JP2003521251A - Methods and materials related to CUB domain polypeptides and polynucleotides - Google Patents

Abstract

  (57) [Summary] The present invention provides polynucleotides corresponding to novel human secretory CUB domain polypeptides and novel polynucleotides and polypeptides encoded by mutants or variants thereof. It is. These polynucleotides consist of a nucleic acid sequence (Hyseq clone recognition number 2924342 (SEQ ID NO: 1)) isolated from a testis cDNA library. Other features of the invention include novel human secreted CUB domain polypeptides and vectors involved in the process of generating antibodies specific for such polypeptides.

Description

Detailed Description of the Invention

1. TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with the uses of those polynucleotides and proteins, particularly in the therapeutic, diagnostic and research fields. It is a thing. The present invention particularly relates to novel CUB domain polypeptides.

2. BACKGROUND OF THE INVENTION Identified polynucleotide and polypeptide sequences can be used, for example, to identify mutations responsible for diagnostics, forensics, genetic mapping, genetic diseases or other characteristics, and many other types of DNA and amino acid sequence dependent sequences. It has many uses, such as creating data and products. A protein has biological activity, for example, by its secretory nature in leader sequence cloning, by its cell or tissue source in a PCR-based approach, or by structural similarity to other genes with known biological activity. It has been known. The present invention relates to encoding polynucleotides and polypeptides. The present invention particularly relates to novel CUB domain polypeptides and polynucleotides.

Receptor / ligand relationships are crucial in many processes, including cell adhesion, communication, development, immune response and the like. Recently, it was proved that the CUB domain has such a ligand recognition function. The CUB domain was initially Complement
(Complement) component, Sea Urchin protein Uegf, and BM
It was so named because it was reported in P-1 (Li et al. (1996) Proc.
Natl. Acad. Sci. USA 93, 5127-5130).
The CUB domain is present in various proteins ranging from complement components and developmental proteins to spermadhesin proteins and renal filtration proteins. C
UB appears to provide an important function in the reaction between proteins. It may be involved in the regulation of ligand recognition and receptor activity. Bone morphogenetic protein-
Both 1 (BMP-1) and spermadhesin have CUB.

Bone morphogenetic proteins (BMPs) are composed of a family of polypeptides that have been shown to induce bone and cartilage growth (Reddi, AH (19
98) Clin Orthop 355S, 566-72). BMP-1 is bone /
It is a cartilage growth promoter and is used as an experimental model for the treatment of osteoporosis or for repair of articular cartilage disease. BMP-1 is identical to the procollagen (precollagen) C proteinase and is a homologue of the Drosophila growth protein toroid (Sieron et al. (2000) Biochemistry 393).
231-3239). BMP-1 is a zinc-requiring astatine family endopeptidase, a large amino-terminal endopeptidase domain, an epidermal growth factor (EGF) -like domain, and three CUs.
It has a B domain.

Astatin family member proteins have been shown to play important roles in embryonic hatching, dorsal / ventral patterning, and early developmental decisions.
Alternative splicing of transcripts of the pre-collagen C proteinase produces three types of enzymes: 1) 100 kDa mammalian toroid protein or C proteinase, 2) 80 kDa splice variant, and 3) 70 kDa BMP-1.
To code. BMP-1 lacks the CUB4 and CUB5 domains present in the 100 kDa C proteinase splicing variant. BMP
The -1 alternative splice isoform is differentially expressed in embryonic and adult tissues. Transgenic mice without the BMP-1 allele are stillborn due to an intestinal hernia displaying barbed filamentous collagen fibrils. It is implied that the process of pre-collagen is incomplete. In Drosophila, the toroid protein disrupts the cordin analog SOG (short gastulation), releasing BMP-4, an essential component for dorsal / ventral patterning.

The enzymatic activities of all three C proteinase isoforms have been shown to be identical. But functionally, they cannot replace each other. This indicates that mutant CUB domains and / or EGF-like domains confer isoform substrate specificity (Sieron et al. (2000) Bioch.
emissory 39, 3231-3239). BMP-1 or 100kD
The authors showed that the a mutant binds to pre-collagen 1, but CUB domains 4 and 5 cannot. So the first three CUB domains of BMP-1 are:
It provides the protein-protein interactions necessary to initiate the activity of proteolytic enzymes.

[0007] Spermadhesin is a family of proteins expressed in the male reproductive tract (Topfer-Petersen E et al. (19
98) Andrologia 30, 217-224). Spamadhesin is a protein of approximately 13 kDa and is composed of a single CUB domain. It is a multifunctional protein that can bind to carbohydrates, proteoglycans, phospholipids, and protease inhibitors and is involved in various stages of fertilization. During ejaculation, spermadhesin forms a protective membrane around the sperm head. It may later function as the first receptor for the oocyte zona pellucida, contributing to initiation and recognition of sperm-egg binding.

Accordingly, the CUB domain polypeptides and polynucleotides of the invention are useful in treating fractures or bone growth, assisting cartilage repair treatment, controlling osteonecrosis, bone / bone
It may be used to control cartilage tumor growth, regulate collagen formation, and regulate growth and development of other tissues or organs. CUB domain polypeptides and polynucleotides could also be used to protect the sperm head and provide induction cues during artificial insemination and in vitro fertilization.

3. SUMMARY OF THE INVENTION The present invention provides novel CUB domain polypeptides, novel isolated polynucleotides encoding such peptides, recombinant DNA molecules, cloned genes as included therein. Or for the discovery of allelic variants thereof, antisense polynucleotide molecules, and antibodies specifically recognizing one or more epitopes present on those polypeptides, as well as hybridomas (hybrid cells) that produce these antibodies. Is based. In particular, the polynucleotide of the present invention is a CUB domain polynucleotide (Hyseq clone recognition number 2 isolated from a cDNA library generated from human ovary).
924342 (SEQ ID NO: 1)).

The composition of the invention further comprises a vector, such as an expression vector, which comprises a polynucleotide according to the invention, a genetically engineered cell containing such a polynucleotide, and such a polynucleotide. It includes cells that have been genetically engineered to express.

The compositions of the present invention provide isolated polynucleotides, including but not limited to: That is, a polynucleotide consisting of the nucleotides designated as SEQ ID NO: 1-3, 5, or 12; or SEQ I
D NO: 1-3, 5 or 12 fragments; SEQ ID NO: 1-3, 5
, Or a polynucleotide consisting of 12 full-length coding sequences (eg,
SEQ ID NO: 4); and any SEQ ID NO: 1-3, 5, or 12 of the mature protein coding sequence. The polynucleotides of the present invention further provide polynucleotides including, but not limited to: That is, (a) SEQ I
D NO: 1-3, 5, or the complement of any nucleotide sequence designated 12; (b) Severe hybridization to a nucleotide sequence encoding either SEQ ID NO: 4 or 6-8 (hybrid Forming) hybridizing (hybridizing) under conditions; an allelic variant of any of the above-mentioned polynucleotides, having a sequence identity of 70% or more to the polynucleotide; any of the above A polynucleotide encoding a homologue (eg, ortholog) of the peptide of SEQ ID NO: 4;

The collection used for this purpose may be a collection of single polynucleotides. A collection of sequence information or unique identifying information for each sequence may be arranged on the sequence of the nucleic acid. In one embodiment, each segment of sequence information is located on the sequence of the nucleic acid to detect the polynucleotide containing that segment. The array can also be configured to detect a perfect match or mismatch to the polynucleotide containing the segment. The collection can also be in a computer-readable format.

The present invention further provides a cloning or expression vector containing at least a fragment of the polynucleotide having the above-described constitution, and a host cell or tissue transformed with the expression vector. Among the useful vectors are plasmids, cosmids, lambda phage variants, phagemids, etc. well known in the art.
Therefore, the present invention provides a vector containing the polynucleotide of the present invention and a host cell containing the polynucleotide. In general, the vector contains at least one origin of replication in the body and contains convenient restriction endonuclease sites and a selectable marker for the host cell. Vectors of the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. The host cells of the invention are prokaryotic or eukaryotic cells, unicellular or multicellular organisms.

The present invention is composed of polypeptides, including but not limited to isolated polypeptides selected from the group comprising the amino acid sequences of SEQ ID NO: 4 or 6-8. The polypeptide of the present invention has (a) SEQ ID NO: 1.
Any polynucleotide having a nucleotide defined in -3, 5, or 12; or (b) hybridizes as a complement of (a) the polynucleotide of (a) under stringent hybridization (hybridization) conditions. A polypeptide having biological activity encoded by any of the above-mentioned polypeptides. SE
Also included in consideration are biological or immunologically active variants of any of the protein sequences listed in Q ID NO: 4 or 6-8, as well as those that retain biologically or immunologically active and approximately equivalent thereto. The polypeptide of the present invention may be chemically synthesized in whole or in part, but the genetically engineered cell of the present invention (eg, host cell)
It is desirable that it is produced by genetic recombination using

The invention also provides compositions comprising the polypeptides of the invention. The pharmaceutical composition of the present invention may include a hydrophilic carrier, that is, a pharmaceutically acceptable carrier, in addition to the polypeptide of the present invention.

The invention also provides a host comprising an expression vector comprising at least a fragment of a polynucleotide encoding a polypeptide of the invention in a suitable culture medium under conditions which allow expression of the desired polypeptide. It relates to a method of producing a polypeptide of the invention by culturing cells and purifying the protein or peptide from the culture or host cells. Preferred embodiments include where the protein made by such a process is a mature protein.

The polynucleotide according to the present invention can be applied to various techniques known to molecular biologists in the art. These techniques include application to hybridization (hybridization) probes, application to oligomers and primers, application to PCR, application to sequences, application to computer-readable media, application to mapping of chromosomes and genes. , Application of proteins to genetically engineered products, and production of antisense DNA or RNA, its chemical analogs, and the like. For example, the polynucleotides of the invention can be used as hybridization probes to detect the presence of mRNA of a particular cell or tissue in a sample using, for example, in situ hybridization (hybridization). .

In another embodiment, as an expressed sequence tag for the identification of expressed genes, and
Well known to those skilled in the art, Volras et al. Science 258: 52-
59 (1992), the polynucleotide can be used diagnostically as an expressed sequence tag for physical mapping of the human genome.

The polynucleotide according to the present invention can also be used in ordinary procedures and methods currently applied to other proteins. For example, the polynucleotides of the invention can be used to generate antibodies that specifically bind the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting and quantifying polypeptides in tissues. The polypeptide of the present invention can be used as a molecular weight marker and as a food supplement.

Also provided are various methods of preventing, treating, or ameliorating a medical condition comprising administering to a subject mammal a therapeutically effective amount of a peptide of the invention and a pharmaceutically acceptable carrier. .

In particular, the polypeptides and polynucleotides of the present invention are useful for treating fractures or bone growth, assisting cartilage repair treatment, controlling osteonecrosis, controlling bone / cartilage tumor growth, regulating collagen formation, and others. It may be used to regulate the growth and development of tissues or organs. The polypeptides and polynucleotides of the present invention include
It may also be used during artificial insemination and in vitro fertilization to protect the sperm head and provide guidance cues.

The method of the present invention also indicates the effective dose of the composition comprising the polynucleotide or polypeptide of the present invention and a pharmaceutically acceptable carrier, as a symptom or sign of a disease as described herein. It also provides a method for treating a disease, which comprises administering to a test mammal. Further, the invention includes methods of treatment for diseases or disorders as described in this application. It also includes the step of administering a compound consisting of a composition or other substance that modulates the overall activity of the target gene construct and the pharmaceutically acceptable carrier. Compositions and other agents can affect the level of target gene / protein expression or such modulation of the target protein activity. Specifically, the effective dose of a composition comprising the polypeptide of the present invention or the binding agent of the CUB domain polypeptide of the present invention (for example, an antibody having particularly reactivity) is not limited to humans, including mammals including humans. Provided is a method for the prevention, treatment or amelioration of medical conditions, including viral diseases, which comprises administering to an animal. For individuals in need of treatment, whether the polypeptide according to the invention or its binding agent (or inhibitor) is beneficial to the individual in need of treatment will depend on the mechanism of the particular condition or pathology. Dependent.

According to this method, the polypeptides of the present invention can be administered to cause inhibition of cell function in vitro or in vivo. Certain polypeptides of the present invention can be administered alone for in vivo use or as an adjunct to other therapeutic modalities.
On the other hand, the protein or other active ingredient of the present invention can be included in a preparation of a specific antihistamine or anti-inflammatory agent to reduce the side effects of the antihistamine or anti-inflammatory agent.

[0024]   The present invention further provides methods of pharmaceuticals useful in the above methods.

The invention further relates to a method of detecting the presence of a polynucleotide or polypeptide of the invention in a sample (eg tissue or sample). Such methods can be utilized, for example, as part of the prognosis and diagnostic evaluation of diseases as described herein, and in identifying subjects who are predisposed to such conditions.

The present invention provides a method for detecting the presence of a polypeptide of the present invention in a sample, which is bound to the polypeptide for a sufficient period under conditions sufficient to form the complex. A method is provided in which a sample is contacted with a compound that forms a complex, and the formation of the complex is detected, thereby detecting the polypeptide when the complex is formed.

The present invention also provides a kit of polynucleotide probes and / or monoclonals, and optionally, quantitative standards, for practicing the methods of the present invention. The invention further provides a method of assessing the efficacy of a drug and monitoring the progress of a patient in making clinical attempts to treat the above mentioned diseases.

The invention also provides methods for identifying compounds that modulate (eg, increase or decrease) the expression or activity of the polynucleotides and / or polypeptides of the invention. Such methods can also be used, for example, in identifying compounds that can reduce the symptoms of the disease, as described above. Such methods also include assays for compounds and other substances that interact with the polypeptides of the invention.

The present invention provides a method for identifying a compound that binds to the polypeptide of the present invention, wherein the polypeptide is contacted with the polypeptide for a sufficient period of time under conditions sufficient to form a complex. A method of detecting a compound that binds to a polypeptide by forming a complex of the compound and detecting the complex of the polypeptide and the compound by detecting the complex is provided.

Also, the compound is contacted with the polypeptide in the cell for a time sufficient to form a complex of the polypeptide and the compound, thereby allowing the compound to express a reporter gene sequence and directing reporter gene sequence expression. Also provided is a method of identifying a compound that binds to a polypeptide, the method comprising detecting the compound by detecting, and determining that a compound that binds to the polypeptide is detected when a complex of the polypeptide and the compound is detected. .

5. DETAILED DESCRIPTION OF THE INVENTION The CUB domain polypeptide of SEQ ID NO: 4 is a secretory protein of about 110 amino acids and has a predicted molecular weight of about 12 kDa unglycosylated. BLA
Protein database search using STX algorithm (Altschul, S
． F. J. et al. Mol. Evol. 36: 290-300 (1993).
And Altschul, S .; F. J. et al. Mol. Biol. 21
: 403-10 (1990) incorporated herein by reference), SEQ ID NO: 4 is homologous to the bone morphogenetic protein 1 (BMP-1) CUB domain. GeneAtlas software (Molecular Simul
ations, Inc. , San Diego, Calif.), SEQ ID NO: 4 is homologous to spermadhesin protein and seminal plasma protein.

FIG. 1 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) A protein encoded by the CUB domain polypeptide and Afri
Figure 3 shows a BLASTX amino acid sequence alignment between the bone morphogenetic protein 1 (BMP-1) precursor SEQ ID NO: 9 of can Clawed Frog (Xenopus laevis), both sequences showing SEQ ID NO: 4 of 11
It shows 57% similarity for two amino acid residues and 40% identity for the same 112 amino acid residues of SEQ ID NO: 4. FIG. 2 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) A protein encoded by a CUB domain polypeptide and human B
Figure 3 shows a BLASTX amino acid sequence alignment with MP-1 CUB2 domain SEQ ID NO: 10, both sequences having 54% similarity for 108 amino acid residues of SEQ ID NO: 4, SEQ ID NO: 4
It shows that 43% of the same 108 amino acid residues of are identical.

FIG. 3 shows that SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) MSI GeneAtla between the protein encoded by the CUB domain polypeptide and bovine spermadhesin SEQ ID NO: 11.
s amino acid sequence alignments, both sequences having SEQ ID NO:
It has 40.4% similarity and 19.2% identity for 4 identical residues. PSI-BLAST has an e value of 4.2e-17, protein database ID number entry = 1sfp (Research Collaborat
ory for Structural Bioinformatics. h
ttp: // www. rcsb. org / pbd), confirmation score = 0.42, S
Present in residues 1-106 of EQ ID NO: 4.

GeneAt, Molecular Simulations, Inc.
las software (Molecular Simulations, Inc.,
CIB domain polypeptide SEQ ID NO: 4 has a region with a motif characteristic of the major seminal plasma glycoprotein of sus scrofa molecule in residues 1-103 by using San Diego, CA). Was confirmed.
PSI-BLAST has an e value of 1.7e-15, protein database ID number entry = 1 sppB (Research Collaboratory for)
Structural Bioinformatics. http: /// w
w. rcsb. org / pbd), confirmation score = 0.24, SEQ ID N
O: Present at residues 1-103 of 4.

GeneAt, Molecular Simulations, Inc.
las software (Molecular Simulations, Inc.,
It was confirmed that the CUB domain polypeptide SEQ ID NO: 4 has a region at residues 2-107 having a motif characteristic of sus scrofa seminal plasma protein by using San Diego, CA). It was PSI
-E value of BLAST is 1.7e-15, protein database ID number entry = 1spp (Research Collaboratory for Str)
Structural Bioinformatics. http: // www. rc
sb. org / pbd), P value = 4.7e-02, present in residues 2-107 of SEQ ID NO: 4.

The predicted signal peptide of about 15 residues has SEQ ID NO: 4 (
It is encoded by residues 1 to 15 of SEQ ID NO: 6). Extracellular potions are effective by themselves. This can be confirmed by mammalian cell expression and degradation product sequencing. Signal peptide site is Kyte-Do
little hydrophobicity prediction algorithm (J. Mol Biol. 155,
pp. 105-31 (982), incorporated herein by reference). Those skilled in the art will appreciate that the actual split site may be different than that predicted by the computer program. In particular, the polypeptides and polynucleotides of the present invention are useful for treating fractures or bone growth, assisting cartilage repair treatment, controlling osteonecrosis, controlling bone / cartilage tumor growth, regulating collagen formation, other tissues or organs. Could be used to regulate growth and development of The polypeptides and polynucleotides of the invention may also be used during artificial insemination and in vitro fertilization to protect the sperm head and provide induction cues.

5.1 Definitions In this specification and the appended claims, the singular forms “a”, “an” and “t”.
“He” includes a plurality of cases unless there is a clear difference in the text.

The term “active” refers to a form of a polypeptide that has biological and / or immunological activity of a naturally occurring polypeptide. According to the invention, "biological activity" or "biological activity" refers to a protein or peptide that has a naturally occurring structural, regulatory or biochemical function.

Similarly, “biological activity” or “biological activity” refers to a natural, recombinant (recombinant) or synthetic CUB domain peptide, or any of those peptides, that is biologically specific to the appropriate animal or cell. Means to elicit a response and bind to a particular antibody. By "CUB domain biological activity" is meant a biological activity similar to that of the CUB domain.

The term “activated cell”, as used herein, refers to those cells that enter or exit the intracellular or extracellular membranes, such as secretory molecular cells as part of a normal or disease process. Includes export of enzyme molecules.

“Complementary” or “complementarity” means the natural binding of polynucleotides by base pairing. For example, the 5'-AGT-3 'sequence is the complementary sequence 3'-TCA-
Combine with 5 '. Complementarity between two single-stranded molecules is either "partial" complement to which some nucleic acids bind, or "complete" complementarity such that total complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization (hybridization) between nucleic acid strands.

The term “embryonic stem cell (ES)” refers to a cell that can give rise to many differentiated cell types in the fetus or adult, including germ cells. "Germline stem cells (GS
“C)” refers to a stem cell derived from a primordial germ cell, which stably and continuously supplies the embryo cell for the production of a reproductive body. “Primordial germ cells (PGCs) are a small group of small cells separated during embryogenesis from other cell lines that have the ability to differentiate into germ cells and other cells, especially the yolk sac, mesentery, or gonad ridge. Refers to cells, PGCs are a source involved in the generation of GSCs and ES cells, and PGCs, GSCs, and ES cells have a self-renewal function, and therefore these cells proliferate the germ line and form adult special organs. Not only do they generate multiple permanent differentiated cells, but they also regenerate themselves.

The term “expression modulatory fragment”, EMF, refers to nucleotides that modulate the expression of an operably associated ORF or other EMF.

As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of EMF. EMFs include, but are not limited to, promoters and sequences that modulate promoters (inducers). One class of EMFs are nucleic acid fragments that induce the expression of an operably linked ORF in response to specific regulatory factors or physiological events.

“Nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonucleotide” are terms used interchangeably and refer to a nucleotide or a heteromultimer of these nucleotide sequences. These terms also refer to genomic or synthetic DNA or RNA, peptide nucleic acid (PNA) or any DNA that may be genomic or single-stranded or double-stranded, and may also be significant or antisense.
-Like or RNA-like substance. When the polynucleotide is RNA, it is believed that T (thymine) in the sequences described in this application can be replaced by U (uracil). Generally, the nucleic acid segments provided by the invention are assembled from fragments of genomic and short oligonucleotide linkers, or from sequences of oligonucleotides, or from individual nucleotides, derived from microbial or viral operons, or eukaryotic genes. A synthetic nucleic acid expressed in a recombinant transcription unit comprising regulatory elements of

“Oligonucleotide fragment” or “polynucleotide fragment”, “portion” or “segment” or “probe” or “primer” are used interchangeably and at least about 5 Of at least about 7 nucleotides, and preferably at least about 7 nucleotides, and most preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides, and most preferably at least about 17 nucleotides. Refers to a string of nucleotide residues. A fragment is less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably about 10 nucleotides.
Refers to those consisting of no more than 0 nucleotides, more preferably no more than 50 nucleotides, and most preferably less than about 30 nucleotides. The probe is
Desirably consisting of about 6 to about 200 nucleotides, more preferably about 15 to about 50 nucleotides, more preferably 17 to 30 nucleotides, and most preferably 20 to 25 nucleotides. Refers to. The fragments are preferably used to identify or amplify similar or related portions of mRNA or DNA molecules in the polymerase chain reaction (PCR), various mating processes or microarray processes. Fragments or segments can unambiguously identify each polynucleotide sequence of the present invention. Fragment is SEQ ID NO: 1
It is desirable to have a sequence that is approximately similar to -3, 5, or 12 portions.

Probes are used, for example, to determine if a particular mRNA molecule is present in cells or tissues, or to separate similar nucleic acid sequences from the chromosomal DNA described by Walsh et al. (Walsh, PS, et al., 1992.
, PCR Methods Appl 1: 241-250) They can be labeled by methods well known to those skilled in the art such as nick translation, Klenow-fill-in reaction, and PCR. The probes of the invention, their preparation and / or labeling are described by Sambrook, J. et al. By 1989 A Mol
Equal Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, NY; or Auchbell, F .; M. Et al., 1989, Current Protoco
ls in Molecular Biology, John Willley
& Sons, New York, NY, both of which are incorporated herein by reference.

