The Applicant hereby states that the donation of a sample of microorganisms should be made prior to the grant of the patent.
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

[1021] (Finland) The applicant hereby states that the application (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

[1022] (UK) Applicants hereby claim that microbial samples are only made available to professionals. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Registration No. 203957 (Denmark) Applicants hereby give samples to Claim that it is done only for experts. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

[1023] (Sweden) The applicant hereby considers that the provision of samples shall be the subject matter of the art until the application has been published (by the Swedish Patent Office) or has been decided by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be filed by the applicant with the International Bureau not later than 16 months from the priority date (preferably PCT Applicant's Gui).
Form PCT / RO / 13 described in Annex Z of Volume I of de
4). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts (lis) prepared by the Swedish Patent Office.
any of the persons listed in to of recognized experts),
Alternatively, it may be any person approved by the applicant in the individual case.

[1024] (Netherlands) Applicants are required to specialize in microorganisms based on the provisions of Patent Act 31F (1) until the date of issue of a Dutch patent or the date of rejection, withdrawal or lapped of an application. Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 5/14 A61P 7/06 4C084 7/02 9/10 4H045 7/06 9/14 9/10 11 / 00 9/14 11/06 11/00 13/12 11/06 17/00 13/12 17/02 17/00 19/02 17/02 21/04 19/02 25/00 21/04 25/02 25 / 00 25/14 25/02 25/16 25/14 25/18 25/16 25/22 25/18 25/24 25/22 25/28 25/24 27/00 25/28 27/02 27/00 29/00 27/02 101 29/00 31/04 101 37/00 31/04 37/04 37/00 43/00 105 37/04 107 43/00 105 C07K 14/47 107 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C12N 15/00 ZNAA 5/00 A 33 / 566 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US , UZ, VN, YU, ZA, ZW (72) Inventor Rosen, Craig Ai. United States Maryland 20882, Leightonsville, Rolling Hill Road 22400 (72) Inventor Ruben, Stephen M. United States Maryland 20882, Leightonsville, Heritage Hills Drive 18528 (72) Inventor Komat Souris, George United States Maryland 20901, Silver Spring, Garwood Street 9518 F Term (reference) 2G045 AA25 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA01 A BA44 CA01 GA11 HA12 4B063 QA05 QQ42 QQ91 4B064 AG01 AG27 CA19 CC24 DA01 DA13 4B065 AA93Y AB01 AC14 BA02 CA25 CA44 CA46 4C084 AA01 AA06 AA07 BA01 BA02 ZA251 ZA201 ZA201 ZA201 ZA201 ZA201 ZA201 ZA201 ZA201 ZA201 ZA201 ZA1 ZA 161 ZA1 ZA 175 ZA541 ZA551 ZA811 ZA891 ZA941 ZA961 ZB051 ZB071 ZB091 ZB111 ZB151 ZB221 ZC211 ZC351 ZC551 4H045 AA10 AA11 AA20 BA10 CA40 EA20 EA50 FA74

Claims (23)

[Claims] 1. An isolated nucleic acid molecule, which comprises: (a) a polynucleotide fragment of SEQ ID NO: X, or a polynucleotide fragment of the cDNA sequence contained in ATCC Deposit No. Z that is hybridizable to SEQ ID NO: X. (B) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: or a polypeptide fragment encoded by the cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X; (c) SEQ ID NO: Y; Or a polynucleotide encoding a polypeptide domain encoded by a cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X; (d) a polypeptide epitope of SEQ ID NO :, or a sequence Hybrida on number X Polynucleotide encoding the polypeptide epitope encoded by the cDNA sequence contained in ATCC Deposit No. Z; (e) a polynucleotide encoding a polypeptide of SEQ ID NO: Y having biological activity, or a sequence CDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to number X; (f) Polynucleotide that is a variant of SEQ ID NO: X; (g) Polynucleotide that is an allelic variant of SEQ ID NO: X; (h) A polynucleotide encoding a species homologue of SEQ ID NO: (i) a polynucleotide capable of hybridizing to any one of the polynucleotides specified in (a) to (h) under stringent conditions, Here, the polynucleotide has a nucleotide sequence consisting of only A residues or T residues. The nucleic acid molecule does not hybridize under stringent conditions, a polynucleotide, comprising a polynucleotide having at least 95% identical to the nucleotide sequence to a sequence selected from the group consisting of isolated nucleic acid molecules. 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. 3. The polynucleotide fragment comprises a sequence identified as SEQ ID NO: Y, or a nucleotide sequence encoding a polypeptide encoded by the cDNA sequence contained in ATCC Deposit No. Z, which is capable of hybridizing to SEQ ID NO: X. 2. The isolated nucleic acid molecule of claim 1, which comprises. 4. The isolated of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: X, or the cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X. Nucleic acid molecule. 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises consecutive 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 consecutive 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, which comprises a vector sequence. 11. An isolated polypeptide comprising: (a) a polypeptide fragment of the encoded sequence contained in SEQ ID NO: Y, or ATCC Deposit No. Z; (b) a biological activity. (C) a polypeptide domain of the encoded sequence contained in SEQ ID NO: Y, or ATCC Deposit No. Z; (c) a polypeptide domain of the encoded sequence contained in SEQ ID NO: Y, or ATCC Deposit No. Z; d) a polypeptide epitope of the encoded sequence contained in SEQ ID NO: Y, or ATCC deposit number Z; (e) a secreted form of the encoded sequence contained in SEQ ID NO: Y, or ATCC deposit number Z; ) SEQ ID NO: Y, or the full length protein of the encoded sequence contained in ATCC Deposit No. Z; (g) a variant of SEQ ID NO: Y (h) Allelic variants of SEQ ID NO: Y; or (i) SEQ ID NO: Y of species homologs, including at least 95% identical to the amino acid sequence to a sequence selected from the group consisting of,
Isolated polypeptide. 12. The isolated polypeptide of claim 11, wherein the secreted form or the full-length protein comprises a contiguous amino acid deletion from either the C-terminus or the N-terminus. 13. An isolated antibody that specifically binds to the isolated polypeptide of claim 11. 14. Expressing the isolated polypeptide of claim 11.
Recombinant host cell. 15. A method for producing an isolated polypeptide, comprising the step of: (a) culturing the recombinant host cell according to claim 14 under conditions such that the polypeptide is expressed; And (b) recovering the polypeptide. 16. A polypeptide produced by the method of claim 15. 17. A method of preventing, treating or alleviating a medical condition, comprising:
13. A method 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 for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising the step of: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1. And (b) diagnosing a pathological condition, or susceptibility to a pathological condition, based on the presence or absence of the mutation. 19. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) the presence or amount of expression of the polypeptide of claim 11 in a biological sample. And (b) diagnosing a pathological condition, or susceptibility to a pathological condition, based on the presence or amount of expression of the polypeptide. 20. A method of identifying a binding partner for the polypeptide of claim 11, comprising: (a) contacting the polypeptide of claim 11 with a binding partner;
And (b) determining whether the binding partner results in activity of the polypeptide. 21. A gene corresponding to the cDNA sequence of SEQ ID NO: Y. 22. A method of identifying activity in a biological assay, comprising:
Wherein the method comprises: (a) expressing SEQ ID NO: X in cells; (b) isolating the supernatant; (c) detecting activity in a biological assay; and (d) ) Identifying a protein in the supernatant that has the activity. 23. A product produced by the method of claim 20.