The nucleic acid sequences of the present invention also include information for any nucleic acid sequence of SEQ ID NO: 1-3, 5, or 12. Sequence information is SEQ ID NO: 1-3, 5
, Or 12 segments, SEQ ID NO: 1-3, 5
, Or 12 sequence information. Such a segment could also be a 20mer nucleic acid because the probability of a perfect 20mer match in the human genome is 3
This is because it is 1/00. For the human genome, there are 3 billion base pairs in a set of chromosomes. 4 because not 20 mer ²⁰ may be present, so that a set number of base pairs present in the chromosomes of human 300 times more 20-mers are present. Using the same analysis, the probability of a 17-mer being a perfect match in the human genome is one-fifth. If these segments are used as arrays in expression studies, 15-mer segments can be used. Similarly, the probability of a perfect match in the expressed sequence for the 15-mer is about 5% of the total genomic sequence for the expressed sequence.
Since it is composed of less than or equal to%, it is almost one fifth.

Similarly, in using sequence information to detect single mismatches, one segment is a 25-mer. The probability that a 25-mer will appear in the human genome with only one mismatch is calculated by multiplying the probability of a perfect match (1 ÷ 425) by the increased probability of mismatch at each nucleotide position (3 × ²⁵ ). The probability of being detected with one array of 18-mers with one mismatch is approximately one-fifth. 1
The probability of detecting a 20-mer with one mismatch in the human genome is approximately one-fifth.

The term “open reading frame”, ORF, refers to a series of nucleotide triplets to amino acids that do not have a stop codon, and is a sequence transcribable into a protein.

“Operably linked” or “operably associated” refers to nucleic acid sequences that are operably associated. For example, a promoter is operably linked to, and operably associated with, a coding sequence when it controls the transcription of the coding sequence. When the operably linked nucleic acid sequences are contiguous and in the same reading frame, a genetic element, eg, a regulatory gene, is not contiguously linked to the coding sequence but does not
You can control the translation.

The “pull reporter (pluripotent)” means a cell capable of differentiating into various kinds of differentiated cells existing in an adult organ. Pluripotent cells have a limited differentiation potential compared to totipotent cells.

“Polypeptide” or “peptide” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide or protein sequence or fragments thereof, both naturally occurring and synthetic. A "fragment,""portion," or "segment" of a polypeptide is a stretch of a series of amino acid residues of at least 5 amino acids, preferably at least about 7 amino acids, more preferably about 17 or more amino acids. Is. Peptides are desirably no more than 200 amino acids, more desirably no more than 150 amino acids, and most desirably no more than 100 amino acids. Desirably, the peptide is about 5 to 200 amino acids. To be active, a polypeptide must be of sufficient length to exhibit biological and / or immunological activity.

The term “naturally-occurring polypeptide” refers to a polypeptide produced by a cell that has not been genetically engineered to produce, and in particular is acetylated, carboxylated, glycosylated, phosphorylated, lipid, It means various polypeptides resulting from post-translational modification of a polypeptide including, but not limited to acetylation, acylation and the like.

The term “translated protein coding sequence” refers to a sequence that encodes a full-length protein and includes any leader or process sequences.

“Mature protein coding sequence” refers to a sequence that encodes a peptide or protein without a leader / signal [information] sequence. The peptide has a leader sequence that has been removed during intracellular processing, or the protein has a leader sequence that has been synthesized or generated using a polynucleotide that has only the mature protein coding sequence. Sometimes.

“Derivative” refers to ubiquitin conjugation, labeling (eg, with respect to radionuclides or various enzymes) covalent polymer attachment, eg pegylation (derivatization with polyethylene glycol) and chemical synthesis of amino acids such as ornithine. A polypeptide that has been chemically modified using such techniques as insertions or substitutions and is one that does not naturally occur in human proteins.

The term “variant” (or “analog”) differs from naturally-occurring polypeptides with the insertion, deletion, and substitution of amino acids, eg, recombinant D
Refers to those generated using the NA method. A guide for deciding which amino acid residues to replace, add or remove without killing the activity of interest is to compare the sequence of a particular peptide with that of a homologous peptide, A) The number of amino acid sequence changes that occurred in (1) can be minimized or replaced by an amino acid having a common sequence.

Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected by making use of the "redundancy" in the genetic information. . Various codon substitutions, eg, silent changes that create various restriction sites, can be introduced into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system to optimize cloning. The properties of any part of the polypeptide are modified to reflect the domains of the polypeptide or other peptides added to it in the polynucleotide sequence, such as ligand binding affinity, interchain affinity, or degradation or metabolism. Characteristics such as turnover change.

Amino acid “substitutions” are the result of replacing one amino acid with another amino acid that has similar structural and / or chemical properties, such as a traditional amino acid alternator. “Conservative” amino acid substitutions are made on bases that have polar, charge, soluble, hydrophobic, and / or amphiphilic similarities of related residues. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine, and neutral polar amino acids include glycine, serine, threonine, cystine, tyrosine, asparagine, and glutamine. Including, positively charged (basic) amino acids include algin, lysine and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertion" or "removal" ranges from about 1 to 20 amino acids, more preferably 1 to 10 amino acids. A given change is determined empirically by systematically inserting, removing, or exchanging amino acids in the polypeptide molecule, using recombinant DNA techniques, and assaying the resulting recombinant variants.

Alternatively, where altered function is desired, modified polypeptides are engineered by insertion, removal or non-conservative alterations. Such alterations are possible, for example, by altering one or more biological function or biochemical properties of the polypeptide of the invention. For example, their alterations alter polypeptide properties such as ligand binding affinity and interchain affinity, or degradation / turnover rate. Moreover, such alterations can be chosen to generate a more suitable polypeptide for expression, scale-up, etc. of the host cell chosen for expression. For example, cysteine residues can be removed or replaced with other amino acid residues to eliminate the disulfide bridge.

The term “purified” or “substantially purified” means the presence of a specified nucleic acid or polynucleotide in the absence of other biopolymers, eg, polynucleotides, proteins and the like. In some embodiments, the polynucleotides or polypeptides are purified to greater than 95% by weight of the designated biopolymer, more preferably greater than 99% by weight (provided that water, buffers, and other Molecules of less than 1000 Daltons may be present).

By “separation” is meant separating a nucleic acid or polypeptide from at least one other component (eg, nucleic acid or polypeptide) present in the nucleic acid or polypeptide obtained from its natural source. . In one example, the nucleic acid or polypeptide is only present (if any) in the presence of solvent, buffer, iron, or other component normally present in the solution. The term "separation" or "purification" is not used for nucleic acids or polypeptides present in natural sources.

The term “recombinant” as used herein in reference to a polypeptide or protein refers to a polypeptide obtained from a recombinant (eg, microbial, insect, or mammalian) expression system. Or it means a protein.
“Microorganism” refers to a recombinant polypeptide or protein produced in a bacterial or fungal (eg, yeast) expression system. By product is meant a polypeptide or protein that is free of natural endogenous substances as a "recombinant microorganism" and has no associated natural glycosylation. For example, E. There are no glycosylation modifications in polypeptides or proteins expressed in most bacterial cultures such as E. coli. Polypeptides and proteins expressed in yeast generally have glycosylated forms that differ from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector for expressing a polypeptide from a DAN (RNA) sequence. The expression medium is (1) a genetic element having a regulatory role in gene expression, such as a promoter or an enhancer, (2) a structural or coding sequence transcribed into mRNA and translated into a protein, and (3) appropriate. It may consist of a transcription unit consisting of a combination with different transcription start and stop sequences. Structural units of interest for use in yeast or eukaryotic expression systems desirably include a leader sequence that enables extracellular secretion of the transcribed protein by the host cell. It may also contain an amino terminal methionine residue if the recombinant protein is expressed without a leader or transport sequence. Residues may or may not be subsequently cleaved from the expressed recombinant protein to obtain the final product.

By “recombinant expression system” is meant a host cell in which a recombinant transcription unit has been stably integrated into chromosomal DNA or which carries the recombinant transcription unit extrachromosomally. A recombinant expression system as defined herein is one that expresses a heterologous polypeptide or protein upon the introduction of regulatory elements linked to the DNA segment or synthetic gene to be expressed. The term also refers to host cells which are stably combined with recombinant genetic elements that play a regulatory role in gene expression, such as promoters and enhancers. A recombinant expression system as defined herein expresses a polypeptide or protein endogenous to a cell by the introduction of regulatory elements linked to the DNA segment or synthetic gene to be expressed. The cell may be either prokaryotic or eukaryotic.

A “secreted” protein is a protein that is transported across or across a membrane, including when delivered as a result of a signal sequence in an amino acid sequence when it is expressed in a suitable host cell. The "secreted protein" includes all proteins secreted completely (eg, soluble protein) or partially from the cells in which it is expressed. "Secreted protein" includes proteins that are transported across the membrane of the endoplasmic reticulum. "Secreted protein" also refers to proteins that contain atypical signal sequences (eg, interleukin-1 beta, Krasney, PA and Young, PR (1.
962) Cytokine 4 (2): 134-143) and factors released from disrupted cells (eg, interleukin-1 receptor / antagonist,
Arend, W. P. Et al. (1998) Annu. Rev. Immunol
． 16: 27-55). If necessary, the expression vector may be designed to contain a "signal or leader sequence" that directs the polypeptide through the cell's membrane. Such sequences may be provided by recombinant DNA by techniques, from heterologous protein sources, or naturally occurring in the polypeptides of the invention.

The term “severe” is used in the sense of stringent, as is commonly understood by those of skill in the art. For severe conditions, extremely stringent conditions (for example, hybridization of filter-immobilized DNA with 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C. (hybridization), 6
8 ° C 0.1X SSC / 0.1% SDS) and moderately harsh conditions (eg 42 ° C 0.2X SSC / 0.1% SDS). Other hybridization (hybridization) conditions are explained by examples in the text.

For deoxyoligonucleotide hybridization (hybridization),
Other hybridization conditions include 37 ° C (for 14-base oligonucleotide), 48 ° C (for 17-base oligonucleotide), 55 ° C (for
Wash with 6X SSC / 0.05% sodium pyrophosphate at 20 bases oligonucleotide) and 60 ° C (for 23 bases oligonucleotide).

The term “approximately equivalent” or “approximately the same” is used for both nucleotide and amino acid sequences, for example, having one or more substitutions, removals or additions from the reference sequence, which results in the reference sequence Refers to mutant sequences that do not result in a functionally unfavorable difference when compared to. Usually, such near equivalent sequences will have 35% or more of those listed herein (ie, of the residues that have been replaced, added, and / or removed in the approximately equivalent sequence relative to the reference sequence). The value obtained by dividing the number by the total number of residues in the sequence that is almost equivalent is 0.35 or more). Such sequences are said to have 65% sequence identity to the listed sequences.

In some embodiments, a substantially equivalent, eg, variant sequence of the invention does not differ by more than 30% (70% of the sequences are identical) compared to the listed sequences; certain variants of this example include: No more than 25% difference (75% sequence identity); no more than 20% difference in another variant of this example (80% sequence identity); 10% in yet another variation of this example No difference (90% of the sequences are the same); 5% or more (95% of the sequences are the same) in another mutation of this example. Nearly equivalent, eg, mutant amino acid sequences according to the present invention preferably have at least 80% sequence identity, more preferably at least 85% sequence identity, and more preferably 90%, as compared to the listed amino acid sequences. It is even more desirable to have a% sequence identity. More desirable is at least 95% sequence identity, even more desirable is at least 98% sequence identity, and most desirable is at least 99% sequence identity. Nearly equivalent nucleotide sequences of the present invention may have a lower percentage of sequence identity, taking into account the redundancy or degeneracy of the genetic code. Nucleotide sequences should have a sequence identity of at least 65%, preferably a sequence identity of at least 75%, a sequence identity of at least 80%, a sequence identity of at least 85%. More desirable is at least 90% sequence identity, more desirable is at least 95% sequence identity, at least 98% sequence identity is even more desirable, and at least 99% sequence identity is most desirable. For purposes of the present invention, sequences that have approximately the same biological activity and approximately the same expression characteristics are considered to be substantially equivalent. For purposes of determining identity, cleavage of the mature sequence (eg, via a mutation that creates a pseudo stop codon) should be excluded. The sequence identity is determined, for example, by the Jotan-Hein method (H
ein, J .; (1990) Methods Enzymol. 183:
626-645)). 183: 626-645). Identity between sequences can also be achieved by other methods well known to those skilled in the art, which are performed by changing hybridization (hybridization) conditions.

“Totipotent” refers to the ability of a cell to differentiate into any type of cell of a mature organ.

By “transformation” is meant introducing DNA into a suitable host cell so that the DNA can be recreated as an extrachromosomal element or as part of a chromosome. "
"Transfection" means the taking up of an expression vector by a suitable host cell whether or not the coding sequence is actually expressed. "Infection" refers to the introduction of nucleic acid into a suitable host cell using a virus or viral vector.

As used herein, “uptake modulating fragment” UMF means a series of nucleotides that mediates uptake of linked DNA fragments into cells. UMF can be easily identified by a known computer system using a known UMF as a target sequence or target motif as described below. The presence and activity of UMF can be confirmed by binding the suspected UMF to a marker sequence. The resulting nucleic acid molecule is cultured under suitable conditions in a suitable host cell and the uptake of the marker sequence is determined. As mentioned above, UMF increases the frequency of incorporation of linked marker sequences.

The above terms have the meanings given above, unless the context clearly dictates otherwise.

5.2 Nucleic Acids of the Present Invention The present invention provides novel secretory CUB domain polypeptides, the discovery of polynucleotides encoding the CUB domain polypeptides, and cancer and other immunological disorders. It is based on the use of these compositions for the diagnosis, treatment or prevention of.

Isolated polynucleotides of the present invention include, without limitation, the following: That is,
SEQ ID NO: 1-3, 5, or 12 polynucleotides forming a sequence of nucleotides; SEQ ID NO: 1-3, 5, or 12 single fragments; SEQ ID NO: 1-3, 5, Or a polynucleotide forming 12 full-length protein coding sequences (eg SEQ ID NO: 4
); And SEQ ID NO: 1-3, 5, or 12, which includes a polynucleotide forming a nucleotide sequence that encodes the mature protein coding sequence of the polynucleotide. Polynucleotides of the invention also include without limitation: A polynucleotide that hybridizes (hybridizes) to the following under stringent hybridization (hybridization) conditions: (a) SEQ ID
NO: the complement of any of the nucleotide sequences 1-3, 5, or 12; (b
) SEQ ID NO: a polynucleotide encoding any one of the 4 or 6-8 polypeptides; (c) a polynucleotide that is an allelic variant of any of the above polynucleotides; (d) above. A polynucleotide encoding a homologue of any species of the protein; or (e) SEQ ID N
A polynucleotide comprising a specificity domain or truncation of the O: 4 or 6-8 polypeptide. The domain of interest depends on the nature of the encoded polypeptide.
For example, the domain of a receptor-like polypeptide may be ligand-binding, extracellular, transmembrane,
Or a cytoplasmic domain, or a combination thereof; a domain of an immunoglobulin-like protein comprises an immunoglobulin-like variable domain; a domain of an enzyme-like polypeptide comprises catalytic and substrate binding domains; and a domain of a ligand polypeptide comprises It contains a receptor binding domain.

The polynucleotide of the present invention includes naturally occurring DNA, or wholly or partially synthetic DNA. For example, cDNA and genomic DNA, and RNA, such as mRNA. The polynucleotide-related portion includes the entire coding region of cDNA or a part thereof.

The present invention also provides a gene corresponding to the sequence of the cDNA disclosed herein.
These corresponding genes can be separated according to known methods using the sequence information disclosed herein. These methods include making probes, or primers, from the disclosed sequence information to identify and / or amplify genes in appropriate genomic libraries, or other sources of genomic material.

Additional 5'and 3'sequences can be obtained by methods known in the art. For example, SEQ
Use any of the polynucleotides of ID NO: 1-3, 5, or 12 or a portion thereof as a probe to select the appropriate cDNA or genomic DNA under appropriate hybridization conditions. By SE
Full-length cDNA or genomic DNA corresponding to any of the Q ID NOs: 1-3, 5, or 12 polynucleotides can be obtained. Alternatively, S
The polynucleotides of EQ ID NO: 1-3, 5, or 12 can be used as the basis for suitable primers to allow the identification and / or amplification of genes in a suitable genomic DNA or cDNA library.

The sequences of the nucleic acids of the invention can be found in one or more public databases, such as dbEST, g
It is assembled from ESTs and sequences (including cDNA and genomic sequences) obtainable from bpri and UniGene. The EST sequences can provide sequence information to identify, representative fragment or segment information, or novel segment information for the full-length gene.

The polynucleotide of the present invention also provides a polynucleotide containing a nucleotide sequence almost equivalent to the above-mentioned polynucleotide).

A polynucleotide according to the present invention may be, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, more typically at least 85%, 86%, 87%, 88%, 89%, more typically at least 90%,
91%, 92%, 93%, 94% and even more typically at least 95%, 9
It has 6%, 97%, 98%, 99% sequence identity to the polynucleotide.

Included within the scope of nucleic acid sequences of the present invention are SEQ ID NO:
A fragment of a nucleic acid sequence that hybridizes (hybridizes) to any of the 1-3, 5 or 12 nucleotide sequences under stringent conditions, or the complement thereof, wherein the fragment is more than about 5 nucleotides, preferably 7 nucleotides. More preferably more than 9, more preferably more than 17 nucleotides. For example, fragments of 15, 17, or 20 or more nucleotides that are selective (ie, specifically hybridize to any one of the polynucleotides of the present invention) are considered. A probe capable of specifically hybridizing to one of the polynucleotides (hybridization) is capable of distinguishing the polynucleotide sequence of the present invention from other polynucleotide sequences in a cognate gene, or from a gene of another species to a human gene. Can be distinguished,
And preferably it is based on a unique nucleotide sequence.

The sequences included within the scope of the present invention are not limited to these particular sequences, but also include allelic (allelic) and species variations. Allele and species variation is SEQ
Sequences obtained under ID NO: 1-3, 5, or 12, representative fragments thereof,
Or SEQ ID NO: 1-3, 5, or 12 with a minimum of 90%, preferably 9
Nucleotide sequences that are 5% identical can be routinely determined by comparing sequences from other isolates of the same species.

Furthermore, in keeping with codon variability, the invention, as well as the specific ORFs disclosed herein, also include nucleic acid molecule codes of the same amino acid sequence. That is, one O
It is clearly conceivable to replace one codon with another codon encoding the same amino acid in the coding region of RF.

Approximate results for the nucleic acids of the invention, including SEQ ID NOs: 1-3, 5, or 12, can be obtained by searching databases using algorithms or programs. Search for alignments of localized sequences, preferably using the basic local alignment search tool BLAST (Alts
hul, S.H. F. J Mol. Evol. 36 290-300 (1
993) and Altschul S. et al. F. et al. J. Mol
． Biol. 21: 403-410 (1990)).

The present invention also provides homologs (homologues, homologs) (orthologs) of the disclosed polynucleotide and protein species. The ortholog of the species can be separated and identified by preparing an appropriate probe or primer from the sequence presented here and selecting an appropriate nucleic acid source from the desired species.

The present invention also includes allelic variants of the disclosed polynucleotides or proteins; ie, alternative forms of naturally occurring isolated polynucleotides, which are also identical to those encoded by the polynucleotides. , Homologous (homologous), or related proteins.

The nucleic acid sequences of the present invention are further directed to sequences encoding variants of the described nucleic acids. Variants of these amino acid sequences can be made by the known method of making appropriate nucleotide changes to the original or variant polynucleotide. There are two variable elements in the construction of amino acid sequence variants. The location of the mutation and the nature of the mutation. Nucleic acids encoding amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode non-naturally occurring amino acid sequences. Modification of these nucleic acids can occur at different sites (variable positions) or highly conserved regions (constant regions) depending on the heterologous nucleic acid. The sites at those positions are usually modified in series; for example, first conservative selections are made (eg, hydrophobic amino acids with different hydrophobic amino acids), and then distant selections (eg. For example, hydrophobic amino acids can be deleted or inserted at the target site (with the amino acid loaded). Amino acid sequence deletion is
Usually about 1 to 30 residues, preferably about 1 to 10 residues and usually contiguous. Amino acid insertions may be amino- and / or carboxyl-terminal fusions, spanning residues 1 to 100 or more in length, as well as intrasequence insertions of single or multiple amino acid residues. Intra-sequence insertions usually span 1 to 10 amino acid residues and are preferably 1 to 5 residues.

Examples of terminal insertions include heterologous signal sequences and FLAG-like sequences necessary for secretion or intracellular targeting in different host cells, or poly-uses useful for purifying the expressed protein. There is a histidine sequence.

In a preferred method, the polynucleotide encoding the novel amino acid sequence is converted via the induction of site-directed mutagenesis. This method involves converting a polynucleotide using an oligonucleotide sequence to encode the amino acid variant of interest, while at the same time producing a sufficient quantity of nucleotides on either side of the converted amino acid, on either side of the site to be converted. Form a stable duplex. In general, the technique of site-directed mutagenesis is well known to those skilled in the art, and this technique is exemplified in the following publication: Edelman et al., DNA 2: 183 ( 19
83). Diverse and efficient methods for creating site-specific changes in polynucleotide sequences are described by Zoller and Smith, Nucleic Acid Res. 10: 6487 65
Published in 00 (1982). PCR can also be used to create amino acid sequence variants from novel nucleic acids. If a small amount of template DNA is used as the starting material, primers with a sequence slightly different from the corresponding portion in the template DNA can be used to create the desired amino acid variants. Amplification by PCR increases the number of DNA fragment constructs that differ from the polynucleotide template that encodes the polypeptide at the position specified by the primer. This DNA fragment construct replaces the corresponding region in the plasmid, resulting in a polynucleotide encoding the desired amino acid mutation.

Another technique for making amino acid variants is the cassette mutagenesis technique, described by Wealth et al., Gene 34: 315 (1985). Other mutagenesis techniques are well known in the art. For example, the techniques described by Sam Brooks, supra, and "Current Protocols in Molecular Biology" by Ausubell et al. Due to the intrinsic degeneracy of the genetic information, other DNA sequences encoding nearly identical or functionally equivalent amino acid sequences were used in the practice of the present invention to clone and express these novel nucleic acids. Good to use. These DNA sequences include those that are capable of hybridizing to a suitable and novel nucleic acid sequence under stringent hybridization conditions.

A polynucleotide encoding a cleavage portion of a desired polypeptide of the invention is
It can be used to create polynucleotides that encode chimeric or fusion proteins and (hetero) heterologous protein sequences that form one or more regions of the invention.

The polynucleotides of the present invention further include the complement of any of the above polynucleotides. The polynucleotide may be DNA (genomic, cDNA, amplified or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art, and include, for example, hybridization (), which can routinely separate identified polynucleotides of a desired sequence. (Hybridization) conditions are included.

According to the present invention, the sequence of a polynucleotide consisting of the mature protein coding sequence corresponding to either SEQ ID NO: 4 or 6-8, or a functional equivalent thereof, is used to generate a nucleic acid in a suitable host cell. It can be used to make expressing recombinant DNA molecules, or their functional equivalents. Also included is the cDNA insert of the clone identified here.

Polynucleotides according to the present invention can bind to any other type of nucleotide sequence by well-established techniques of recombinant DNA (see J. Sambrook et al. 10 (1989) Molecular cloning: laboratories). Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences that bind to polynucleotides include a variety of vectors; eg, plasmids, cosmids, lambda phage derivatives, phagemids, and others well known in the art. Therefore, the present invention also provides a vector containing the polynucleotide of the present invention and a host cell carrying the polynucleotide. Generally, the vector will contain at least one
It contains a source of replication that functions in the individual organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. The vectors according to the invention include expression vectors,
Included are replication vectors, probe generation vectors, and sequence vectors. According to the invention, the host cell may be a prokaryotic cell or a eukaryotic cell and may be a unicellular organism or part of a multicellular organism.

The invention further comprises recombinantly produced nucleic acids having the nucleotide sequence of SEQ ID NO: 1-3, 5, or 12 or a fragment thereof, or any of the other polynucleotides of the invention. Provide things. In one embodiment, the recombinant constructs of the invention are comprised of a vector such as a plasmid or virus, in which the nucleotide sequence of SEQ ID NO: 1-3, 5, or 12 or a fragment thereof is included. The nucleic acid possessed is inserted either forward or backward. In the case of a vector of the invention or comprising one of the ORFs, the vector may further comprise regulatory sequences: eg a promoter operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are available from commercial sources for making the recombinant constructs of the invention. The following vectors are given as examples. Bacteria: pBs, Phagescript, PsiX174, pBluesc
ript SK, pBs KS, pNH8a, pNH16a, pNH1
8a, pNH46a (Stratagene); pTrc99A, pK
K223-3, pKK233-3, pDR540, pRIT5 (Pha
rmacia). Eukaryotic cells: pWLneo, pSV2cat, pOG44, PXTI,
pSG (Pharmacia) pSVK3, pBPV, pMSG,
pSVL (Pharmacia).

The isolated polynucleotides of the present invention have been isolated from nucleic acids Res. It may be functionally linked to expression control sequences such as the pMT2 or pED expression vectors disclosed in 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known, and R. Kaufman, "Methods in Enzymology" 185, 537-5.
66 (1990). As defined herein, "operably linked" means that the isolated polynucleotide of the invention and the expression control sequence are placed in a vector or cell and the protein is linked. It is designed to be expressed by a transformed (transfected) host cell with a polynucleotide / expression control sequence.

The promoter region can be selected from any desired gene using the CAT (chloramphenicol transferase) vector or other vector with a selectable marker. Two suitable vectors are pKK232-8 and pCM7.
Individually named bacterial promoters are lacI, lacZ, T3, T7
, Gpt, lambda, PR, and trc. Eukaryotic promoters include: CMV immediate early, HSV thymidine kinase early and late SV40, LTR from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. In general, recombinant expression vectors contain an origin of replication and a selectable marker that enables the transformation of host cells, for example, the E. coli ampicillin antibody gene and the S. cerevisiae TRP1 gene and a promoter that directs the transcription of a downstream structural sequence extracted from a highly expressed gene. The promoters are obtained from operons which encode glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), factors, acid phosphatase, or heat shock proteins, among others. The heterologous sequence is assembled in appropriate phase with translation initiation and termination sequences, preferably a leader sequence that enables secretion of the translated protein into the periplasmic space or extracellular medium. .
The option is that the heterologous sequence can encode a fusion protein that includes an amino-terminal identifying peptide that exerts the desired properties. For example, stabilizing or simply purifying the expressed recombinant product. Useful expression vectors for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein, with appropriate translation initiation and termination signals, in an active reading step with a functional promoter. The vector is formed from one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, amplification in the host. Prokaryotic hosts suitable for transformation include the following bacteria: Escherichia coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces, and various species of Staphylococcus. Others can be selected and used.

As a representative but not limiting example, useful bacterial expression vectors include selectable markers and the well known cloning vector pBR322 (ATCC).
37017) and is composed of a bacterial prototype of replication derived from a commercially available plasmid. Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM 1 (Promega Biote)
ch, Madison, WI, USA). These pBR322 "backbone" sections are linked to the appropriate promoter and structural sequences to be expressed. After transformation of the appropriate host strain and growth to the appropriate cell density, the selected promoter is induced or repressed by appropriate means (eg by temperature alteration or chemical induction) and the cells are cultured for an additional period of time. . Cells are usually harvested by centrifugation, disrupted by physical or chemical means, and the remaining crude extract retained for further purification.

The polynucleotides of the present invention can also be used to elicit an immune response. For example,
Fans and others. , Nat. Biotech. 17: 870-872 (1999), and as used herein as a reference, the nucleic acid sequence encoding the polypeptide is preferably D
After intramuscular injection of NA, it is used to raise antibodies against the encoded polypeptide. The nucleic acid sequence is preferably inserted into a recombinant expression vector and may be in the form of naked DNA.

5.3 Antisense Another aspect of the invention is that it is hybridizable or complementary to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1-3, 5, or 12 or a fragment, analogue or derivative thereof. Related to isolated, antisense nucleic acid molecules. An "antisense" nucleic acid consists of a nucleotide sequence that is complementary to a "sense" nucleic acid that encodes a protein: for example, the coding strand or mR of a double-stranded cDNA molecule.
It is complementary to the NA sequence. In particular aspects, at least about 10,25,50,1
An antisense nucleic acid molecule complementary to 00, 250 or 500 nucleotides, or all or part of the coding strand is provided. SEQ ID NO: 1
A nucleic acid molecule encoding a fragment, homologue, analog or derivative of the protein of any of -3, 5, or 12, or complementary to the nucleic acid sequence of SEQ ID NO: 1-3, 5, or 12 Further provided is an antisense nucleic acid.

In one embodiment, the antisense nucleic acid molecule is antisense to the “coding region” of the coding strand of the nucleotide sequences of the invention. The term "coding region" refers to the region of nucleotide sequence which consists of codons translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to the "non-coding region" of the coding strand of a nucleotide sequence of the invention.

The term “non-coding region” refers to the 5 ′ and 3 ′ sequences which flank the coding region and are not translated into amino acids. (That is, the 5'and 3'untranslated regions).

Given the sequences of the coding strands (eg, SEQ ID NOs: 1-3, 5, or 12) encoding nucleic acids disclosed herein, the antisense nucleic acids of the invention are Watson. And click, or Hoogsteen base pairing rules. The antisense nucleic acid molecule can be complementary to the entire coding region of the mRNA, although minority nucleotides that are antisense to only a portion of the coding or noncoding region of the mRNA are more desirable. For example, the antisense minor nucleotide can be complementary to the region surrounding the translation start site of mRNA. Antisense minor nucleotides include, for example, about 5, 10, 15, 20,
It can be 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acid of the present invention can be prepared by chemical synthesis or enzyme-bonding reaction known in the art. For example, antisense nucleic acids (eg, antisense minor nucleotides) are naturally occurring nucleotides or to increase the biostability of the molecule, or of double-stranded double-stranded structures formed between the antisense and sense nucleic acids. It can be chemically synthesized using various modified nucleotides to increase physical stability. For example, a nucleotide in which a derivative of phosphorothioic acid and acridin are substituted can be used.

Examples of modified nucleotides that can be used to generate antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxylhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thioyuridin, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosilicuocine, inosine, N6-isopentenailadenine, 1-methylguanine, 1-methylinosin, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- Manosiliquacin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2
-Methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v
), Wibutoxosine, pseudo uracil, chyosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (
v), 5-methyl-2-thiouracil, 3 (3-amino-3-N-2-carboxylpropyl) uracil, (acp3) w, and 2,6-diaminopurine.
Alternatively, antisense nucleic acids can be generated biologically using an expression vector in which the nucleic acid is inserted as a subclone in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid becomes the target nucleic acid). In contrast, it is antisense-oriented and will be discussed in more detail in the next subsection).

The antisense nucleic acid molecules of the invention are usually administered to a subject or are generated in situ and hybridize (hybridize) with mRNA or genomic DNA encoding the protein of the invention. Alternatively, it binds and suppresses protein expression; for example, suppresses transcription and translation. The hybridization may be by conventional nucleotide complementarity or, in the case of an antisense nucleic acid molecule that binds to the double strand of DNA, for example, a specific interaction within the large groove of the double helix. By this, a stable double structure may be formed. One example of a route of administration of antisense nucleic acid molecules according to the present invention is direct injection at the tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and administered systemically. For example, for systemic administration, the antisense molecule is modified to specifically bind the receptor or an antigen expressed on the surface of selected cells. For example, a method of binding an antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen is used. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to obtain a sufficient intracellular concentration of the antisense molecule, construction of the vector in which the antisense nucleic acid molecule is placed under the control of the strong Pol II or Pol III promoter is desirable.

In another embodiment, the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule. Alpha-anomeric nucleic acid molecules specifically form double-stranded hybrids with complementary RNA in which, unlike normal ones, the strands are parallel to each other (Gautier et al. (1987) Nucleic acid Res. 15:
6625-6641). In addition, the antisense nucleic acid molecule is 2'-o-methylribonucleotide.
Nucleotides (Inoue et al. (1987) Nucleic Acids Res 15: 6131-61
48) or a chimeric RNA-DNA analogue (Inoue et al., Inoue et al. (1987) FEBS Lett 215: 32).
7-330). 5.4 Ribozyme and PNA Half In yet another embodiment, the antisense nucleic acids of the invention are ribozymes. Ribozymes are catalytic RNA molecules having RNase activity and can divide a single-stranded nucleic acid such as mRNA having a complementary region into two. Therefore, ribozymes (eg, Hummerhead ribozymes; (Haselhoff and Gerach (1
988) Nature 334: 585-591) can catalytically divide mRNA transcription into two and be used to suppress mRNA translation. Ribozymes with specificity for the nucleic acids of the invention can be designed based on the nucleotide sequences of the DNAs disclosed herein (ie SEQ ID NO: 1-3, 5, or 12).

For example, the derivative of Tetrahymena L-19 IVS RNA can be structured such that in the SECX-encoding mRNA, the nucleotide sequence of its active site is complementary to the nucleotide sequence bisected. Reference example: Check, US patent. No. 4,987,071, and check others. US patent. No. 5,
116, 742.

As an alternative, mRNA can be used to extract specific RNA from a pool of RNA molecules.
Catalytic RNA with degradative enzymatic activity can be selected. Reference example: Bartel and others, (
1993) Science 261: 1411-1418. Alternatively, regulatory regions (eg promoter and / or enhancer)
Gene expression can be suppressed by forming a triple-helical structure that prevents transcription of the gene in the target cell by targeting a nucleotide sequence complementary to. Reference: Helen. (1991) Anticancer drug Des. 6: 569-84; Helen et al. (1992)
) Ann. N. Y Acad. Sci. 660: 27-36; and Mehra (1992) Bioassay 14: 807-15. .

In various embodiments, the nucleic acids of the invention can be modified in half the bases, half the sugars, or the phosphate backbone to improve, for example, the stability, hybridization, or solubility of the molecule. is there. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (see: Hylap et al. (1
996) Bio or g Med Chain 4: 5-23). As used herein, the term "peptide nucleic acid" or "PNA" means a mimic of a nucleic acid, such as DNA, in which the backbone of deoxyribose phosphate is replaced by the backbone of a pseudopeptide, and four natural nucleobases. Only retained. The neutral backbone of PNA has been shown to hybridize specifically to DNA and RNA under low ionic strength conditions. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols, as described by Hylap et al., Supra (1996); Perry O'Keefe et al. (1996) PNAS 93: 14670-675. As it is. The PNAs of the invention can be used in therapeutic and diagnostic applications. For example, PNA can be used as an antisense or antigene agent that induces repression of transcription or translation arrest or replication to regulate sequence-specific gene expression. Applications of the PNAs of the present invention include analysis of single base pair mutations, eg by PCR tightening directed by PNA, combined with other enzymes such as S1 nucleolytic enzymes and artificial restriction enzymes (Hylap B. 1996), supra), and probes or primers for DNA sequences and hybrid formation (Hylap et al. (1996), supra;
Perry O'Keefe (1996), supra).

In another embodiment, PNAs of the invention can be modified to improve stability or cellular uptake. The method is, for example, the addition of lipophilic or other auxiliaries to the PNA,
Such as forming PNA-chimeras, using liposomes and other drug delivery techniques known in the art. For example, PN that combines the advantageous properties of PNA and DNA.
An A-DNA chimera can be generated.

Such chimeras allow DNA recognition enzymes, such as the RN enzyme H and the DNA polymerase, to interact with the DNA portion, while at the same time the PNA portion provides a high degree of binding affinity and specificity. The PNA-DNA chimera can be ligated using a linker of an appropriate length selected in consideration of the stacking of bases, the number of bonds between bases, and the orientation (Hylap (1996) supra). Synthesis of PNA-DNA chimera
Hilap (1996) supra and Fin et al. (1996) Nucleic acid Res 24:33.
This can be done as described in 57-63. For example, the DNA strands were synthesized on a fixed support using standard phosphate amidite coupling chemistry and modified nucleoside analogs such as 5 '-(4-methoxyoxytyl) amino-5'-deoxy-thymidin. Phosphate amidites can be used between PNA and the 5'end of DNA (Mug et al. (1989) Nucleic Acids Research Nucl Acid Res 17:59).
73-88). The PNA monomers are then stepwise linked to produce a chimeric molecule with a 5'PNA segment and a 3'DNA segment (Fin et al. (1
996) above). Alternatively, the chimeric molecule may include a 5'PNA segment and a 3 '
It can also be synthesized using the DNA segment. Reference: Peterson et al. (1975) Bio Org Med Chem Lett 5: 111.
9-11124. In other embodiments, minority nucleotides include other accessory groups such as peptides (eg, peptides that target host cells in the body) or cell membrane layers (Reference: Letzinger et al., 1989, Proc. Natl. Acad. Sci. U
． S. A. 86: 6553-6556; Rumeter et al., 1987, Pro.
c. Natl. Acad. Sci. 84: 648-652; PCT.
Publication No. W088 / 09810) or blood-brain barrier (reference example: PCT Publication No. W089 / 10134) are also included in the drug. Furthermore, minority nucleotides are used as hybridizing-inducing resolving agents (Reference Example: Kroll et al., 19
88, BioTechniques 6: 958-976) or an intercalating agent (Reference Example: Son, 1988, Pharm. Res. 5: 539-549). For this purpose, minority nucleotides may be conjugated to other molecules such as peptides, hybridisation-inducing crosslinkers, carriers, hybridisation-triggered resolving agents and the like.

5.5 Host The present invention further provides host cells genetically engineered to include the polynucleotides of the present invention. For example, the host cell may contain a nucleic acid of the invention that has been introduced using known transformation, transfection or infection methods. The invention further provides host cells genetically engineered to express the polynucleotides of the invention, wherein the polynucleotides are heterologous to the host cell in which the expression of the polynucleotide is engineered. Functionally associated with regulatory sequences.

Knowledge of the CUB domain DNA sequence can regulate cells to allow or increase expression of CUB domain polypeptides. The cells are conditioned (eg, by cognate recombination) and cells of the CUB domain polypeptide are replaced by replacing all or part of the naturally occurring CUB domain promoter with all or part of a heterologous promoter. Can be expressed at higher levels. The heterologous promoter is inserted so that it is functionally linked to the CUB domain coding sequence. Reference example: PCT International Publicici
on No. WO94 / 12650, PCT International
Publication No. WO92 / 20808, and PCT I
international Publication No. WO91 / 09
955. In addition to a heterologous promoter DNA, an amplifiable marker DNA (
Examples: ada, dhfr, and multifunctional CAD encoding carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase.
It is also contemplated that the gene) and / or intron DNA be inserted with the heterologous promoter DNA. If linked to the CUB domain coding sequence, amplification of the marker DNA by standard selection methods leads to co-amplification of the CUB domain coding sequence within the cell.

The host cell may be a highly eukaryotic host cell, such as a mammalian cell, a low eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. To introduce recombinant constructs into host cells, calcium phosphate transfection, DEA
E, Dextran-mediated transfection, or electroporation. (Davis, L. et al., Basic Methods of Molecular Biology (1986)). A host cell carrying one of the polynucleotides of the present invention will produce the gene product encoded by the segregated fragment in the conventional manner (in the case of the ORF) or produce a heterologous protein under the control of EMF. Can be used for

Any host / vector system can be used to express one or more ORFs of the invention. These include without limitation: eukaryotic hosts, such as: HeLa cells, Cv-1 cells, COS cells, 293 cells, Sf9 cells, and prokaryotic hosts, such as E. coli and B. Subtilis. The most desirable cells are usually those that do not express a particular polypeptide or protein, or those that express the polypeptide or protein at natural low levels. The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.
RNA extracted from a DNA construct of the invention using a cell-free translation system
Can also be used to produce such proteins. Suitable cloning and expression vectors using prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition Cold Spring Harbor, NY (1).
989), the disclosure of which is incorporated herein by reference.

A variety of mammalian cell culture systems can be used to express recombinant protein. For an example of an expression system in mammalian cells, see Gluzman, Cell, 23: 175 (1981).
And the COS-7 line of monkey kidney fibroblasts as described in 1. Those capable of expressing compatible vectors in other cell lines are, for example, C127, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermal A.
431 cells, human Colo2O5 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary organisms Explants, HeLa cells, mouse L cells, BHK, H
L-60, U937, HaK or Jurkat cells. Mammalian cell expression vectors include origins of replication, appropriate promoters and necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5 '.
It is formed by a lateral non-transfer arrangement. For example, DNA sequences taken from the viral genome of SV40, the SV40 progenitor, early promoters, enhancers, splices, and polyadenylation sites can be used to provide the necessary nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually separated from cell pellets by first extraction, followed by one or more salting out, water ion exchange, or size exclusion chromatography. If desired, the protein can be refolded to complete the organization of the mature protein. Finally, high performance liquid chromatography (HPLC) is used to complete the purification procedure. The microbial cells used to express the protein can be disrupted by conventional methods, such as freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

Alternatively, the protein can be produced in lower eukaryotes such as yeast or insects, or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schi.
zosaccharomyces pombe, Kluyveromyces
Strain, Candida, or any yeast strain capable of expressing a heterologous protein. Potentially suitable bacterial strains include E. coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made of yeast or bacteria, it may be necessary to modify the protein produced there in order to obtain a functional protein, for example by phosphorylation or glycosylation of the appropriate sites. Absent. Attachment of such covalent bonds can be accomplished by known chemical or enzymatic methods. ． In another embodiment of the invention, cells and tissues can be engineered to express an endogenous gene comprising a polynucleotide of the invention under the control of inducible regulatory elements, in which case the endogenous gene Regulatory sequences are replaced by cognate recombination. As described herein, gene targeting techniques are used to replace an existing regulatory region of a gene with a regulatory sequence isolated from a different gene or with a novel regulatory sequence synthesized by genetic engineering techniques. be able to. The regulatory sequences are composed of promoters, enhancers, backbone attachment regions, negative regulatory elements, transcription initiation sites, regulatory protein binding sites, or a combination of these sequences. Alternatively, sequences that affect the structure or stability of the RNA or protein produced can be replaced, removed, attached, or otherwise modified by targeted methods. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences to improve or modify the delivery or secretion properties of proteins, or alter or improve the function or stability of protein or RNA molecules, Including other sequences.

The targeting method may be to simply insert the regulatory sequences, for example inserting a new promoter or enhancer or both upstream of the gene to bring the gene under the control of the new regulatory sequences. Alternatively, the targeting method is to simply delete regulatory elements, eg, delete tissue-specific negative regulatory elements. Alternatively, the targeting method replaces existing elements; for example, tissue-specific enhancers are replaced with enhancers that have specificity for cell types that are broader or different than the naturally occurring elements. Here, naturally occurring sequences are deleted and new sequences are added. In any case, targeting method identification is facilitated by the use of one or more selectable marker genes flanking the target DNA,
Cells can be selected in which the exogenous DNA has bound to the host cell genome. Target method identification is
It is also facilitated by using one or more marker genes that exhibit negative selection properties. That is, the negative selectable marker is linked to the exogenous DNA, but is flanked by the target sequences, and those that carry out the correct cognate recombination with sequences in the host cell genome are negatively selectable. Does not have stable binding with the marker. Markers useful for this purpose include the herpes simplex virus thymidine kinase (TK) gene, or the bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene.

Gene targeting or gene activation techniques that can be used in accordance with this aspect of the invention are described in more detail below: US Pat. 5,272,071 addressed to Chapel; US Patent No. 5,578,461 to Sherwin et al .; International Application No. PCT / US
92/09627 (WO93 / 09222) Selden et al .; and International Application No.
． PCT / US90 / 06436 (WO91 / 06667) School et al., As well as the entire contents thereof, are incorporated herein by reference.

5.6 Polypeptides of the Invention Isolated polypeptides of the invention include, without limitation, polypeptides consisting of: SEQ ID NO: 4 or an amino acid sequence set as one of 6-8. , Or the amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NO: 1-3, 5, or 12, or the corresponding full-length or mature protein. It is also desirable that the polypeptide of the invention is a biologically or immunologically active polypeptide encoded by: (a) SEQ ID
Polynucleotide having one of the nucleotide sequences set to NO: 1-3, 5, or 12 or (b) encoding any one of the amino acid sequences set to SEQ ID NO: 4 or 6-8 Or (c) a polynucleotide which hybridizes (hybridizes) to the complement of the polynucleotide of (a) or (b) under conditions of severe hybridization (hybridization). The present invention also provides
Biologically or immunologically active mutants of any of the amino acid sequences set forth as SEQ ID NO: 4 or 6-8, or the corresponding full-length or mature proteins; and those with biological activity And "almost equivalent" (eg, amino acid identity is at least 65%, at least 70%, at least 75%, at least 80%, at least 8)
5%, 86%, 87%, 88%, 89%, minimum 90%, 91%, 92%, 93
%, 94%, typically at least 95%, 96%, 97%, more typically at least 98%, or most typically at least 99%) with biological activity . The polypeptide encoded by the allelic variant has SEQ ID NO: 4 or 6
The activity may be similar, higher, or lower compared to a polypeptide composed of -8.

Also included in the invention are fragments of the proteins of the invention capable of exhibiting biological activity, which may be linear or may be made circular using known methods. For example, these are H.264. U. Saragovi et al., Bio / Techno
LOGY 10, 773-778 (1992) and R.M. S. McDowell et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992)
), And both are added here for reference. Such fragments can also be fused to carrier molecules such as immunoglobulins for various purposes, such as increasing the valency of the protein's binding site.

The present invention also provides two forms of the disclosed proteins, full length and mature (eg, without a signal sequence or precursor sequence). Protein coding sequences are identified in the sequence listing by translation of the disclosed nucleotide sequences. Mature forms of such proteins can be obtained by expressing full length polynucleotides in a suitable mammalian cell, or other host cell. The sequence of the mature form of the protein can also be determined from the full length amino acid sequence. Soluble proteins are also provided when the proteins of the invention are membrane bound. In such a form, some or all of the region that binds the protein to the membrane is deleted so that the protein is fully secreted from the cell in which it is expressed.

Further, the protein composition of the present invention can include an acceptable carrier such as, for example, a pharmaceutically acceptable hydrophilic carrier.

The invention further provides an isolated polypeptide encoded by a nucleic acid fragment or a degenerate variant of a nucleic acid fragment of the invention. A "degenerate variant" is a nucleotide fragment that differs in nucleotide sequence from a nucleic acid fragment of the invention (eg, ORF), but encodes the same polynucleotide sequence due to the degeneracy of the genetic code. A preferred nucleic acid fragment of the present invention is a protein-encoding ORF.

[0128] The isolated polypeptide or protein. You can get any of At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. The synthesized protein sequence is
It may share primary, secondary, or tertiary structure or conformational features with the protein, and thus possess biological properties, including the activity of the protein. This technique is particularly useful for producing small peptides and fragments of large polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. Therefore, it can be used as a biologically active or immunological substitute for naturally purified proteins in the selection of therapeutic compositions and in the immunological process of antibody development.

The polypeptides and proteins of the invention can be alternatively purified from cells that have been modified to express the desired polypeptide or protein. "Modified to express a desired polypeptide or protein" means herein that a cell is genetically engineered to express a polypeptide or protein that it normally does not produce or normally produces at low levels. Means to be changed to. One of skill in the art can readily follow the procedures for introducing and expressing either recombinant or synthetic sequences in eukaryotic or prokaryotic cells to produce cells which produce either the polypeptides and proteins of the invention. It can be adjusted.

The present invention also refers to a method of producing a polypeptide. For example, a method of growing a host cell in an appropriate culture medium, a method of purifying a protein from cells or a culture medium in which the cells grow, and the like. For example, in the process of producing a polypeptide included in the method of the present invention, a host cell having an appropriate expression vector containing the polynucleotide of the present invention can be used.
There are also methods of culturing under conditions that allow the expression of the encoded polypeptide.

The polypeptide is readily removed from culture or from lysates made from host cells for further purification. A preferred embodiment is that the protein produced by the process is in full-length or mature form.

In an alternative method, the polypeptide or protein is purified from bacterial cells that naturally produce the polypeptide or protein. One of skill in the art can readily utilize known methods for separating polypeptides and proteins to obtain one of the isolated polypeptides or proteins of the invention. These methods include, without limitation, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, and immunoaffinity chromatography. Reference example:
Scopes, Protein Purification Theory and Practice, Springer-Fairlag (1994)
Sambrook et al., Molecular Cloning: Laboratory Manual; Ausbel et al.,
Current protocols in molecular biology. Polypeptide fragments that retain biological and immunological activity include fragments that consist of about 100 or more amino acids, or about 200 or more amino acids, and fragments that encode a domain of a particular protein.

The purified polypeptide can be used in an in vitro binding assay, a method well known in the art for identifying molecules that bind to a polypeptide. These molecules are
Including without limitation: small molecules, molecules from combinatorial libraries, antibodies or other proteins. Molecules identified in binding assays are tested in body tissue culture or animal models for antagonistic or hostile activity, which methods are well known in the art. Briefly, the molecule is injected into multiple cell cultures or animals and then tested for cell / animal length of death or survival.

Furthermore, the peptides or molecules capable of binding to the peptides of the present invention may be complexed with toxins such as ricin, cholera or cell toxic compounds. The toxin-bound molecular complex is used to target tumors or other cells depending on the specificity of the SEQ ID NO: 4 or 6-8 binding molecule.

The proteins of the invention may also be expressed as a component of transgenic animal products, eg, transgenic milk of cows, goats, pigs, sheep. These animals are characterized by having somatic cells or germ cells containing a nucleotide sequence encoding a protein.

The proteins referred to herein also include proteins that are characterized by an amino acid sequence similar to that of the purified protein, but the amino acids are naturally modified or artificially designed. For example, modification of a peptide or DNA sequence can be done by one of ordinary skill in the art using known techniques. Modifications of the protein sequence of interest include altering, substituting, replacing, inserting or deleting selected amino acid residues of the coding sequence.

For example, one or more cystine residues can be deleted or replaced with another amino acid to alter the molecular structure. Such changes, substitutions,
The techniques of shuffling, inserting or deleting are well known to those skilled in the art. (Reference example: US Patent No. 4,518,584). Desirably, such changes, substitutions,
The replacement, insertion or deletion is to retain the desired activity of the protein. The regions of the protein that are important to the function of the protein can be generated by a variety of methods well known in the art: alanine scanning methods systematically replace one or a series of amino acids with alanine, resulting in The resulting alanine-containing variants are tested for biological activity. This type of assay determines the importance of the replaced amino acid in biological activity. Protein regions important for protein function can be determined with the eMATRIX program.

Other fragments and derivatives of protein sequences, which are expected to retain all or part of the activity of the protein and which are useful for selection or other immunological methods, may be identified using the disclosure herein. Made easily by industry experts. Such modifications are included in the present invention.

The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors and the insect expression system used to produce the protein. Baculovirus / insect cell expression system materials and methods are available as kits from commercial vendors. For example, Invitrogen, San Die
go, Calif. , U. S. A. (The MaxBat ^™ kit), such methods are well known in the art, and are described by Summers and Smith, Texas Agriculture Experiment Station Bulletin No. The description of 1555 (1987) is added here as a reference. Using the expression used herein, insect cells capable of expressing the polynucleotides of the invention are "transformed". The protein of the present invention can be prepared by culturing transformed host cells under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein is purified from its culture medium (ie, culture medium or cell extract) using known purification processes such as gel filtration and ion exchange chromatography. Protein purification also includes: Affinity columns containing agents that bind to proteins; Concanavalin A-agarose, Heparin-Toyopearl ^™ or Cibachrome Blue 3GA.
One or more column steps overlying an affinity resin such as Sepharose ^™ ; one or more for performing hydrophobic interaction chromatography using a resin such as phenyl ether, butyl ether, or propyl ether The above steps; or immunoaffinity chromatography.

Alternatively, the protein of the invention may be expressed in a form that facilitates purification. For example, it can be expressed as a fusion protein such as: maltose binding protein (MBP),
Glutathione-S-transferase (GST) or thioredoxin (TRX), or his label. Kits for the expression and purification of such fusion proteins are available from the following vendors respectively: New England BioLab (Beverly, Mass.
), Pharmacia (Piscataway, NJ) and Invitr.
ogen. The protein is also epitopically labeled and then purified using an antibody directed against the epitope. One of such episodes ("F
LAG® ”) is sold by Kodak (New Haven, Conn.).

Finally, one or more reversed phase high performance liquid chromatography (RP-HPL)
Q) (hydrophobic R such as silica gel with pendant methyl or other aliphatic groups)
The protein is further purified using the step (using P-HPCL media). By using some or all of the above-mentioned purification procedures in various combinations, almost the same recombinant recombinant protein can be produced. The protein thus purified contains almost no other mammalian proteins, and is defined as "isolated protein" in the present invention.

Polypeptides of the invention include analogs (variants). The polypeptide of the present invention is C
Includes UB domain analogs. This is a CUB consisting of a fragment of the CUB domain polypeptide of the invention and one or more deleted, inserted or substituted amino acids.
Contains a domain polypeptide. CUB domain polypeptide analogs of the invention also include fusions of CUB domain polypeptides, or modifications of CUB domain polypeptides, wherein CUB domain polypeptides or analogs are other half or Fused to multiple halves, eg, target halves or other therapeutic agents. Their analogues may show improved properties such as activity and / or stability.
Examples of halves fused to a CUB domain polypeptide, or analogs, are target halves that carry the polypeptide to neurons, eg, antibodies to the central nervous system, or to receptors and ligands expressed on neuronal cells. It is an antibody to carry. Other halves fused to the CUB domain polypeptide include therapeutic agents, such as antidepressants, or other agents for neurological disorders. The CUB domain polypeptide can also be fused to neuronal growth regulators and other targeted delivery chemokines.

5.6.1 Determining Identity and Similarity between Polypeptides and Polynucleotides Desirable identities and / or similarities are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs, including without limitation the following: GCG program packages including GAP (Deverau, J., et al., Nucleic Acid Research 12 (1): 387 (1984);
Genetics Computer Group, University
of Wisconsin, Madison, WI), BLASTP, BRAS
TN, BLASTX, FASTA (Altschul, SF, et al., Molecular Biology. 215: 403-410 (1990), PSI-BLAST (Altsc.
hul S. F. et al. , Nucleic acid research. vol. 25, pp. 3389-
3402, included herein by reference), eMatrix software (e.g.
Wu et al., J. Comp. Biol. , Vol. 6, pp. 219
-235 (1999), incorporated herein by reference), eMotif software (Nevill-Manning et al., ISMB-97, vol 4,
pp. 202-209, herein incorporated by reference), GeneAtlas software (Molecular Simulations Inc., (MS
I), San Diego, California) (Sanchez and Sali (1)
998) Proc. Natl. Acad. Sci. , 95, 135
97-13602; Kitson DH et al. (2000) "Remote homology detection using structural modeling-evaluation"submitted; Fischer and Eisenberg (
1996) Protein Sci. 5, 947-955), and Kyt.
e-Dolittle Hydrophobicity Prediction Algorithm (J. Mol Biol,
157, pp. 105-31 (1982), incorporated herein by reference). BLAS
T program is National Center for Biotechno
logy Information [National Center for Biotechnology Information] (
NCDI) and other sources (BLAST Manual, Arthur, S., et al. NCB NLM NIH Bethesda, MD 20894; Arthur, S., et al., J. Mol. Biol. 215: 403-410 (1990).
) Is generally sold by

5.7 Chimeras and Fusion Proteins The present invention also provides chimeras or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" is composed of a polypeptide of the invention operably linked to another polypeptide. Within the fusion protein, the polypeptide of the invention corresponds to all or part of the protein of the invention. In one embodiment, the fusion protein comprises at least one biologically active portion of a protein of the invention. In another example,
The fusion protein comprises at least two biologically active moieties of the proteins of the invention. Within the fusion protein, the term "operably linked" indicates that the polypeptide of the invention and the other polypeptide are fused in frame with each other.
The polypeptide can be fused N-terminal, C-terminal, or in the middle.

For example, in one embodiment, the fusion protein comprises a polypeptide of the invention operably linked to the extracellular domain of a second protein.

In another embodiment, the fusion protein is a GST-fusion protein, wherein the polypeptide sequence of the invention is fused to the C-terminus of the GST (ie glutathione S-transferase) sequence.

In another embodiment, the fusion protein is an immunoglobulin fusion protein, wherein the polypeptide sequence of the invention is composed of one or more domains fused to sequences derived from a member of the immunoglobulin protein family. To be done.

The immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between the ligand of the invention and the protein of the invention at the surface of the cell, thus Suppress the conversion of the signal. Immunoglobulin fusion proteins can be used to influence the bioavailability of cognate ligands. Inhibition of ligand / protein interactions is therapeutically useful both in treating reproductive and differentiation disorders (eg, cancer) and in regulating (eg, promoting or inhibiting) cell survival. Further, the immunoglobulin fusion protein of the present invention is also used as an immunogen, which produces an antibody in a subject, purifies the ligand, and suppresses the interaction between the polypeptide of the present invention and the ligand during the screening assay. Identify.

Chimeric or fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, the code for DNA fragments for different polypeptide sequences is:
They are connected in a frame according to conventional techniques. The conventional technique is, for example, to use a terminal having a blunt end or a corrugated end for connection, to make an appropriate terminal by digestion of an inhibitory enzyme, to fill an adhesive terminal appropriately, and to avoid an undesired bond. It is a method of treating with an alkaline phosphatase and then enzymatically ligating. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments is accomplished using anchor primers. The anchor primer creates a complementary overhang between two contiguous gene fragments and the two fragments are then annealed and reamplified to generate a chimeric gene sequence (reference example: edited by Ausbel et al., " Current Protocols in Molecular Biology ", John Wiley & Sons, 1992). In addition, many expression vectors are commercially available as fusion half encodings (eg, GST polypeptides). A nucleic acid encoding a polypeptide of the invention can be cloned into an expression vector such that the fusion half is linked in-frame to the protein of the invention.

5.8 Gene Therapy Mutations in the polynucleotide genes of the invention result in loss of the normal function of the encoded protein. Accordingly, the invention provides gene therapy for restoring the normal activity of a polypeptide of the invention or treating a condition associated with the polypeptide of the invention. Delivery of the functional gene encoding the polypeptide of the present invention to a suitable cell is carried out ex vivo, in vivo, or in vivo using a vector.
More specifically, it can be performed using viral vectors (eg, adenovirus, gland-associated virus, or retrovirus) or ex vivo using physical DNA transfer methods (eg, liposomes or chemotherapy). It Reference example: Anderson, Na
true [natural], replenishment vol. 392, no. 6679, pp.
25-20 (1998), as an additional reference material for gene therapy technology, Friedman, Science [Science], 244: 1275-1281 (1989).
); Vama, Scientific American [Scientific American]: 68-84 (1990); and Miller, Nature.
e, 357: 455460 (1992). An introduction to the genes encoding the nucleotides of the invention or the polypeptides of the invention can be achieved using extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). It is also possible to culture the cells ex vivo in the presence of the protein of the invention in order to proliferate the cells and impart the desired effect or activity to the cells. The treated cells are introduced into the body for therapeutic purposes. Alternatively, in other human conditions, preventing expression of the polypeptide of the invention or suppressing its activity would be useful in treating the condition. The application of antisense or gene therapy to negatively regulate the expression of the polypeptides of the invention is also contemplated.

Another method of suppressing protein expression is the method known in the art by introducing antisense molecules, their complements, or translated RNA sequences into the nucleic acids of the invention. In addition, the polypeptides of the invention are suppressed using targeted deletion methods or by inserting negative regulatory elements such as tissue-specific silencers.

The invention further provides cells engineered in the body to express the polynucleotides of the invention. Such polynucleotides are operably associated with host cell heterologous regulatory sequences which facilitate expression of the polynucleotide in the cell. Such a method can be used for the purpose of increasing or decreasing the expression of the polypeptide of the present invention.

The knowledge of the DNA sequences provided by the present invention allows the regulation of cells to effect, increase or decrease the expression of endogenous polypeptides. The cell is tunable (eg, by cognate recombination) and all or part of the naturally occurring promoter is replaced with all or part of the heterologous promoter, causing the cell to express the protein at higher levels. To do so. The heterologous promoter is inserted so that it is operably linked to the desired protein encoding sequence. Reference example: PCT Internet
nal Publication No. WO 94/12650, PCT
International Publication No. WO 92
120808, and PCT International Publicatio
n No. WO 91/09955. In addition to heterologous promoter DNA, amplifiable marker DNA (eg, ada, dh which encodes carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase).
It is also considered that the fr and the multifunctional CAD gene) and / or the intron DNA are inserted together with the heterologous promoter DNA. Once linked to the coding sequence for the desired protein, amplification of the marker DNA by standard selection methods leads to co-amplification of the desired protein coding sequence within the cell.

In another embodiment of the invention, cells and tissues can be designed to express one endogenous gene consisting of a polynucleotide of the invention under the control of inducible regulatory elements. In that case, the regulatory sequences of the endogenous gene are replaced by cognate recombination. As described herein, gene targeting is used to replace an existing regulatory region of a gene with a regulatory sequence isolated from a different gene or with a novel regulatory sequence synthesized by genetic engineering methods. To be Such regulatory sequences are composed of promoters, enhancers, backbone attachment regions, negative regulatory elements, transcription initiation sites, regulatory protein binding sites, or a combination of these sequences. Alternatively, the RNA produced
Alternatively, sequences that affect the structure or stability of the protein can be replaced, removed, or
Added or modified. These sequences include polyadenylation signals, mRNA stabilizing elements, splice sites, leader sequences that improve or modify the transport or secretion properties of proteins, or other sequences that alter or improve the function or stability of protein or RNA molecules. Composed of.

The targeting method may be to simply insert the regulatory sequences, for example to insert a new promoter or enhancer or both upstream of the gene and bring the gene under the control of the new regulatory sequences. Alternatively, the targeting method is to simply delete regulatory elements, eg, delete negative regulatory elements that are tissue specific. Alternatively, the targeting method replaces an existing element; for example, a tissue-specific enhancer is replaced with an enhancer that is specific for a cell type that is wider or different than the naturally occurring element. Here, naturally occurring sequences are deleted and new sequences are added. In any case, targeting method identification is facilitated by the use of one or more selectable marker genes flanking the target DNA to select cells in which the exogenous DNA binds to the host cell genome. I can. Targeted method discrimination is also facilitated by the use of one or more marker genes that exhibit negative selection properties. That is, the negative selectable marker is linked to the exogenous DNA, but is flanked by the target sequences, and those that carry out the correct cognate recombination with sequences in the host cell genome are negatively selectable. Does not have stable binding with the marker. Markers useful for this purpose include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene.

Gene targeting or gene activation techniques that can be used in accordance with this aspect of the invention include:
More specifically, US Pat. 5,272,071 addressed to Chapel; US Patent No.
5,578,461 to Sherwin et al .; International Application No. PCT / US92 /
09627 (WO93 / 09222) Selden et al .; and International Application No.
PCT / US90 / 06436 (WO91 / 06667) from Skoulchi and others;
These are hereby incorporated by reference in their entirety.

5.9 Transgenic Animals A preferred method of determining the biological function of a polypeptide of the present invention in the body is one of the More genes are overexpressed or inactive [Capekki, S
science 244: 1288-1292 (1989)]. Animals in which the gene is overexpressed under the regulatory control of exogenous or endogenous promoter elements are known as transgenic animals. An animal with an endogenous gene that has been inactivated by cognate recombination is called a "knockout" animal. Knockout animals are preferably non-human mammals, which are described in US Pat. It can be prepared as described in 5,557,032. Transgenic animals serve to determine the role that the polypeptides of the present invention play in biological processes, preferably disease states. The transgenic animal is useful as a model system for identifying compounds that regulate the metabolism of fats and oils. Transgenic animals, preferably non-human mammals, are included in US Pat.
5,489,743 and PCT Publication No. WO94 /
It can be created using the method described in 28122.

A transgenic animal in which all or part of the promoter of the polynucleotide of the present invention is activated or inactivated to change the level of expression of the polynucleotide of the present invention can be prepared. Deactivation can be performed using the homologous recombination method described above.
Activation either recruits cognate promoters to increase protein expression, or
Or it can be achieved by replacing it. A cognate promoter can be supplemented by the insertion of one or more heterologous enhancer elements known to confer promoter activity in a particular tissue.

The polynucleotides of the invention may also be expressed in animals that do not express the function of the CUB domain polypeptide, eg, using cognate recombination or knockout methods, or CUB.
Allows the development of animals expressing variants of the domain polypeptide.
Such animals serve as models to study the in vivo activity of CUB domain polypeptides and their modulators.

In a preferred method of determining the biological function of the polypeptide of the present invention in the body, one or more genes provided by the present invention are overexpressed in the reproductive system of animals using homologous recombination. Have been or have been deactivated [Capekki, S
science 244: 1288-1292 (1989)]. Animals in which the gene is overexpressed under the regulatory control of exogenous or endogenous promoter elements are known as transgenic animals. Animals with endogenous genes that are inactivated by cognate recombination are called "knockout" animals. Knockout animals are preferably non-human mammals, which are described in US Pat. It can be prepared as described in 5,557,032. Transgenic animals serve to determine the role that the polypeptides of the present invention play in biological processes, preferably disease states. Transgenic animals are useful as model systems for identifying compounds that regulate fat metabolism. Transgenic animals, preferably non-human mammals, are included in US Pat.
5,489,743 and PCT Publication No. WO94 / 2
It can be created using the method described in 8122.

When all or part of the polynucleotide (mom) of the promoter of the present invention is activated or inactivated to change the expression level of the polynucleotide of the present invention, the transgenic animal can be prepared. Deactivation can be performed using the homologous recombination method described above. Activation can be accomplished by supplementing or replacing cognate promoters to increase protein expression. A cognate promoter can be supplemented by the insertion of one or more heterologous enhancer elements known to confer promoter activity in a particular tissue.

Human CUB Domain Polypeptide Uses and Biological Activities The polynucleotides and proteins of the invention include one or more of the uses or biological activities identified herein (related to the assays listed herein). Are included). The use or activity described for the protein of the present invention is provided by administration or use of the protein or administration or use of a polynucleotide encoding the protein (eg, gene therapy or vector suitable for introducing DNA). . The reaction mechanism or pathology in a particular condition determines whether a polypeptide of the invention, a polynucleotide of the invention or a modulator thereof (activator or inhibitor) is effective in the subject in need of treatment. Accordingly, "therapeutic composition of the invention"
Is a composition comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof), or polypeptides of the invention (full length protein, mature protein and truncated or (Including its domain), or a compound or other substance that modulates the overall activity of the target gene product either in target gene / protein expression or in the activity level of the target protein. Such modulators are polypeptides, analogs, (variants), fragments and fusion proteins, antibodies and other binding proteins, compounds that directly or indirectly activate or inhibit the polypeptides of the invention (described herein). As identified, eg, a compound identified in a drug screening assay); antisense polynucleotides and polynucleotides suitable for triple helix formation, and, in particular, one or more epitopes of a polypeptide of the invention. Antibodies or other binding agents. The polypeptides of the present invention may likewise be involved in cell activation, or may be involved in one of the other physiological pathways mentioned herein.

5.10.1 Research Use and Practicality The polynucleotide provided by the present invention can be used by researchers for various purposes. For example, it has the following uses: To express a recombinant protein for analysis, characterization, or treatment; As a marker of tissue in which the corresponding protein is preferentially expressed (structurally, or tissue differentiation, development, Or at a particular stage of the disease); as a gel molecular weight marker; a chromosomal marker for chromosomal identification or mapping of relevant gene locations,
Or as a marker; to identify potential genetic disorders, to compare with the patient's endogenous DNA sequence; to use as a probe for hybridization and to discover new related DNA sequences; in gene fingerprinting methods As a source of information to guide PCR primers; as a probe to "exclude" known sequences in the process of finding other novel polynucleotides; oligomers for attachment to "gene chips" or other supports For selection and production; using DNA immunization technology to produce anti-protein antibody; and as an antigen for producing anti-DNA antibody or eliciting other immune response. The polynucleotide encodes a protein that binds or may bind to other proteins (eg, in receptor-ligand interactions).
, Using the polynucleotide in an interaction trap assay (eg Guris et al.,
Cell 75: 791-803 (1993)), it is also possible to identify the polynucleotide encoding the protein of the binding partner or to identify the inhibitor of the binding interaction.

The polypeptides provided by the invention can also be used in assays to determine biological activity as follows: in a multiple protein panel for large-scale screening; to raise antibodies or to elicit other immune responses. For; as a reagent in an assay for quantitatively determining the protein (or its receptor) level in the liquid of an organism; as a tissue marker in which the corresponding polypeptide is preferentially expressed (structural or tissue differentiation) , At specific stages of development or disease); and, of course, to separate correlated receptors or ligands. Proteins involved in these binding interactions can also be used to screen peptides, small molecule inhibitors of binding interactions, or agonists.

The polypeptides of the present invention also serve to make antibody substances that specifically immunoreact with CUB domain proteins. Antibodies that bind to the polypeptides of the invention and portions thereof (
Fab fragments, for example) can be used to confirm the presence of such polypeptides in a sample. Such confirmation is carried out using a suitable immunoassay and the polypeptide of the invention specifically bound by the antibody can be used as a positive control.

Any of the research materials can be developed to the level of reagents or kits and commercialized as a research product.

Methods for carrying out the above-indicated applications are well known to those skilled in the art. References disclosing such methods include, without limitation, the following: "Molecular Cloning: Laboratory Manual," Second Edition, Cold Spring Ha.
rbor Laboratory Press, Sambrook, J .; , E.
F. Flitch and T. Maniatis, 1989, and Enzymology Methodology: A Guide to Molecular Cloning Techniques, Academic Press, Burger, S. et al. L. And A. R. Kimmel, 1987.

5.10.2 Nutritional Use The polynucleotides and polypeptides of the present invention can also be used as nutritional sources or nutraceuticals. Such applications can be used without limitation as a protein or amino acid nutritional supplement and as a source of carbon, nitrogen or carbohydrate. In that case,
The polypeptide or the polynucleotide of the present invention can be added to the feed of a specific organism or can be administered in the form of solid or liquid such as powder, tablet, solution, suspension or capsule. In the case of a microorganism, the polypeptide or polynucleotide of the present invention can be added to the culture medium in which the microorganism is cultivated.

Furthermore, the polypeptides of the invention can be used as molecular weight markers and as dietary supplements. For example, the polypeptide comprising SEQ ID NO: 4 has a molecular weight of approximately 12 kDa in the untreated, non-glycosylated state. Protein supplements are well known, and a suitable method of formulating a dietary supplement containing the polypeptide of the present invention can be applied at the technical level of the food manufacturing industry.

5.10.3 Cytokines and Cell Proliferation / Differentiation Activities The polypeptides of the invention exhibit cytokine, cell proliferation (either induction or inhibition), cell differentiation (either induction or inhibition) activity. , Or may induce the production of other cytokines in some cells. The polynucleotide of the present invention can encode a polypeptide exhibiting such characteristics. Many protein factors discovered to date, including all known cytokines, showed activity in one or more factor dependent cell proliferation assays. Therefore, the assay is convenient as a method for confirming cytokine activity. The activity of the therapeutic composition of the present invention has been demonstrated by a factor dependent cell proliferation assay, the cell lines of which include: 32D, DA2.
DA1G, T1O, B9, B9 / 11, BaF3, MC9 / G, M + (preB
M +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2
, TF-1, Mo7e, CMK, HUVEC, and Caco.

Therapeutic compositions of the present invention may be used in the following cases: T-cell or thymocyte proliferation assays include, without limitation, those set forth below:
Current Protocols in Immunology, Editing J. E. Corrigan, A. M
． Klewis Beek, D.C. H. Margries, E. M. Shevac, W. Strobe, published. Greene Publishing A
associates and Wiley-Interscience (3rd
In vitro assay of murine lymphocyte function 3.1-3.19; Chapter 7, Immunology studies in humans); Takai et al. Immunol. 137: 3494-
3500, 1986; Bertanioli et al., J. Immunol. 145
: 1706-1712, 1990; Bertanioli et al., Cell immunology 133.
: 327-341, 1991; Bertanioli et al. Immunol.
149: 3778-3783, 1992; Bowman et al. Immu
nol. 152: 1756-1761, 1994.

Cytokine production and / or pancreatic, lymphoid, or thymocyte proliferation assays include, without limitation, those described below: Stimulation of Polyclonal T Cells, Klewisbeek, A .; M. and Shebach, E .; M. Current protocols in immunology. J. E. e. a. Corrigan edit. Vol I pp. Three
． 12.1-3.12.14, John Wiley and Sons,
Toronto. 1994; and measurement of mouse and human interleukin-γ, Schreiber, R. et al. D. Current protocols in immunology. J
． E. e. a. Corrigan Editing, Vol 1 pp. 6.8.1-6.8
． 8, John Wiley and Sons, Toronto. 19
94.

Assays for hematopoietic and lymphopoietic cell proliferation and differentiation include, without limitation, those set forth below: “Measurement of human and murine interleukins 2 and 4”, Bottomley, K. et al. Davis, L.A. S. And Lipsky P.C. E. "Current Protocols in Immunology" J. E. e. a. Corrigan eds. Vol 1 pp
． 6.3.1-6.3.12, John Wiley and Sons,
Toronto. 1991; Defreys et al., J. Exp. Med. 1
73: 1205-1211, 1991; Morrow et al., Nature 336: 690.
-692, 1988; Greenberger et al., Proc. Natl. Ac
ad. Sci. U. S. A. 80: 2931-2938, 1983;
Measurement of mouse and human interleukin 6-Nordane, R .; Current proto in immunology. Cole J. E. Corrigan eds. Vol 1 pp. 6.6
． 1-6.6.5, John Wiley and Sons, Toronto
． 1991; Smith et al., Proc. Natl. Aced. Sci
． U. S. A. 83: 1857-1861, 1986; Measurement of human interleukin II-Bennett, F .; , Gianotti, J.M. , Clark, S.K. C. And Turner, K .; J. Current protocols in immunology. J. E. Corrigan edit. Vol 1 pp. 6.15.1 Joh
n Wiley and Sons, Toronto. 1991; Measurement of mouse and human interleukin 9-Cialetta A .; , Gianotti, J.M.
, Clark, S.K. C. And Turner, K .; J. Current protocols in immunology. J. E. Corrigan eds. Vol 1 pp-6
． 13.1, John Wiley and Sons, Toronto.
1991.

Antigen reaction assays of T-cell clones, which specifically identify proteins that affect APC-T cell interactions and T cell direct effects by measuring proliferation and cytokine production, Including without limitation: Current Protocols in Immunology, Compilation: J. E. Corrigan, A. M. Chris Beak, D.D. H. Margries, E. M. Shevac, W.
Strober, Publisher Green Publishing Associ
ates and Wiley-Interscience (Chapter 3, in vitro assay of mouse leukocyte function; Chapter 6, cytokines and their cell receptors; Chapter 7)
Chapter, Immunology Studies in Humans); Weinberger et al., Proc. Nad
． Acad. Sci. USA 77: 6091-6095, 1980;
Weinberger et al., Eur. J. Immun. 11: 405411,
1981; Takai et al., J. Immunol. 137: 3494-35
00, 1986; Takai et al., J. , Immunol. 140: 508-
512, 1989.

5.10.4 Stem Cell Growth Factor Activity The polypeptides of the present invention exhibit stem cell growth factor activity and include primordial germ cells, fetal stem cells, hematopoietic stem cells and / or germ cell stem cells in a pull reporter (pluripotent). ) And totipotent (totipotent) stem cells may be involved in proliferation, differentiation and survival. Administration of the polypeptides of the present invention to stem cells in vivo or in vitro may maintain or expand the total number of cells in the totipotent or pullpotent state. It may be useful in the reconstruction of damaged or diseased tissue, transplantation, biopharmaceutical manufacturing, biosensor development. The ability to produce large quantities of human cells has important applications in such areas as: Human protein production. Currently, to treat Parkinson's disease, Alzheimer's disease, and other neurodegenerative diseases must come from non-human sources, donors, cell transplants, etc .; tissues for transplantation, eg bone marrow, skin, cartilage. , Tendons, bones, muscles (including myocardium), blood vessels, retinas, nerve cells, gastrointestinal cells; and organs for transplantation, such as kidney, liver, pancreas (including pancreatic islet cells), heart and lung.

It is contemplated that a plurality of different exogenous growth factors and / or cytokines may be administered with a polypeptide of the invention to achieve the desired effect,
Growth factors include: any of the growth factors mentioned in this document,
Other stem cell maintenance factors, and especially stem cell factor (SCF), leukemia inhibitory factor (LI
F), Flt-3 ligand (Flt-3L), interleukin, recombinant soluble IL-6 receptor fused to IL6, macrophage inflammatory protein I-alpha (M
IP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO)
), Platelet factor 4 (PF-4), growth factor derived from platelets (PDGF), nerve growth factor, and fibroblast basal growth factor (bFGF).

Totopotent stem cells give rise to almost all mature cell types, so proliferating these cells in culture can facilitate the production of large amounts of mature cells. Techniques for culturing stem cells are known in the art, and the administration of the polypeptide of the present invention may be administered in combination with other growth factors and / or cytokines, but is expected to enhance the survival rate and proliferation of stem cells. ing. This can be accomplished by administering the polypeptides of the invention directly to the culture medium. Alternatively, stromal cells transfected with a polynucleotide encoding a polypeptide of the invention can be used as a feeder layer for stem cells in culture or in the body. Stromal cells of the feeder layer include embryonic body bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see US Pat. No. 5,690,926).

The stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This allows the generation of an undifferentiated totipotent / pull retentive stem cell line, which is beneficial as it is, but can also be differentiated into the desired mature cell type. These stable cell lines can be used as a source of undifferentiated totipotent / pull repotent mRNA for the construction of cDNA libraries and templates for polymerase chain reaction experiments. These studies allow the isolation and identification of differentially expressed genes in stem cells that regulate stem cell proliferation and / or maintenance. Proliferation and maintenance of totipotent stem cells is useful in the treatment of many pathological conditions. For example, the polypeptides of the present invention manipulate stem cells in culture to produce neuroepithelial cells that supplement cells damaged by disease, autoimmune disease, accidental injury, or genetic disorders, It can be used for replacement. The polypeptide of the present invention is useful for inducing the proliferation of nerve cells and regenerating nerve and brain cell tissues. That is, it is used for the treatment of diseases and neuropathy of central and peripheral nerves, and mechanical and traumatic disorders accompanied by degeneration, death, or injury of nerve cells and nerve tissues. Further, the expanded stem cells can be genetically modified for gene therapy and after transplantation to reduce host rejection of the replacement tissue.

The expression of the polypeptides of the invention and its effect on stem cells can also be manipulated to direct the controlled differentiation of stem cells into more differentiated cell types. A widely applicable method of taking a pure population of a particular differentiated cell type from an undifferentiated stem cell population is to use a cell type-specific promoter that drives a selectable marker. The selectable marker only allows cells of the desired type to survive. For example, stem cells are cardiomyocytes (Woobas et al., Differentiation, 48:17.
3-182, (1991); Krug et al., J. Clin. Invest, 9
8 (1): 216-224, (1998)) or skeletal muscle cells (Browder,
L. W. Tissue Engineering Theory eds. Lanza and others, Academic Pre
ss (1997)). As an alternative method, the stem cells are cultivated in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the present invention that promotes differentiation by suppressing the activity of endogenous stem cell factor, thereby imparting directionality. Differentiation of the established stem cells can be achieved.

In vitro cultures of stem cells can be used to determine whether the polypeptides of the invention exhibit stem cell growth factor activity. Stem cells have been isolated from a variety of cell sources, including hematopoietic stem cells and embryonic stem cells, and have been identified by Thompson et al. In the presence of the polypeptides of the invention alone or in combination with other growth factors or cytokines.
roc. Natl. Acad. Sci, U.S.S. S. A. , 92: 784
Incubated in feeder layers as described in 4-7848 (1995). The ability of the polypeptides of the present invention to induce stem cell proliferation is determined by colony formation on a semi-solid support, as described, for example, in Bernstein et al., "Blood," 77: 2316-2321 (1991). It

5.10.5 Hematopoiesis-regulating activity The polypeptides of the present invention are associated with hematopoiesis-regulating and thus of treatment of bone marrow or lymphoid cell disorders. It has a minor biological activity in supporting colony-forming cells or in supporting factor-dependent cell lines, indicating its involvement in the regulation of hematopoiesis, for example, supporting growth and proliferation of erythroid progenitor cells alone or with other cytokines. Thereby exhibiting utility, for example, in combination with various anemia treatments or irradiation / chemotherapy to stimulate the production of erythroid precursors and / or erythrocytes, and For example, supporting the growth and proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie conventional CSF activity), which are useful in combination with chemotherapy to prevent or treat the resulting myelosuppression. In, the growth and proliferation of megakaryocytes, and hence the growth of platelets
Supports proliferation and thereby various platelet disorders (diseases) such as thrombocytopenia
In general, instead of or as an adjunct to platelet transfusion, allowing the prevention or treatment thereof, and / or the growth of hematopoietic stem cells to allow maturation to any or all of the above hematopoietic cells. And in favor of proliferation, therefore
Various stem cell disorders, such as those usually treated by transplantation, including but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria, and in vivo or in vitro (ie, bone marrow transplantation, or Limited to, for example, repopulation of post-irradiation / chemotherapy stem cell fractions as normal cells or as genetically engineered cells for gene therapy in either peripheral progenitor cell transplantation (in conjunction with homologous or xenogeneic) No therapeutic utility is demonstrated.

[0182]   The therapeutic composition of the present invention can be used in:

[0183]   Suitable assays for proliferation and differentiation of various hematopoietic cell lines have been cited above.

Embryonic stem cell differentiation (to identify other proteins that affect embryonic differentiation hematopoiesis)
For the assay, Johannsson et al. Cellular Biolog
y 15: 141-151, 1995; Keller et al. , M
olecular and Cellular Biology 13: 473
-486, 1993; McClanahan et al. , Blood
81: 2903-2915, 1993, but is not limited thereto.

Assays for stem cell survival and differentiation (identifying proteins that regulate lympho-hematopoiesis among others) are based on the methylcellulose colony form.
ing assays, Freshney, M .; G. In Cultu
re of Hematopoietic Cells. R. 1. Fre
shney, et al. eds. Vol pp. 265-268,
Wiley-Liss, Inc. , New York, N .; Y. 199
4; Hirayama et al. , Proc. Natl. Acad
． Sci. USA 89: 5907-5911, 1992; Primi.
five hernatopoietic colony forming c
ells with high proliferative potenti
al, McNiece, I.M. K. and Bridgedell, R.A.
A. In Culture of Hematopoietic Cells
． R. I. Freshney, et al. eds. Vol pp
． 23-39, Wiley-Liss, Inc New York, N.
． Y. 1994; Neben et al. , Experimental
Hematology 22: 353-359, 1994; Cobble.
Stone area forming cell assay, Ploem
acher, R.A. E. In Culture of Hematopoi
tic Cells. R. I. Freshney, et al. e
ds. Vol pp. 1-21, Wiley-Liss, Inc. ，
New York, N.M. Y. 1994; Long term bone
marrow cultures in the presence of s
temporal cells, Spooner, E.I. , Dexter,
M. and Allen, T .; In Culture of Hemat
opetic Cells. R. 1. Freshney, et a
l. eds. Vol pp. 163-179, Wiley-Liss,
Inc. , New York, N .; Y. 1994; Long ter
m culture initiating cell assay, Sut
herland, H .; J. In Culture of Hematop
oietic Cells. R. 1. Freshney, et al.
eds. Vol pp. 139-162, Wiley-Liss, I
nc. , New York, N .; Y. There are, but not limited to, the tests described in 1994.

5.10.6 Tissue Growth Activity The polypeptides of the present invention also provide for growth or regeneration of bone, cartilage, tendon, ligament and / or nerve tissue, and wound healing and tissue repair or replacement or burns, incisions or ulcers. Is also involved in the healing of.

Polypeptides of the present invention that promote the growth of ligaments and / or bone when bone is not properly formed can be used in the healing of fractures and ligament injuries or defects in humans and other animals. A polypeptide of the present invention, an antibody,
Compositions of binding partners or other modulators can be used prophylactically in mitigating subcutaneous fractures as well as open fractures and may also improve prosthesis fixation. New bone formation induced by osteogenic substances helps repair congenital, traumatic, or oncological resection craniofacial defects, and is also useful in cosmetic surgery.

The polypeptides of the present invention are also involved in attracting osteogenic cells, stimulating osteogenic cell growth, and inducing osteogenic cell precursor differentiation. Tissue destruction processes mediated by stimulation of bone and / or ligament repair, or both, or by inflammation or inflammatory processes (
Osteoporosis due to the inhibition of collagenase activation, osteoclast activation, etc.)
Treatment of osteoarthritis (osteoarthritis), bone degenerative diseases, or periodontal disease may also be possible using the compositions of the present invention.

Another category of tissue regeneration activity involving the polypeptides of the invention is tendon / ligament-like tissue formation. Induction of tendon / ligament-like tissue or other tissue formation can lead to healing of tendon or ligament damage, malformations and other tendon or ligament defects in humans and other animals when these tissues are not properly formed. Can be used. A formulation using such a tendon / ligament-like tissue-inducing protein can be used not only for improving fixation of a tendon or a ligament to bone or other tissue, for repairing a defect of the tendon or a ligament tissue, but also for tendon or a ligament tissue. It may also be used as a preventive measure to prevent damage. De novo tendon / ligament-like tissue formation induced by the composition of the present invention contributes to the repair of congenital, traumatic, or other causes of tendon or ligament defects, and also for tendon or ligament attachment and repair. It can also be used for cosmetic surgery. The composition of the present invention may be used to induce tendon or ligament forming cells, stimulate growth of tendon or ligament forming cells, induce differentiation of tendon or ligament forming cell precursors, or act in vivo on tendon / ligament forming cells or tissue repair. To provide an environment for inducing the growth of in vitro precursors. The compositions of the present invention are also useful for treating tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The composition may also include a sequestering agent as a suitable substrate and / or carrier as is known in the art.

The compositions of the present invention are not only useful for the proliferation of nerve cells and regeneration of nerve and brain tissue, ie, the treatment of central and peripheral nervous system diseases and disorders, but also for nerve cells and nerve tissue. It is also useful for mechanical and traumatic disorders including degeneration, death or trauma.
More precisely, peripheral nervous system diseases such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, as well as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Drager syndrome. It can also be used for the treatment of central nervous system diseases such as. Further, the conditions treated by the present invention include spinal cord disorders, mechanical, traumatic disorders such as brain trauma, and cerebrovascular disorders such as stroke.

The compositions of the present invention also include pressure ulcers, ulcers associated with vascular dysfunction,
It may help promote better and faster closure of non-healing wounds, including but not limited to surgical and traumatic wounds.

The compositions of the present invention also include organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, myocardium) and vascular (including vascular endothelium) tissue and the like. It is also involved in the production or regeneration of other tissues such as, or in promoting the proliferation of cells forming such tissues. Part of the desired effect is due to the inhibition or modulation of fibrotic scars that allow the regeneration of normal tissue. The polypeptides of the present invention may also exhibit angiogenic effects.

The compositions of the present invention are also useful for enteroprotection, or treatment of lung and liver fibrosis, conditions resulting from reperfusion injury of various tissues and systemic cytokine injury.

The compositions of the present invention are also useful for promoting or inhibiting the differentiation of the aforementioned tissues from precursor tissues or precursor cells, or for inhibiting the growth of said tissues.

[0195]   The therapeutic composition of the present invention can be used in:

The International Patent Publ was used for the tissue production activity assay.
ication No. WO95 / 16035 (bone, cartil
age, tendon); International Patent P
publication No. WO95 / 05846 (never, ne
uronal); International Patent Public
ation No. WO91107491 (skin, endother
ium), and is not limited to this.

For the wound-healing activity assay, Winter, Epidermal Wound H
ealing, pp. 71-112 (Maibach, H. I. a.
nd Rovee, D.N. T. , Eds. ), Year Book Me
digital Publishers, Inc. , Chicago, as
modified by Eaglestein and Mertz, J.M.
Invest. There is a test described in Dermatol 71: 382-84 (1978), but not limited thereto.

5.10.7 Immune Function Stimulating or Suppressing Activity The polypeptides of the present invention also exhibit immunostimulating or immunosuppressive activity, including, but not limited to, the activity according to the assays described herein. The polynucleotide of the present invention can encode a polypeptide exhibiting such activity. The protein can be used in the treatment of various immunodeficiencies and immune disorders such as severe combined immunodeficiency disease (SCID), eg, regulation of growth and proliferation of T and / or B lymphocytes (up or down) as well as NK. It is also useful in affecting the cytolytic activity of cells and other cell populations. These immunodeficiencies are not only due to bacterial or fungal infections but also genetically or virally (eg HI
V), or may be caused by an autoimmune disease. More precisely, HINT, hepatitis virus, herpes virus, actinomycetes, Leishman
ia spp (Leishmania), malaria spp. Infectious diseases caused by viral, bacterial, fungal or other infectious diseases, including various fungal infections such as infections by Candida and candidiasis, etc. may be treatable with the proteins of the invention. Of course, in this respect, the proteins of the invention are also useful when boosting the immune system is generally desired, ie in the treatment of cancer.

Autoimmune diseases treated with the proteins of the invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunity. Includes thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease, and autoimmune inflammatory eye disease.
Such proteins of the invention (or antagonists thereof, including antibodies) are useful for allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory disorders (eg, anaphylaxis, serum sickness, drug reactions, Food allergy, insect venom allergy, mastocytosis, allergic rhinitis, hypersensitivity pneumonia, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, polymorphic erythema, Stevens-Johnson syndrome , Allergic conjunctivitis, atopic keratoconjunctivitis, sexually transmitted keratoconjunctivitis, giant papillary conjunctivitis, contact allergy). Other conditions in which immunosuppression is desired (including, for example, organ transplantation) also include proteins of the invention (
Or its antagonists). The therapeutic effect of the polypeptide or its antagonist on the allergic reaction is evaluated by the cumulative contact increase test (Lastbomb et al., Toxicology 125: 59-).
66, 1998), skin puncture test (Hoffmann et al.,
Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501).
-9), and mouse local lymph node test assay (Kimber et a.
l. , J. Toxicol. Environ. Health 53: 5
63-79) and other in vivo animal models.

When using the proteins of the invention, it is also possible to modulate the immune response in a number of ways. Down-regulation is done in a manner that suppresses or blocks an already ongoing immune response or is involved in the defense of the induction of an immune response. The function of activated T cells is suppressed by the suppression of the T cell response, the induction of specific tolerance in the T cells, or both. Immunosuppression of the T cell response is generally an active, non-antigen specific process that requires continuous exposure of T cells to suppressors. Tolerance associated with induction of non-responsiveness or no response in T cells is distinguished from immunosuppression in that it is generally antigen-specific and persists after tolerant exposure is discontinued. . Operationally, tolerance is indicated by the lack of a T cell response upon re-exposure to a specific antigen in the absence of tolerizing agent.

Down-regulation or protection of one or more antigen functions, including but not limited to B lymphocyte antigen function (such as B7), for example protection of high levels of lymphokine synthesis by activated T cells , In tissue, skin and organ transplant conditions, and in graft-versus-host disease (GVHD). For example, blocking T cell function should result in reduced tissue destruction in tissue transplants. Generally, in tissue transplantation, transplant rejection is initiated by the recognition of T cells as foreign by an immune reaction that destroys the graft. Administration of the therapeutic compositions of the present invention interferes with the synthesis of cytokines by immune cells such as T cells and thus acts as an immunosuppressant. Moreover, the lack of simultaneous stimulation
The T cells will be desensitized (anergized), which may induce tolerance in the subject. Induction of long-term tolerance by B lymphocyte antigen blocking reagents obviates the need for repeated administration of these blocking agents. In order to obtain sufficient immunosuppression or tolerance in a subject, it may be necessary to block the function of the B lymphocyte antigen combination.

The efficacy of certain therapeutic compositions in the prevention of organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of suitable systems that can be used are allogeneic heart transplants in rats and xenogeneic islet cell transplants in mice, both of which are described in Len.
schow et al. , Science 257: 789-792 (1
992) and Turka et al. , Proc. Natl. Ac
ad. It has been used to test the immunosuppressive effect of in vivo CTLA4Ig fusion proteins as described in Sci USA, 89: 11102-11105 (1992). Further, a mouse model of GVHD (graft-versus-host disease) (Paul ed., Fundamental Immunology,
Raven Press, New York, 1989, pp. 84
6-847) can be used to measure the effect of the therapeutic composition of the present invention on the development of this disease.

Blocking antigen function is also therapeutically useful for the treatment of autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells in response to self-tissues, and enhanced production of cytokines and autoantibodies involved in the pathology of the disease. Protection of activation of autoreactive T cells reduces or eliminates pathology.
Administration of agents that block T cell stimulation can be used to suppress T cell activation and prevent the production of autoantibodies or T cell-derived cytokines involved in the process of this disease. Furthermore, the potency of blocking reagents may induce antigen-specific tolerance of autoreactive T cells leading to long-term remission from this disease. The efficacy of blocking reagents in preventing or ameliorating autoimmune diseases can be measured using well characterized animal models of human autoimmune diseases. Examples of this include mouse experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD and BB rats, mouse experimental myasthenia gravis. Disease (Paul ed., Fundamental Immunology,
Raven Press, New York, 1989, pp. 840
-856)).

Upregulation of antigen function (eg, B lymphocyte antigen function) as a means of raising an immune response is also useful in therapy. Up-regulation of the immune response is done either by enhancing the existing immune response or by eliciting an early immune response. For example,
Enhanced immune responses may be useful in the case of viral infections, including systemic viral diseases such as influenza, colds, and encephalitis.

Alternatively, the antiviral immune response of an infected patient may result in the removal of T cells from the patient and expression of a peptide of the invention, or with a stimulatory form of a soluble peptide of the invention together with a viral antigen pulse. It may be enhanced by co-stimulating T cells ex vivo with activated APC and then reintroducing activated T cells into the patient. Another method of enhancing the anti-viral immune response is to isolate infected cells from a patient and allow them to express the proteins of the invention described herein such that the cells express all or part of the protein on their surface. One would transfect with the encoding nucleic acid and reintroduce the transfected cells into the patient. Here, the infected cells would be able to send costimulatory signals and thereby activate T cells in vivo.

The polypeptides of the invention provide the stimulatory signals necessary to induce a T cell mediated immune response against transfected tumor cells. In addition, MHC class I
Or no MHC class II molecule or sufficient amount of MHC
Tumor cells that are unable to re-express class I or MHC class II molecules are treated with MHC class I alpha chain proteins and β ₂ microglobulin proteins, or all or part of MHC class II alpha chain proteins and MHC class II beta chain proteins ( For example, it can be transfected with a nucleic acid encoding all or part of the (cytoplasmic domain truncation), thereby expressing the MHC class I or MHC class II protein on the cell surface. B lymphocyte antigen (eg, B7-1, B7-
The expression of the appropriate class I or class II MHC with a peptide having the activity of 2, B7-3) induces a T cell-mediated immune response against the transfected tumor cells. Optionally, MH such as invariant chain
A gene encoding an antisense construct that blocks the expression of C class II binding proteins is co-transfected with DNA encoding a peptide having B lymphocyte antigen activity to promote presentation of tumor-associated antigens and tumor-specific antigens. Can induce specific immunity. In this way, induction of a T cell-mediated immune response in a human subject is
The tumor-specific tolerance in the subject can be sufficiently overcome.

The activity of the protein of the present invention, although there are other methods, is measured by the following method.

[0208] A suitable thymus or splenocyte cytotoxicity assay may be performed using the Current Protocol.
ols in Immunology, Ed by J.M. E. Colig
an, A. M. Kruisbeek, D .; H. Margulies
, E. M. Shevach, W.M. Strober, Pub. Gr
eene Publishing Associates and Wiley
-Interscience (Chapter 3, In Vitro a
says for Mouse Lymphocyte Function
3.1-3.19; Chapter 7, Immunological stu
Dies in Humans); Herrmann et al. , Pr
oc. Natl. Acad. Sci. USA 78: 2488-249.
2, 1981; Herrmann et al. , 1. Immunol
． 128: 1968-1974, 1982; Handa et al. ，
J. Immunol. 135: 1564-1572, 1985; Ta.
kai et al. , 1. Immunol. 137: 3494-350
0, 1986; Takai et al. , J. Immunol. 1
40: 508-512, 1988; Bowman et al., J. Am.
Virology 61: 1992-1998; Bertagnollie.
t al. , Cellular Immunology 133: 327-34.
1, 1991; Brown et al. , J. Immunol. 1
53: 3079-3092, 1994, but not limited thereto.

For assaying T cell-dependent immunoglobulin responses and isotype switching (identifying proteins that modulate the T cell-dependent antibody response and affect the Thl / Th2 profile from others), see Maliszewski, J. et al. Immunol
． 144: 3028-3033, 1990; and Assays for.
B cell function: In vitro antibody
production, Mond. J. and Brunswick
k, M. In Current Protocols in Immuno
logy. J. E. e. a. Coligan eds. Vol 1
pp. 3.8.1-3.8.16, John Wiley and Son
s, Toronto. There are, but not limited to, the tests described in 1994.

The Current Protocols in Immm was used for mixed lymphocyte reaction (MLR) assays (mainly identifying proteins that produce Th1 and CTL reactions from others).
unology, Ed by J. et al. E. Coligan, A .; M.
Kruisbeek, D .; H. Margulies, E.I. M. Sh
evach, W.E. Strober, Pub. Greene Publi
shing Associates and Wiley-Interdie
nce (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19;
Chapter 7, Immunological studies in Hu
mans); Takai et al. , J. Immunol. 137
: 3494-3500, 1986; Takai et al. , J. I
mmunol. 140: 508-512, 1988; Bertagnol.
li et al. , J. Immunol. 149: 3778-3783
, 1992, but is not limited to them.

A dendritic cell-dependent assay (identifying the protein expressed by dendritic cells that activate naive T cells from others) is described by Guery et al. ，
J. Imimmol. 134: 536-544, 1995; Inaba.
et al. , Journal of Experimental Med
icine 173: 549-559, 1991; Macatonia e.
t al. , Journal of Immunology 154: 507.
1-5079, 1995; Porgado et al. , Journa
l of Experimental Medicine 182: 255-2
60, 1995; Nair et al. , Journal of Vi
LOGY 67: 4062-4069, 1993; Huang et.
al. , Science 264: 961-965, 1994; Maca.
tonia et al. , Journal of Experimenta
I Medicine 169: 1255-1264, 1989; Bhar.
dwaj et al. , Journal of Clinical Inv
and Eg. 94: 797-807, 1994; and Ina.
ba et al. , Journal of Experimental M
edicine 172: 631-640, 1990, but not limited thereto.

Lymphocyte survival / apoptosis assays (identifying proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) are described by Darzynkiewicz et al. , Cytomet
ry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca.
et al. , Cancer Research 53: 1945-195.
1, 1993; Itoh et al. , Cell 66: 233-24.
3, 1991; Zacharchuk, Journal of Immu.
145: 4037-4045, 1990; Zamai et.
al. , Cytometry 14: 891-897, 1993; Go.
rczyca et al. , International Journal
of Oncology 1: 639-648, 1992, but not limited thereto. To assay for proteins that affect T cell commitment and early stages of development:
Antica et al. , Blood 84: 111-117, 19
94; Fine et al. , Cellular Immunology
155: 111-122, 1994; Galy et al. , Blo
od 85: 2770-2778, 1995; Toki et al. ，
Proc. Nat. Acad Sci. USA 88: 7548-755.
1, 1991, but not limited to these.

5.10.8 Activin / Inhibin Activity The polypeptides of the present invention also exhibit activin or inhibin related activity. The polynucleotide of the present invention encodes a polypeptide exhibiting such properties. Inhibin is characterized by its ability to suppress the release of follicle stimulating hormone (FSH), while activin is characterized by its ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the polypeptide of the present invention is
Alone or as a heterodimer with a member of the inhibin family, it is useful as a contraceptive based on the function of inhibin to reduce fertility in female mammals and reduce spermatogenesis in male mammals. is there. Infertility in these mammals can be induced by administering a sufficient amount of another inhibin. Alternatively,
The polypeptide of the present invention can be used as a homodimer or as a heterodimer having an inhibin group subunit of another protein as an FS from anterior pituitary cells.
Based on the function of activin molecules in the stimulation of H (follicle-stimulating hormone) release,
It is useful as a fertility-inducing therapeutic agent. For example, US Pat. 4,798,8
See 85. The polypeptides of the present invention are also useful for advancing fertility initiation in sexually immature mammals, such as in cows, sheep and pigs, to increase lifelong fertility of such livestock. Is.

The activity of the polypeptide of the present invention can be measured by the following method, although there are other methods.

Assays for activin / inhibin activity are described in Vale et al. , End
ocrinology 91: 562-572, 1972; Ling et
al. , Nature 321: 779-782, 1986; Vale.
et al. , Nature 321: 776-779, 1986; M.
ason et al. , Nature 318: 659-663, 198.
5; Forge et al. , Proc. Natl. Acad.
Sci. USA 83: 3091-3095, 1986, but is not limited thereto.

5.10.9 Chemotaxis / chemomotility activity Polypeptides of the invention are, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells or / and It is involved in chemotactic or chemokinetic activity of mammalian cells including endothelial cells. The polynucleotide of the present invention can encode a polypeptide exhibiting such an attribute. The activity of chemotactic and chemokinetic receptors can be used to recruit or attract a desired cell population to a desired site of action. Chemotactic / chemokinetic compositions (eg proteins, antibodies,
Binding partners, or modulators of the invention, provide particular benefits not only in the treatment of localized infections, but also in the treatment of wounds and other tissue trauma. For example,
The attraction of lymphocytes, monocytes or neutrophils to the tumor or site of infection may improve the immune response to the tumor or infectious agent.

A protein or peptide has chemotactic activity for a particular cell population if it can directly or indirectly stimulate the direction or movement of such cell population.
Preferably, the protein or peptide has a function of directly stimulating directed movement of cells. Whether a particular protein has chemotactic activity on a cell population
It can be easily determined by subjecting the protein or peptide to a well-known cell chemotaxis assay.

[0218]   The therapeutic composition of the present invention can be used in:

An assay for chemotactic activity (identifying proteins that induce or prevent chemotaxis) is determined not only by the ability of the protein to induce the attachment of one cell population to another.
It consists of a test that also tests the ability of a protein to induce cell migration within a membrane.

A suitable protocol for migration and attachment is the Current Protocols in
Immunology, Ed by 1. E. Coligan, A .;
M. Kruisbeek, D .; H. Marguiles, E .; M
． Shevach, W.M. Strober, Pub. Greene P
Publishing Associates and Wiley-Inter
science (Chapter 6.12, Measurement o
f alpha and beta Chemokines 6.12.1-6
． 12.28; Taub et al. J. Clin. Invest.
95: 1370-1376, 1995; Lind et al. APM
IS 103: 140-146, 1995; Muller et al E.
ur. J. Immunol. 25: 1744-1748; Gruber.
et al. J. of Immunol. 152: 5860-5867
, 1994; Johnston et al. J. of Immuno
l. 153: 1762-1768, 1994, but is not limited thereto.

5.10.10 Chemotaxis and Thrombolytic Activity The polypeptides of the present invention may also be involved in hemostasis, thrombolysis or thrombosis. The polynucleotide of the present invention can encode a polypeptide exhibiting such an attribute. The composition is useful for promoting coagulation and other hemostasis in the treatment of various coagulation disorders, including genetic disorders such as hemophilia, or in the treatment of wounds resulting from trauma, surgery or other causes. Is. The compositions of the present invention are also useful in the treatment or prevention of thrombotic lysis or formation inhibition and conditions resulting therefrom (such as heart or central nervous vessel infarction (eg, stroke)).

[0222]   The therapeutic composition of the present invention can be used in:

For assaying hemostasis and thrombotic activity, see Linet et al. , J. Clin.
Pharmacol. 26: 131-140, 1986; Burdic.
k et al. , Thrombosis Res. 45: 413-419
, 1987; Humphrey et al. , Fibrinolysi
s 5: 71-79 (1991); Schaub, Prostaglan.
dins 35: 467-474, 1988, but is not limited thereto.

5.10.11 Cancer Diagnosis and Treatment The polypeptides of the invention are also involved in the production, growth or metastasis of cancer cells. Detection of the presence or amount of a polynucleotide or polypeptide of the invention is useful in diagnosing and / or prognosing one or more cancers. For example, the polynucleotide of the present invention /
Increased presence or expression of the polypeptide may indicate a hereditary risk of cancer, a precancerous condition or a progressive malignancy. Conversely, a deficiency in the gene or the absence of this polypeptide may be associated with a cancerous condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be diagnostic or predictive.

The treatment of cancer involves the inhibition of tumor cell proliferation, inhibition of angiogenesis (the growth of new blood vessels required to support tumor growth) and / or reducing tumor cell motility and invasiveness to prevent metastasis. Promotes tumor regression. The therapeutic compositions of the present invention are effective in adult and pediatric tumors, including solid / malignant tumors, locally advanced tumors, human soft tissue sarcomas, metastatic cancers such as lymphatic metastases, multiple bone marrow. Hematopoietic malignancies such as tumors, acute / chronic leukemia, lymphoma, etc. Breast cancer including cell carcinoma and ductal cancer, esophageal cancer, gastric cancer, colon cancer, gastrointestinal cancer including colorectal cancer, and urinary organs including polyps associated with colorectal abnormal growth, pancreatic cancer, liver cancer, bladder cancer and prostate cancer Cancer, ovarian cancer, uterine (including endometrium) cancer, malignant tumor of female reproductive organs including solid tumor of follicle, renal cancer including renal cell carcinoma, endogenous brain tumor, neuroblastoma,
Astrocyte (astrocytic) brain tumor, glioma, brain tumor including metastatic tumor cell invasion in the central nervous system, bone cancer including osteoma, melanoma, human skin keratinocyte tumor progression, squamous cell carcinoma , Basal cell carcinoma, hemangiopericytoma and K
Includes arposi sarcoma.

A polypeptide, polynucleotide or modulator of a polypeptide of the invention, including inhibitors and stimulators of biological activity of the polypeptide of the invention, may be administered for the treatment of cancer. The therapeutic composition can be administered alone or in combination with adjunctive therapies for cancer, such as surgery, chemotherapy, radiation therapy, hyperthermia and laser therapy, in a therapeutically effective dose, with beneficial effects such as , May reduce the size of the tumor, reduce the growth rate of the tumor, inhibit metastasis, or improve the overall clinical status without necessarily eradicating the cancer.

The composition can be administered in a therapeutically effective amount as part of an anti-cancer cocktail.
The anti-cancer cocktail is a combination drug comprising the polypeptide or modulator of the present invention and a pharmaceutically acceptable carrier for transportation, and one or more anti-cancer agents. The use of anti-cancer cocktails as a treatment for cancer has become routine. Anticancer agents known in the art that can be used therapeutically in combination with the polypeptides or modulators of the invention include actinomycin D, aminoglutethimide, asparaginase, bleomycin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, (cis-DDP). , Cyclophosphamide, cytarabine hydrochloride (cytosine
Arabinoside), dacarbazine, dactinomycin, daunomycin, daunorubicin hydrochloride, doxorubicin hydrochloride, estramustine sodium phosphate, etoposide, (V16-213), floxuridine, 5-fluorouracil (5-F)
u), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon alpha-2s, interferon alpha-2b,
Leuprolide acetate (LHRH-releasing factor analog), lomustine, mechlorethamine hydrochloride (nitrogen mustard), melphalan, mercaptopurine,
Mesna, methotrexate, (MTX), mitomycin, mitoxantrone hydrochloride, octreotide, plicamycin, procarbazine hydrochloride, streptozocin, tamoxifene citrate, thioguanine, thiotepa, vinblastine sulfate,
There are vincristine sulfate, amsacrine, azacitidine, hexamethylmelanin, interleukin-2, mitoguazone, pentostatin, semustine, teniposide, and vindesine sulfate.

Furthermore, the therapeutic compositions of the present invention can be used for the prophylactic treatment of cancer. There are genetic and / or environmental conditions (eg, exposure to carcinogens) known in the art that predispose an individual to cancer. In these circumstances, it may be advantageous to treat these individuals with a therapeutically effective amount of a polypeptide of the invention to reduce the risk of developing cancer.

In vitro models can be used to determine effective amounts of the polypeptides of the invention as treatments for potential cancer. These in vitro models include assays of cultured tumor cell proliferation, proliferation of cultured tumor cells in soft agar (Preshney, (1987
) Culture of Animal Cells: A manual
of Basic Technology, Wily-Liss, New Y
ork, NY Ch 18 and Ch 21), Giovanell
a et al. , J. Natl. Can. Inst, 52: 921.
-30 (1974) tumor system in nude mice, Pilkin
gton et al. , Anticancer Res. , 17: 410
7-9 (1997) in the Boyden Chamber assay and possible migration and invasiveness of tumor cells, and Ribatta et al. , I
ntl, J.N. Dev. Biol. , 40: 1189-97 (1999).
And Li et al. , Clin. Exp. Metastasis,
17: 423-9 (1999), there is an assay for angiogenesis such as induction of angiogenesis of chicken chorioallantoic membrane or induction of migration of vascular endothelial cells. Suitable tumor cell lines are, for example, the American Type Tissue Culture Co.
Available from the reflection catalog.

5.10. 12 Receptor / Ligand Activity The polypeptides of the present invention also include receptors for receptor / ligand interactions,
It exhibits activity as a receptor ligand, an inhibitor, or an agonist (agonist). The polynucleotide of the present invention encodes a polypeptide exhibiting such properties. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (selectins, integrins and their There are, but is not limited to, cell adhesion molecules (such as ligands) and receptor / ligand pairs involved in antigen display, antigen recognition and the development of cellular and humoral immune responses. Receptors and ligands may also be useful in screening peptides or small molecule inhibitors for potential related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor fragments and ligands, are themselves useful as inhibitors of receptor / ligand interactions.

The activity of the polypeptide of the present invention can be measured by the following methods, although there are other methods.

A suitable assay of receptor ligand activity is the Current Protoc
ols in Immunology, Ed by J.M. E. Colig
an, A. M. Kruisbeek, D .; H. Margulies
, E. M. Shevach, W.M. Strober, Pub. Gr
eene Publishing Associates and Wiley
-Interscience (Chapter 7.28, Measurem)
ent of Cellular Adhesion under stati
c conditions 7.28. 1-7.28.22), Takai
et al. , Proc. Natl. Acad. Sci. USA 8
4: 6864-6868, 1987: Bierer et al. , J.
Exp. Med. 168: 1145-1156, 1988; Rosen.
Stein et al. , J. Exp. Med. 169: 149-1
60 1989; Stollenburg et al. J. Immuno
l. Methods 175: 59-68, 1994; Stitt et.
al. , Cell 80: 661-670, 1995,
The test is not limited to these.

By way of example, a polypeptide of the invention is used as a receptor for one or more ligands, which mediates the biological activity of that ligand. Ligands can be identified by binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays or other methods known in the art.

To characterize whether a drug or protein is an agonist (agonist), or antagonist (antagonist), or partial agonist or partial antagonist, other proteins are used as competitive ligands There is a need. The polypeptide of the present invention or its ligand is labeled by being bound to a radioisotope, a colorimetric molecule or a toxin molecule by a conventional method ("Guide to
Protein Purification "Murray P. Deutsch
scher (ed) Methods in Enzymology Vol
． 182 (1990) Academic Press, Inc. San D
iego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14. Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluoresamine, or rhodamine and other colorimetric molecules. An example of a toxin is, but is not limited to, ricin.

5.10.13 Drug Screening The present invention is useful for screening compounds with new polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptide or fragment used in such a test may be free in solution, attached to a solid support, raised on the cell surface, or intracellular. One method of drug screening utilizes eukaryotic or prokaryotic host cells, which are stably transformed with recombinant nucleic acid expressing the polypeptide or fragment thereof. Drugs are screened against such transformed cells in a binding protein competition assay. Viable or immobilized forms of such cells can be used for standard binding assays. For example, determining the complex formation between the polypeptide or fragment of the invention and the substance tested, or examining the reduction in complex formation between the new polypeptide and an appropriate cell line known in the art. As a source of test compounds that can be screened for their ability to bind, or the ability to modulate (ie, increase or decrease) the activity of the polypeptides of the invention (1
) Inorganic and organic compound libraries, (2) natural product libraries and (3)
There are combinatorial libraries consisting of random or mimetic peptides, oligonucleotides, or organic molecules.

Chemical libraries are easily synthesized or purchased from many commercial sources, and
This includes structural analogs of known compounds, or via natural product screening.
Compounds identified as "hits" or "leads" are included.

Sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation or marine organisms, and mixed libraries for screening include (1) soil, plants or marine organisms. It can be prepared by fermentation and extraction of broth from a microorganism, or (2) extraction from the microorganism itself. Natural product libraries include polyketides, non-ribosomal peptides and (non-naturally occurring) variants thereof.
For review, see Science 282: 63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily generated by conventional automated synthetic methods, PCR, cloning or patented synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Note that other libraries of interest include peptide, protein, peptidomimetic, multi-parallel synthetic collection, recombinant, and polypeptide libraries. Myer for a review of combinatorial chemistry and libraries created from it
s, Curr. Opin. Biotechnol. 8: 701-707 (1
997). A review and examples of peptidomimetic libraries can be found in Al-
Obeidi et al. , Mol. Biotechnol, 9 (3)
: 205-23 (1998); Hruby et al. , Curr O
pin Chem Boil, 1 (1): 114-19 (1997): Dor
ner et al. , Bioorg Med Chem, 4 (5): 70.
9-15 (1996) (alkylated dipeptides).

By identifying modulators using the various libraries described herein, "hits" (or "reads") to optimize the ability of "hits" to bind to polypeptides of the invention. ) Can modify (change) the candidate. The molecules identified in the binding capacity assay are then assayed for antagonist or agonist activity in in vivo tissue culture or in animal models well known in the art. Briefly, the molecule is titrated into a large number of cell cultures or animals and then tested for cell / animal death or cell / animal survival prolongation.

Thus, the binding molecule forms a complex with a toxin, eg ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex then targets the tumor or other cell by virtue of the specificity of the binding molecule for the polypeptides of the invention. Alternatively, the binding molecule is complexed with an imaging agent for targeting and imaging purposes.

5.10.14 Assay for Receptor Activity The present invention also provides methods for detecting the specific binding of a polypeptide, eg, ligand or receptor. This technique provides a number of assays, previously unknown, that are particularly useful for identifying binding partners of the receptor polypeptides of the invention. For example, mammalian or bacterial cells, or expression cloning using a dihybrid screening assay, can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with an appropriately immobilized polypeptide of the invention can be used to recognize the polypeptide of the invention and to separate the binding polypeptides. Modulate (ie, increase or decrease) the biological activity of a polypeptide of the invention
There are many different libraries used to identify compounds, especially small molecules. The ligand for the receptor polypeptide of the present invention can be identified by adding an exogenous ligand or a cocktail of ligands to two genetically identical cell populations except for the expression of the receptor of the present invention. One cell population expresses the receptor of the present invention, while the other cell population does not. The response of the two cell populations to the added ligand is compared. Alternatively, the expression library can be co-expressed intracellularly with a polypeptide of the invention and the autocrine reaction can be assayed to identify potential ligands. Further, as another example, BIA core assays, gel overlays or other methods known in the art can be used to identify binding partner polypeptides, including (1) organic and inorganic chemical libraries, Included are (2) natural product libraries and (3) combinatorial libraries such as random peptides, oligonucleotides or organic molecules.

The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptides of the present invention can be determined. For example, the cytoplasmic domain of a polypeptide of the invention is
A chimeric protein fused to the extracellular portion of the protein whose ligand has been identified is produced in the host cell. The cells are then incubated with a ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. The predicted modification, or phosphorylation, of a known downstream protein involved in intracellular signal transduction can then be measured. Also, other methods known in the art can also be used to identify signaling molecules involved in receptor activity.

5.10.15 Anti-inflammatory Activity The compositions of the present invention also exhibit anti-inflammatory activity. Anti-inflammatory activity stimulates cells involved in the inflammatory response, suppresses or promotes cell-cell interactions (such as cell adhesion), suppresses chemotaxis of cells involved in the inflammatory process, or It is obtained by promoting, suppressing or promoting cell extravasation, or stimulating or suppressing the production of other factors that more directly suppress or enhance inflammation.
Compositions having such activity are useful for infections (septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin killing, arthritis, complement-mediated hyperacute rejection, nephritis, Not limited to these, it can be used for the treatment of cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or chronic or acute inflammatory conditions that are considered to be caused by overproduction of cytokines such as TNF and IL-1. . The compositions of the invention are also useful in treating anaphylaxis or hypersensitivity to an antigenic substance or material. The composition of the present invention comprises sepsis, acute pancreatitis, endotoxin shock, cytokine shock, rheumatoid arthritis, chronic inflammatory rheumatism, pancreatic cell disorder due to diabetes mellitus type 1, graft-versus-host disease, inflammatory bowel disease, lung disease. For the prevention or treatment of conditions such as inflammation, other autoimmune diseases or inflammatory diseases associated with, as an antiproliferative agent for acute or chronic myelogenous leukemia, or prevention of preterm labor in the pre-stage of intrauterine infection. Can be used in, but is not limited to.

5.10.16 Leukemia Leukemia and related disorders may be treated or prevented by administering therapeutic agents that enhance or inhibit the function of the polynucleotides and / or polypeptides of the invention. Such leukemias and related disorders include acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic Myelocytic (granulocyte) leukemia, and chronic lymphocytic leukemia (for a review of these diseases, see Fishman et al.
al. , 1985, Medicine, 2d Ed. , J. B. L
ippincott Co. , Philadelphia), and is not limited to these.

Nervous System Diseases Neurons that are associated with cell types that can test the efficacy of interventions of compounds that modulate the activity of the polypeptides and / or polynucleotides of the invention, and thus can be treated with an indication of therapeutic utility. The systemic diseases include, but are not limited to, nervous system damage and nervous system diseases, or nervous system disorders that cause disconnection of axons, regression of nerve cells, degeneration, or demyelination. Nervous system lesions to be treated in patients (including humans and non-human mammals) according to the present invention include the following central (
Including but not limited to lesions of either the spinal cord, brain, or peripheral nervous system.

(I) Traumatic lesions, including those caused by physical injury or surgery, eg, lesions that cut a part of the nervous system, or pressure injuries. Or ischemic lesions resulting in nerve cell damage or death due to oxygen deprivation in a part of nervous system such as ischemia (iii) As a result of infection by abscess, or infection by human immunodeficiency virus, herpes zoster, herpes simplex virus Infectious lesions that are destroyed or damaged as a result of infection in part of the nervous system in combination with or with Lyme disease, tuberculosis, syphilis (iv) Parkinson's disease, Alzheimer's disease, Huntington Butoh Disease or degeneration associated with amyotrophic lateral sclerosis, as well as degeneration processes involving them that result in the destruction or damage of parts of the nervous system. (Regression) lesion (v) Vitamin B12 deficiency, folate deficiency, Wernicke's disease, tobacco Includes, but is not limited to, alcoholic amblyopia, Marcaferva Vinami disease (primary degeneration of the corpus callosum) and alcoholic cerebellar degeneration.

(Vi) Neurological lesions associated with systemic diseases, including but not limited to diabetes (diabetic neuropathy, facial paralysis), systemic lupus erythematosus, cancer or sarcoma.

(Vii) Lesions caused by toxic substances including alcohol, lead or certain neurotoxins (viii) Demyelinating lesions in which part of the nervous system is destroyed or damaged by demyelinating disease, multiple sclerosis, human Includes immunodeficiency virus-associated myelopathy, transverse myelopathy, various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis,
Not limited to these.

Useful therapeutic agents for nervous system diseases according to the present invention can be selected by testing biological activity for promoting survival or differentiation of neural cells. For example, but not by way of limitation, therapeutic agents that induce any of the following effects are useful according to the invention. Increased neuronal survival time in culture.

[0250]   (Ii) Increased sprouting of nerve cells in the culture medium or in vivo.

(Iii) Increased production of nerve cell-related molecules such as choline acetyltransferase or acetylcholinesterase associated with motor neurons in culture or in vivo.

[0252]   (Iv) The symptoms of in vivo nerve cell dysfunction were reduced.

Such effects can be measured by any method known in the art. Preferably, but not limited to the examples, the increase in the survival rate of nerve cells is described by Arakawa et al.
(1990, J. Neurosci. 10: 3507-3515), and the increase in the sprouting of nerve cells can be measured by the Pestronk et.
al. (1980, Exp. Neurol. 70: 65-82) or B.
row et al. (1981, Ann. Rev. Neurosci.
4: 17-42), and the increase in the production of nerve cell-related molecules can be measured according to the molecule to be measured by bioassay, enzymatic assay, binding antibody, Northern blot assay, or the like. Motor neuron dysfunction can also be measured by the assessment of physical symptoms such as motor neuron disorders, eg weakness, motor neuron transmission velocity or functional disability.

In certain embodiments, motor neuron disorders treated according to the invention include infarcts, infections, exposure to toxins, trauma, surgical injuries, degenerative diseases or other components of motor neurons and the nervous system. Diseases that affect components, such as malignant tumors, and diseases that selectively affect nerve cells, such as amyotrophic lateral sclerosis, as well as progressive spinal muscular atrophy, progressive bulbar paralysis, and primary side Chordosclerosis, infantile, juvenile muscular atrophy, progressive bulbar paralysis in early childhood (Fatio-Ronde syndrome), poliomyelitis and post-polio syndrome, and hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease)
Not limited to, but included.

5.110.18 Other Activities The polypeptides of the present invention still further exhibit one or more of the following activities or actions. That is, height, weight, which suppresses or arrests the growth, infection or function of infectious agents, including but not limited to bacteria, viruses, fungi and other parasites.
Hair color, eye color, skin, percentage of fat on small areas or other tissue pigmentation,
Or acts on physical characteristics including (but not limited to) the size or shape of an organ or body part (eg, increase or shrinkage of the breast, or changes in bone morphology and shape) (inhibition or enhancement), male or Affects the metabolism, catabolism, assimilation, processing, utilization, storage, or excretion of dietary fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, and other nutritional factors or components that affect female fertility, Hematopoiesis, which provides an analgesic or other pain-reducing effect on behavioral characteristics including, but not limited to, appetite, libido, stress, cognition (including cognitive impairment), depression (including depressive disorder) and violence. In the case of enzymes, hormones, endocrine activation, in the case of enzymes that promote the differentiation and growth of embryonic stem cells in non-systems, correct enzyme deficiency,
Treating deficiency-related disorders, treating hyperproliferative disorders (such as psoriasis), immunoglobulin-like activities (such as the ability to bind antigen or complement), and acting as antigens in vaccine components Then, an immune reaction is promoted to such protein or other substance or entity that cross-reacts with such protein.

5.10.19 Identification of Polymorphisms Demonstration of polymorphisms allows the identification of such polymorphisms in human subjects, and this genetic pharmacological information can be used for diagnosis and therapy. Such polymorphisms can be associated, for example, with various disease states (such as diseases involving inflammation or immune responses).
Associated with a specific predisposition or susceptibility to or specific response to drug administration, and this genetic information can be used to tailor prophylactic or therapeutic treatments as appropriate. For example, if there is a polymorphism associated with a predisposition to inflammation or an autoimmune disease, identifying the presence of the polymorphism can diagnose this condition in humans.

Polymorphisms can be identified in various ways well known in the art, and in all cases, a sample from a patient will generally be obtained and the DNA analyzed from this sample, optionally isolating or amplifying the DNA. To identify the presence of polymorphisms in DNA. For example, PCR is used to amplify a fragment of the appropriate genomic DNA, which is then sequenced. Alternatively, the DNA is an allele-specific oligonucleotide
Subject to hybridization (hybridize appropriate oligonucleotides under conditions that allow detection of single base mismatches) or single nucleotide extension assay (hybridize immediately adjacent to the polymorphic position ( The hybridizing oligonucleotide is extended with one or more labeled nucleotides).
In addition, conventional restriction enzyme fragment length polymorphism analysis (using restriction enzymes that digest genomic DNA differently depending on the presence or absence of the polymorphism) may be performed. The array of nucleotide sequences of the invention can be used to detect polymorphisms. This array may use a modified version of the nucleotide sequence of the present invention to detect the nucleotide sequence of the present invention. In a substitute, any one of the nucleotide sequences of the invention
One can be replaced on this array to detect changes in these sequences.

Alternatively, polymorphisms resulting from changes in the amino acid sequence can also be detected by detecting the corresponding changes in the amino acid sequence of the protein, eg, antibodies specific for the mutant sequence.

5.10.20 Arthritis and Inflammation The immunosuppressive effect of the inventive composition on rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant-induced arthritis in rats. The experimental design is described in Holoshitz et al. (1983, Scie)
nce, 219: 56) or B. Waksman (1963, Int
． Arch. Allergy Appl. Immunol. , 23:
129). This disease is a complete Freund adjuvant (
It can be caused by a single injection (usually an intradermal injection) of dead Mycobacterium tuberculosis suspended in CFA). There are various injection methods,
In the case of mice, a mixture of supplements may be injected into the base of the tail. The polypeptide is administered in phosphate buffer (PBS) in an amount of about 1-5 mg / kg. The control standard solution uses only PBS.

[0260] A method of testing the efficacy of a test composition is by intradermal injection of dead Mycobacterium tuberculosis in CFA followed immediately by administration of the test composition and then every other day for 2 days.
Continue it until 4th. 14, 15, 1 after injection of mycobacterial CFA
On days 8, 20, 22, and 24, the above J. An overall score for arthritis as described by Holoshitz is obtained. Data analysis should show that the test compounds have a dramatic effect on joint swelling with a reduction in the arthritis score.

Therapeutic Methods The inventive compositions (including polypeptide fragments, analogs, variants, antibodies, and other binding agents and modifiers such as antisense polynucleotides) can be applied to a variety of therapeutic methods. Therapeutic applications include the following examples.

5.11.1 Examples One application of the invention is a method of administering an effective amount of a CUB domain polypeptide or other composition of the invention to an individual suffering from a disease or disorder. In this case, it is possible to adjust the administration of an effective amount by controlling the peptide of the present invention. The mode of administration is not critical, but parenteral administration is preferred. An exemplary method of administration is an intravenous bolus. The dosage of CUB domain polypeptides or other compositions of the invention will usually be determined by the prescribing physician. Dosage should depend on age, weight, patient health and response. Usually, the amount of polypeptide per unit dose is 0.01 μg to 100 m / kg of patient weight.
g, the most preferred amount is 0.1 μg to 10 mg. The CUB domain polypeptide of the present invention is injectable with a pharmaceutically approved parenteral carrier for parenteral use. Such carriers are well known in the art, and correspond to, for example, water, saline, Ringer's solution, dextrose solution, and a solution containing a small amount of human serum albumin. The carrier may contain minor amounts of additives and other active ingredients that maintain isotonicity and stability of the polypeptide. Preparation of such solutions is within the skill of the art.

5.12 Pharmaceutical Formulations and Methods of Administration Proteins or other compositions of the invention (including recombinant and non-recombinant sources, regardless of source, antibodies and other binders of polypeptides of the invention) ) To patients in need of treatment, as it is, or to treat and cure various diseases,
It can be administered as a pharmaceutical composition in admixture with a suitable carrier or vehicle. Such compositions include, in addition to protein or other active ingredient or carrier, diluents, fillers, salts, buffers,
Stabilizers, solubilizers, and other materials well known in the art can also be included. "
"Pharmaceutically acceptable" means a non-toxic material that does not interfere with the biological effect of the active ingredient. The characteristics of the carrier will depend on the method of administration. Among the pharmaceutical compositions of the invention are cytokines, lymphokines, or hematopoietic factors such as M
-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11
, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TN
F-1, TNF-2, G-CSF, Meg-CSF and the like, thrombopoietin, stem cell factor, erythropoietin and the like can also be included. Furthermore, it is possible to combine the protein of the invention with other substances which are effective in the treatment of diseases or disorders. Some of these substances include the cytokines mentioned in the text, as well as epidermal growth factor (E
GF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), and various other growth factors.

In addition, other pharmaceutical agents which activate the activity of the protein or which supplement its therapeutic activity or use can be included in the pharmaceutical composition. Such addenda and / or agents may be included in the pharmaceutical composition in order to elicit a synergistic effect with the protein of the invention or other active ingredients, or to minimize side effects. Conversely, clotting factors, cytokines, lymphokines, other hematopoietic factors, thrombolytics, antithrombotic factors, or anti-inflammatory drugs (eg, IL-1Ra, IL-1Hy1, IL-1Hy).
2, anti-TNF, corticosteroids, immunosuppressants) for the purpose of minimizing the side effects of the protein of the present invention and other active ingredients specific clotting factors, cytokines, lymphokines, other hematopoietic factors, thrombolytic, It can also be included in antithrombotic factors, or anti-inflammatory drugs. The proteins of the present invention are multimers (eg heterodimers or homodimers).
It may be active in or in complex with itself or with other proteins. As a result, the pharmaceutical composition of the invention may include the protein of the invention in the form of multimers or complexes.

Instead of being included in the pharmaceutical composition of the present invention (which includes the first protein), the second protein or therapeutic agent can also be administered together with the first protein (eg, substances that are simultaneously or in combination). When the therapeutic concentration varies depending on the condition to be achieved at the treatment site). Formulation and dosing techniques for instant application compounds are described in Mack Publishing Co., Easton, PA. Publishing "Remington Pharmaceutical Sciences"
May be used as a reference. A therapeutically effective dose refers to the amelioration of symptoms, for example treatment,
By an amount of the compound sufficient to effect a cure, prophylaxis, amelioration of associated medical conditions, or a treatment, cure, prophylaxis, or an increase in the recovery rate of such conditions. When the individual active ingredients are administered alone, a therapeutically effective dose refers to that active ingredient only.
When used in combination, therapeutically effective dose refers to the total amount of active ingredient, which results in a therapeutic effect, whether the combined administration is sequential or simultaneous.

In practicing the methods of treatment or use of the present invention, a protein or other active ingredient of the present invention is administered to a mammal having a therapeutic condition in a therapeutically effective amount. The protein or other active ingredient of the present invention can be administered alone in the method of the present invention or in combination with other therapeutic methods, for example, a therapeutic method using cytokines, lymphokines and other hematopoietic factors. Is also possible. Cytokines,
When administered in combination with one or more of lymphokines and other hematopoietic factors, the protein or other active ingredient of the present invention is a cytokine, lymphokine,
The other hematopoietic factor, thrombolytic factor, or antithrombotic factor may be administered simultaneously or continuously. In the case of continuous administration, the order of administration of the protein of the present invention or other active ingredient in combination with cytokine, lymphokine, other hematopoietic factor, thrombolytic factor, or antithrombotic factor is The doctor will determine its suitability.

5.12.1 Route of Administration Suitable administration methods include oral, rectal, transmucosal, enteral administration, endothelial injection (intramuscular, subcutaneous, intramedullary injection, etc.), subarachnoid, direct intraventricular administration. Injection, intravenous injection, intraperitoneal injection,
Intranasal injection, intraocular injection, etc. are included. Administration of the proteins of the invention or other active ingredients used in pharmaceutical compositions, or practice of the methods of the invention, can be accomplished in a variety of conventional ways. For example, oral administration, inhalation, topical or dermal application, subcutaneous, intraperitoneal, parenteral injection and the like. Intravenous injection is preferred for administration to patients.

It is also possible to administer the compounds locally rather than systemically. For example, direct injection into a joint or a fibrous tissue suffering from arthritis, in which case the depot or sustained release drug is often used. The compounds can also be administered topically, eg as eye drops, to prevent scarring, which is a common complication of glaucoma surgery. It can also be administered using a targeted drug delivery system. For example, it is a method of using a liposome coated with a specific antibody or the like by targeting a tissue or a fibrous tissue at a site causing arthritis. Liposomes are selectively targeted and absorbed by the afflicted cellular tissue.

The polypeptides of the invention can be used in any manner that delivers an effective amount to the required site of action. Determination of an appropriate administration method and effective amount is
Included within the skill of the art. As with the treatment of injuries, the therapeutic compound may be administered directly to the area. Appropriate dose ranges for the polypeptides of the invention may be extrapolated from doses using appropriate animal models or similar studies using such models. Thereafter, the dose may be adjusted by the clinician as necessary to enhance the maximum therapeutic effect.

Compositions / Formulations The pharmaceutical compositions used according to the invention can thus be formulated in the customary manner using one or more physiologically effective carriers which consist of media and adjuvants. Vehicles and adjuvants help shape the active compound into pharmaceutically acceptable forms. Such pharmaceutical compositions can be manufactured by well known methods. For example, common methods such as mixing, dissolving, granulating, dragee (sugar coating), micronizing, emulsifying, encapsulating, entrapping or lyophilizing. Proper formulation depends on the mode of administration. When administered orally in a therapeutically effective amount, the protein or active ingredient of the invention is administered in the form of tablets, capsules, powders, solutions or elixirs. When administered in tablets, the pharmaceutical compositions of this invention may also include solid carriers such as gelatin and adjuvants. Tablets, capsules, and powders contain from about 5% to 95% protein or active ingredient of the invention. Ideally, it is preferable that the protein or active ingredient of the present invention is contained in about 25% to 95%. When administered as a liquid, a liquid carrier such as water, petroleum, animal or vegetable oils such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oil may be added. Physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol and polyethylene glycol may be further included in the liquid pharmaceutical composition. In the case of a liquid, the protein or active ingredient of the present invention is contained in an amount of about 0.5 to 90% by weight. Ideally, it is preferable that the protein or active ingredient of the present invention is contained in an amount of about 1 to 50% by weight.

When a therapeutically effective amount of a protein or active ingredient of the invention is administered by intravenous, cutaneous or subcutaneous injection, the protein or active ingredient of the invention is pyrogen-free and parenterally acceptable. It is administered in the form of a possible liquid. The preparation of such a parenterally acceptable protein or other active ingredient solution in consideration of pH, isotonicity, stability, etc.
It belongs to the technical category of the industry. Preferred pharmaceutical compositions for intravenous, cutaneous or subcutaneous injection include, in addition to the protein or active ingredient of the invention, isotonic carriers such as sodium chloride injection, Ringer's injection, glucose injection, glucose and chloride. Sodium injection, lactated Ringer's injection, or other carrier well known in the art should also be included. The pharmaceutical composition of the present invention includes a stabilizer, a preservative,
Buffers, antioxidants and other additives well known in the art may be included. For injection, the substances of the invention may be formulated in liquid form. If possible, physiologically compatible buffers such as Hank's solution, Ringer's solution and physiological saline are preferred. For transmucosal administration, specific penetrants are used. Such penetrants are generally well known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers include the compounds of the present invention in tablets, pills, dragees, capsules, liquids, gels,
Allows for formulation into syrups, slurries, suspensions, etc., for oral ingestion by the patient. Pharmaceutical preparations for oral use include the addition of suitable auxiliaries, if necessary, to obtain the core of a tablet or dragee, then a solid carrier is added and the mixture is powdered,
It is obtained by processing the granular mixture. Particularly suitable carriers are sugar (including lactose, sucrose, mannitol, sorbitol, etc.) and cellulose (eg corn starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy). Methyl cellulose and / or polyvinylpyrrolidone (PVP))
And the like. If necessary, a cross-linking polyvinylpyrrolidone, agar, alginic acid, or a disintegrating agent (Mama) such as a salt thereof such as sodium alginate may be added. Appropriate coatings are provided for the core of the Drazy. Concentrated sugar solutions may be used for this purpose. It may include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution, and suitable organic or mixed solvents. Dyestuffs or pigments may be added to the tablets or dragee coatings for emphasis or to identify content combinations of active compound content.

For pharmaceutical preparation of products for oral use, push-fit capsules made of gelatin and gelatin, and soft shield capsules made of plasticizer such as glycol and sorbitol are used. Push-fit capsules contain the active ingredient in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc, magnesium stearate and, if desired, stabilizers. You can also In the case of soft capsules, the active ingredient may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycols. Further, a stabilizer may be added. For oral administration, the preparation should be in an amount suitable for the purpose. For buccal administration, tablets or lozenges formed by a usual method can be used.

For administration by inhalation, the compounds of the invention are administered by spraying from a pressurized pack or nebulizer. In that case, suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide and other suitable gases are used. In the case of pressurized spray, the dosage unit can be determined by using a valve to deliver a metered amount. Capsules and sachets such as gelatin used in inhalers may be formed by mixing the compound in powder form with a suitable powder base such as lactose or starch. The composition may be formulated for parenteral administration such as by bolus injection or continuous infusion. Formulations for injection may contain preservatives in unit dosage form such as ampoules or multi-dose containers. The composition is suspended or dissolved in an oil or liquid carrier,
It may be emulsified or may contain substances for formulation such as a suspending agent, a stabilizer, a dispersant and the like.

Some pharmaceutical compositions for parenteral administration include liquid forms in which the active compound is contained in soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers also include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate, triglycerides, liposomes and the like. Liquid injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran and the like. If necessary, it is also possible to prepare a highly concentrated solution by including a stabilizer or a substance that enhances the solubility of the compound in the suspension. Alternatively, the active ingredient may be powdered before use and then disinfected and combined with a suitable carrier, such as pyrogen-free water.

The compounds can also be in rectal compositions such as conventional suppository bases, eg suppositories containing cocoa butter and other glycerides and fixed enemas. In addition to the above-mentioned formulation method, a composition for a depot can be prepared. Such long-term sustained methods can be achieved by implants (eg endothelium or intramuscular) or intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric agents and hydrophobic substances (eg substances emulsified in a suitable oil), ion exchange resins, derivatives such as sparingly soluble salts.

The pharmaceutical carrier for the hydrophobic compounds of the present invention is a common solvent system composed of benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The common solvent system can also be a VDP common solvent system. VDP is 3% w / v benzyl alcohol solution, 8% w / v nonpolar surfactant polysorbate 80, and 65% w / v polyethylene glycol 300 in absolute alcohol. VDP common solvent system (VDP: 5W)
VDP was diluted 1: 1 with a 5% glucose aqueous solution. This VDP common solvent system dissolves hydrophobic compounds well and is less toxic for systemic administration.
Naturally, the proportions of the VDP common solvent system are variable and do not destroy the solubility or toxicity properties. Furthermore, the constituent components of the common solvent are also diverse. In addition, other common solvents, such as polysorbate 80, can be replaced by other low toxicity non-polar surfactants and there are various possibilities for the polyethylene glycol fraction size. Other biocompatible polymers can be used in place of polyethylene glycol, such as polyvinyl pyrrolidone. Other sugars or polysaccharides may be used instead of glucose. Alternatively, another delivery system may be used for hydrophobic pharmaceutical compounds. As hydrophobic drugs, liposomes and emulsifiers are well known as carriers or media for transportation. Certain organic solvents such as dimethylsulfoxide can be used, although they are slightly more toxic. In addition, compounds may be delivered using sustained release systems, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
Various types of sustained release materials have been established and are well known in the art. Sustained-release capsules may, depending on the nature of their chemical nature, release the compounds over 2-3 weeks and sometimes over 100 days. Depending on the scientific properties and biological stability of the therapeutic agent, additional methods may be added that may be necessary for the stability of the protein or other active ingredient.

The pharmaceutical composition may be composed of a carrier which comprises a suitable solid or gel phase. Examples of such carriers include calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, polymers such as polyethylene glycol, and the like. Many of the active ingredients of the present invention can also be provided as pharmaceutically compatible counter-ionic salts. Such pharmaceutically acceptable salts are salts which retain their biological effectiveness and the properties of free acids, such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamines, dialkylamines, monoalkylamines, It can be obtained by reacting with an inorganic or organic base such as a dibasic amino acid, sodium acetate, potassium benzoate or triethanolamine.

The pharmaceutical composition of the invention may also be in the form of a complex of the protein or other active ingredient of the invention and the protein or peptide antigen. Protein and / or peptide antigens send stimulatory signals to B and T lymphosites. B-lymphosites respond to antigens through immunoglobulin receptors on their surface. T-lymphosite reacts with an antigen through the T cell receptor (TCR) after the antigen is released by the MHC protein. MHC and structurally related proteins (including those encoded on host cells by class I and class II MHC genes)
Acts to provide the peptide antigen to T lymphosite. The antigenic component may also be supplied by the purified MHC peptide complex alone or with co-stimulatory molecules that directly signal T cells. Alternatively, an antibody capable of binding to surface immunoglobulin or other molecules on B cells, and an antibody capable of binding to TCR or other molecules on T cells,
It is also possible to combine with the pharmaceutical compositions of the invention.

The pharmaceutical composition of the invention may also be in the form of liposomes. In that case, in a liposome, the protein of the present invention is combined with an amphipathic substance such as a lipid, apart from other pharmaceutically acceptable carriers. Lipids are micelles (colloidal dispersion state)
It exists as an insoluble monolayer, or in aqueous solution in the form of an aggregate as a layered phase.
Lipids necessary for liposome formation are monoglyceride, diglyceride, sulfatide, lysolecithin, phospholipid, saponin, bile acid and the like. The method for producing such liposomes is included in the publicly-known art in the art. For example, U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; 4,73.
7, 323, etc., all of which are included in this document as references.

The amount of the protein of the invention or other active ingredient used in the context of the pharmaceutical composition of the invention will be determined by the nature of the condition being treated and its severity. Ultimately, in treating each patient, the attending physician will decide the amount of protein or other active ingredient of the present invention. The physician initially administers a small dose of the protein of the invention or other active ingredient,
Observe patient response. The amount of the protein or other active ingredient of the present invention is increased until the maximum therapeutic effect is obtained, at which point the dosage is no longer increased. Various pharmaceutical compositions used to practice the method of the present invention include from about 0.01 μg / kg to about 100 μg / kg of protein or other active ingredient of the present invention.
It is believed that it should contain mg (preferably about 0.1 μg to about 10 mg, more ideally about 0.1 μg to about 1 mg). Methods of treatment with the compositions of the present invention that are effective in degenerating bone, cartilage, tendons, or ligaments include local, systemic, or local use in the form of implants or devices. Upon administration, the method of treatment of the present invention is carried out in a physiologically acceptable form free of pyrogens. In addition, it may be preferable to deliver the composition to bone, cartilage, or damaged cellular tissue by encapsulation or injection in various ways. Topical administration may be appropriate for wound healing and cellular repair. A therapeutically active substance other than the protein or other active ingredient of the present invention, which may also be included in the above composition, instead of or in addition to the composition of the present invention, either simultaneously or sequentially. You may use it. To promote the formation of bone or cartilage, a composition containing a protein or other active ingredient can be delivered to the damaged site of bone or cartilage, which has the structure necessary for bone or cartilage development and is reabsorbed by the body. Desirably, a substrate is included in the composition. Such substrates may be made from materials currently used in other transplant procedures.

The choice of matrix material is based on biocompatibility, biodegradability, physical properties, appearance and interactive properties. The application method of the composition determines the preparation method. Potential substrates for the composition are biodegradable, such as synthetic calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid,
And polyanhydrides. Other potential materials are biodegradable and biologically defined collagens such as bone and skin collagen. Other substrates are composed of pure proteins or extracellular matrix components. Other possible substrates are non-biodegradable, chemically synthesized, for example, sintered hydroxyapatite, bioglass, aluminates, or other ceramics. The substrate may be composed of a combination of a plurality of the above substances. For example, polylactic acid and hydroxyapatite or collagen and tricalcium phosphate may be used. Bioceramics can also vary in composition. For example, calcium, aluminate, phosphate, processed to make the hole size,
It is also possible to change the size and shape of the particles or the biodegradability. Currently preferred is a copolymer of lactic acid and glycolic acid 50:50 (molecular weight) consisting of porous particles of diameter 150 to 800 microns. In some cases, sequestering agents such as carboxymethyl cellulose and autologous blood may be utilized to prevent the protein composition from dissociating from the substrate.

Preferred as sequestrants are cellulosic materials such as alkyl celluloses (including hydroxyalkyl celluloses). Among these, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose and the like are also included. Most preferred among them is the cation salt of carboxymethyl cellulose (CMC). Other preferred sequestering agents are hyaluronic acid, sodium alginate, polyethylene glycols, polyoxyethylene oxides, carboxyvinyl polymers, and polyvinyl alcohol. The optimum amount of sequestering agent is 0.5-20% by weight based on the total weight of the formulation,
Ideally, it is 1-10% by weight, which represents the amount required to prevent the protein from desorbing from the polymer substrate and at the same time simplify the handling of the composition. The protein is able to assist the osteogenic activity of progenitor cells, in an amount that does not interfere with. In addition, the proteins or other active ingredients of the present invention may be combined with other substances useful in the treatment of bone or cartilage defects, injuries, or problematic tissue. Such substances include various growth factors. For example epidermal growth factor (
EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF) and the like.

Therapeutic compositions are also currently finding value in veterinary medicine. The treatment with the protein or other active ingredient of the present invention can be particularly applied to domestic animals and thoroughbred horses as well as humans. When using a pharmaceutical composition containing a protein for the regeneration of cellular tissue, the dosage is determined by a doctor who makes a diagnosis in consideration of various factors affecting the activity of the protein. For example, the weight of the tissue to be formed, the damaged part, the condition of the damaged tissue, the size of the injury, the type of damaged tissue (eg bone),
The patient's age, sex, diet, degree of infection, administration time, and other clinical factors. The dose will depend on the type of substrate used for regeneration and also on the presence or absence of other proteins in the pharmaceutical composition. For example, IGF in the final composition
When adding other known growth factors such as I (insulin-like growth factor I),
Dosage may be affected. The degree of recovery can be checked by periodically evaluating the growth and / or repair of cellular tissue or bone by a method such as X-ray, histomorphometric determination method, or tetracycline labeling method.

The polynucleotides of the invention can also be used in gene therapy. Such polynucleotides can be introduced into cells in vivo or in vitro to study mammalian expression. The polynucleotides of the invention can also be administered using other methods known to introduce nucleic acids into cells or organisms (eg, virus-mediated or naked DN).
A etc.). The cells can also be cultured in vitro containing the protein of the invention to propagate or to obtain a desired effect or activity. The cells thus treated can be introduced into a living body for therapeutic purposes.

5.12.3 Effective Dosage Pharmaceutical compositions suitable for use in the present invention are compositions which contain an effective amount of the active ingredient to achieve the purpose. More specifically, a therapeutically effective amount means an amount required to slow or reduce the progression of current symptoms in the subject being treated. Determining the effective amount is within the skill of the art and will be further appreciated as described in detail in this document. The therapeutically effective amount of the composition used in the method of the invention can be estimated initially by in vitro analysis. For example, animal models are used to prescribe dosages, range in circulating concentrations, and then use them to more accurately determine human dosages. For example, an animal model is used to prescribe a dose and define a range of circulating concentrations in which an IC50 determined in cell culture (ie, a test compound corresponding to half the maximum value that inhibits the biological activity of the protein). Concentration) is also included. Such information can be used to more accurately determine human usage.

[0287]   Therapeutically effective amount is a compound that achieves recovery of symptoms or prolongation of patient survival.
Means the amount of. The toxic and therapeutic effects of such compounds may be
It can be determined by a standard pharmaceutical method using a substance. For example, LD_Fifty(The number of individuals is
50% die dose) and ED_Fifty(Amount therapeutically effective for 50% of the population) etc.
Is the decision. The dose ratio between toxic and therapeutic effects is called the therapeutic index, LD_FiftyAnd ED _Fifty It is expressed by the ratio of. Compounds with a high therapeutic index are more preferred compounds. this
Data obtained from cell culture analyzes and animal studies are used to determine the range of human usage.
You can The dose of such a compound is ED_FiftyWithin the circulating concentration including
It is preferred that there is no or little toxicity. Dosage is the form or administration method
It changes within that range based on the law. The exact prescription, method of administration, and dosage will depend on the patient.
The doctor decides by looking at the condition. For example, Fingl et al., 197.
5 years, see Chapter 1, page 1 of "Pharmacological basis of therapeutics". Dosage and period
, Individually adjustable to provide a condition where plasma activity levels are at half
. Such a condition is necessary to maintain a favorable effect, or the minimum effective concentration (ME
Is sufficient to maintain C). MEC depends on the type of compound, but in vitro
It can be inferred based on the data. The dose required to achieve MEC is individual
Determine plasma concentration using HPLC analysis or bioassay, depending on sex and administration method
It is possible to do so.

Dosage intervals can also be determined using the MEC value. Compounds are 10-90% of the time
, Should be administered using methods that maintain plasma levels above MEC. It is preferably 30-90%, most preferably 50-90%. In the case of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The dosage of the polypeptides or other compositions of this invention will vary between adults and children, but will range from about 0.01 μg to 100 mg / kg body weight daily. More preferred is a range of about 0.1 μg to 25 mg per kg body weight daily.
The dosing may be once a day, or if the dose is the same, the interval may be shortened or lengthened.

[0290] Of course, the dosage will vary with the age, weight, severity of symptom, mode of administration and judgment of the prescribing subject of the subject being treated.

5.12.4 Packaging The compositions can also be administered using packs or dispensers. There may be included one or more units of the drug containing the active ingredient therein. The pack may be, for example, metal or a plastic foil such as a blister pack. The pack or dispenser may be accompanied by a guide for administration methods. It is also possible to formulate a composition containing a compound of the invention in a pharmaceutically compatible carrier, which can be packaged in a suitable container and labeled with a particular method of treatment.

5.13 Antibodies The present invention also includes antibodies to proteins or fragments of the proteins of the present invention.
As used in this document, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, that is, antigen binding that specifically binds an antigen (produces an immune reaction). Means a molecule that has a site. Such antibodies include polyclonal, monoclonal, chimeric, single chain, Fab, Fab ′ and F (ab ′) 2 fragments, Fab expression libraries and the like. Generally, human antibody molecules are associated with any of the classes IgG, IgM, IgA, IgE, and IgD. These molecules differ from each other by the nature of the heavy chains they contain. Some classes have subclasses such as IgG1 and IgG2. In humans, the light chain may be a kappa or lambda chain. References to antibodies in this document include all such classes, subclasses, and human antibodies.

The isolated protein related to the protein of the present invention can be used as an antigen, or a part or fragment thereof. It can then be used as an immunizing antigen to produce antibodies that immunospecifically bind to the antigen using standard techniques for preparing polyclonal and monoclonal antibodies. The whole protein can be used as an immunizing antigen, or the peptide fragment of the antigen used as an immunizing antigen can be provided by the present invention. The antigenic peptide fragment consists of at least 6 amino acid residues in the amino acid sequence of the whole protein as shown in SEQ ID NO: 4 or 6-8, and surrounds its antigenic determinant (epitope). I'm out. In that case, the antibody of the peptide is
It forms a specific immune complex with the whole protein or a fragment containing the epitope. If possible, the antigenic peptide preferably consists of at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. The epitope surrounded by the antigenic peptide is preferably located on the surface of the protein. The site is usually hydrophilic.

For certain parts of the invention, at least one of the epitopes surrounded by the antigenic peptide is a CUB domain protein located on the surface of the protein, eg a hydrophilic site. Hydrophobic analysis of human protein sequences shows which sites in related proteins are hydrophilic and encode surface residues for antibody production. As a method for producing an antibody, a method showing hydrophilic and hydrophobic sites is already well known in the art. For example, the Kyte Dooliter method or Hopp
The Woods method (whether the Fourier transform is used or not) is used. Ho
pp and Woods, 1981, Proc. Nat. Acad.
Sci. USA 78: 3824-3828; Kyte and Do.
little 1982, J. Mol. Biol. 157: 105
See -142. Both are fully incorporated by reference into this document. Antibodies specific for one or more regions of the antigenic protein, its derivatives, fragments, analogs, or homologues are also provided in this document.

The protein of the present invention, its derivative, fragment, analog, homologue, or ortholog can be used as an immunogen in the production of antibodies that immunospecifically bind to these protein components.

The term “specifically” means that the variable region of an antibody of the invention exclusively recognizes and binds a polypeptide of the invention (ie, other similar polypeptides have sequence identity, A homologue or an analog of the polypeptide of the present invention can be distinguished from the homologue or analog, but it interacts with the outside of the variable region of the antibody, particularly the sequence of the constant region of the molecule, so that other proteins (for example, S. aureus protein A and other antibodies of the ELISA technique). Screening assay tests that determine the binding specificity of an antibody of the invention are well known and routinely practiced in the art. For details of such certification tests, see Halow et al. (Eds)
, Antibodies / Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, New York (1988), Chapter 6. Antibodies that recognize and bind the polypeptide fragments of the invention are predictable provided that they are specific for the full-length polypeptide of the invention as defined above, above all others. . An antibody specific for the full-length polypeptide of the invention,
The antibody of the present invention capable of discriminating a fragment is capable of discriminating a polypeptide from a homologous polypeptide (even if they have the same sequence and are homologs or analogs).

The antibody of the present invention is, for example, for therapeutic purposes (by adjusting the activity of the polypeptide of the present invention), diagnostic purposes by detecting and quantifying the polypeptide of the present invention, or purification of the polypeptide of the present invention. It can be used for etc. Also contemplated are kits (kits) containing the antibodies of the present invention and used for the various purposes described in this document. Generally, the kit of the invention also includes a control antigen to which the antibody is immunospecific. The invention further provides hybrid cells which produce the antibody. The antibodies of the present invention are useful for detecting and purifying the polypeptides of the present invention.

The monoclonal antibody bound to the protein of the present invention may be effective as a diagnostic agent for immunodetection of protein. Neutralizing monoclonal antibodies that bind to proteins may also be effective in treating certain cancers associated with protein-related conditions and abnormal protein expression. For cancer cells or leukemic cells, protein-antagonizing neutralizing monoclonal antibodies may be useful in detecting or preventing protein-mediated cancer cell metastasis.

[0299] The labeled antibody of the present invention can be used for an in vitro, in vivo, and in situ in situ test for identifying cells or tissues expressing a polypeptide fragment. The antibody may also be useful for direct therapy or other diagnostics. In the present invention, it is also possible to immobilize the above antibody on a solid support. Examples of solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®, acrylic resins such as polyacrylamide and latex beads. Techniques for immobilizing antibodies on such solid supports are well known in the art (Weir, DM, et al., "Experimental Immunology Handbook", 4th Edition, Blackwell Scientific Publishing, Oxford. , United Kingdom, Chapter 10 (1
986); Jacoby, W .; D. Et al., Meth. Enzym. 34 Academic Press, New York (1974). The immobilized antibody of the present invention can also be used for in-vitro, in-vivo and in-vivo tests, and for immunoaffinity purification of the protein of the present invention.

Various methods already known in the art can be used to produce a polyclonal or monoclonal antibody against the protein of the present invention, its derivative, fragment, analog, homologue, or ortholog. . (For example, Antibodies: Laboratory Handbooks, Halow E. and Lane D, 19 as referenced in this document.
88, Cold Spring Harbor laboratory Pr
See ess, Cold Spring Harbor, NY. ) Some of these antibodies are discussed below.

5.13.1 Polyclonal Antibodies In the production of polyclonal antibodies, various experimental animals, such as rabbits, can be prepared by injecting the natural protein, synthetic variants thereof, or their derivatives one or more times. Mammals such as goats and mice can be immunized. The preparation of the immunizing antigen may include a natural immunizing antigen protein, a synthetic polypeptide representing the immunizing antigen protein, or a recombinantly expressed immunogenic protein. Furthermore, it is also possible to combine a second protein, which is already known as an immune antigen in the body of the mammal to be immunized, with that protein to form a complex protein. Such immune antigen proteins include keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor and the like. Further, an auxiliary substance may be included. Among the various auxiliary substances used to enhance the immune response are Freund's auxiliary substances (complete and incomplete), mineral gels (eg aluminum hydroxide), surface-active substances (eg lysolecithin, pluronic polyols, Polyanion,
Peptides, oil emulsions, dinitrophenol, etc.). Auxiliary substances used in humans include Calmette Guerlain, Corynebacterium parvum, or similar immunostimulants. Other auxiliary substances that can be used are MPL-TDM auxiliary substances (monophosphoryl lipid A, synthetic trehalose dicorynomycolic acid).

Polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal, eg, blood, and purified by well-known techniques such as affinity chromatography using protein A or protein G. It mainly provides the IgG fraction of immune sera. Thereafter, or as an alternative method, it is also possible to immobilize a specific antigen or an epitope thereof, which is a target of immunoglobulin, on a column, and purify an immune antibody by immunoaffinity chromatography. Regarding the purification of immunoglobulins, see, for example, D. Discussed by Wilkinson (The Scientist Publishing, Philadelphia, PA, The Scientist, 14th).
Vol. 8 (April 17, 2000) 25-28).

5.13.2 Monoclonal Antibodies “Monoclonal antibodies” (MAbs) or “monoclonal compositions” as used in this document.
Means an antibody molecule population that contains only one type of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the monoclonal antibody sites (CDRs) that determine complementarity are the same for all molecules of the population. Therefore M
Ab has a specific affinity for an antigen and contains an antigen binding site capable of causing an immune reaction with an epitope of the antigen.

Monoclonal antibodies are available from Kohler and Milstein, Nature.
e, 256: 495 (1975), and can be prepared using a hybridoma (hybrid cell method). In the hybrid cell method, mice, hamsters and other experimental animals are immunized with an immunizing agent. It is to elicit lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent. Lymphocytes can also be immunized in vitro.

The immunizing substance usually includes a protein antigen, a fragment thereof, a fusion protein thereof and the like. Generally, peripheral blood lymphocytes are used when human cells are required, and spleen cells or lymphoid cells are used when non-human mammals are required. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybrid cells (Goding, Monoclonal Antibodies: Principles and Practices, Academic Press, (1986) 59. -Page 103). Immortalized cell lines are usually transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Usually, murine or murine myeloma cell lines are used. The hybrid cells can be cultured in a suitable medium. The medium preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, the parent cell has the enzyme hypoxanthine guanine phosphoribosyl transferase (HGP).
In the absence of RT or HPRT), medium for hybrid cells usually contains HGP.
It contains hypoxanthine, aminopterin, and thymidine (HAT medium) that inhibit the growth of RT-deficient cells.

The immortalized cells are preferably those having a good fusion rate, those which support a stable high level of antibody expression in the selected antibody-producing cells, and those which are sensitive to a medium such as HAT medium.
More preferred immortalized cells are murine myeloma cell lines. The murine myeloma cell line is the Silk Institute C of San Diego, California.
It is available from the Dell Distribution Center and the American Type Culture Collection in Manassas, VA. Also myeloma and heteromyeloma cell lines into mouse-human person, have been described for monoclonal antibody production of human (Kozbor, J. Immuno l, 133 :. 3001 (1984); Brodeur , etc., monoclonal antibodies produced Technology and Applications , Marcel Dekker, Inc., New York, (1987) 51-63).

The presence of monoclonal antibodies against the antigen is then analyzed in the medium in which the hybrid cells are cultured. The binding specificity of the monoclonal antibody produced by the hybrid cells is determined by immunoprecipitation reaction or by in vitro binding analysis such as radioisotope-labeled immunoassay (RIA) or enzyme-linked specific antibody adsorption assay (ELISA). It is preferable to determine. Such techniques and analytical methods are well known in the art. The binding affinity of the monoclonal antibody is determined, for example, by Manson and Pollard Scatchard analysis, Anal Biochem. , 107: 220 (1980). Separation of an antibody having high specificity and binding affinity to an antigen is desired.

Once the desired hybrid cell is identified, the clone is then subcloned by the limiting dilution method and grown by standard methods. A suitable medium for this purpose is
For example, Dulbecco's Modified Eagle medium and RPMI-16
40 medium. Hybrid cells can also be grown as ascites in the mammalian body.

The monoclonal antibody secreted by the subclone can be isolated from the medium or ascites by a common immunoglobulin purification method such as protein A sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography. It can be separated and purified.

Monoclonal antibodies can also be made by recombinant DNA methods as described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be easily separated and sequenced using conventional methods (eg, specific for the genes encoding the heavy and light chains of the murine antibody). With an oligonucleotide that can bind). The hybrid cells of the present invention are a preferred source of such DNA. The isolated DNA is then loaded into an expression vector, which is then used to synthesize monoclonal antibodies in recombinant host cells, usually in monkey COS cells, Chinese hamster ovary (CHO) cells, myeloma cells, etc. Transfected into host cells that do not produce immunoglobulin proteins. The DNA may, for example, use the encoded sequence of the human heavy and light chain constant regions in place of the homologous murine sequences (US Pat. No. 4,816,567; Morrison, Nature 3 68 , 812-12). (1994)), it is also possible to modify the non-immunoglobulin polypeptide encoded sequence by covalently linking it to the immunoglobulin encoded sequence. It is also possible to use such a non-immunoglobulin polypeptide in place of the constant region of the present invention or in place of the variable region of the antigen binding site of the antibody of the present invention to produce a chimeric bivalent antibody.

5.13.3 Humanized Antibody Furthermore, the antibody against the protein antigen of the present invention can be constituted by a humanized antibody or a human antibody. Such an antibody is suitable for administration to humans because it does not cause an immune reaction in humans even when immunoglobulin is administered. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (
For example, Fv, Fab, Fab ', F (ab') 2, etc. are subsequences of an antibody that binds to an antigen), which are mainly composed of human immunoglobulin sequences and were obtained from non-human immunoglobulins. Contains a minimum of arrays. Humanization of antibodies is
Followed the Winter and methods of his colleagues (Jone, etc., Nature, 32 1: 522-525 ( 1986); Riechmann , etc., Nature, 332: 323-327 (988 ); Verhoeyen , etc., Scien ce, 239: 1534- 1536 (1988)), can be achieved by substituting the corresponding rodent CDRs or CDR sequences for the human antibody sequences. (See also US Pat. No. 5,225,539) In some cases, the Fv substrate residue of human immunoglobulin is replaced with the corresponding non-human residue. Humanized antibodies can be made up of residues that are found neither in the receptor antibody, nor in the import CDRs nor in the substrate sequence. Generally, humanized antibodies are organized into at least one, and usually two, variable regions. In that case, all or substantially all of the CDR portions correspond to that of the non-human immunoglobulin and all or substantially all of the substrate portion is that of the human immunoglobulin consensus sequence. Humanized antibodies ideally comprise at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin (Jones et al. 1986; Riechmann et al., 198).
8; and Presta, Curr. Op. Stgruct. Biol . 2 : 593-596 (1992)).

5.13.4 Human Antibodies Human antibodies relate to antibody molecules in which substantially all of the light and heavy chain sequences, including the CDRs, are of human genetic origin. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies have been described by trioma technology, human B cell hybrid cell technology (Kozbor et al., 1983 Immuno).
l Today 4: 72), and EBV hybrid cell technology (Col.
E. et al., Monoclonal Antibodies and Cancer Treatment, Alan R. et al. Liss, Inc
． , Pp. 77-96)). Human monoclonal antibodies can be used in the practice of the invention, and by using human hybrid cells (Cote et al.
1983, Proc Natl Acad Sci USA 80: 202.
6-2030) or by transforming human B cells with EBV (Epstein-Ber-Wiels) in vitro (Cole.
Et al., Monoclonal Antibodies and Cancer Treatment, Alan R. et al. Liss, Inc.
, Pp. 77-96)).

[0313] In addition, human antibodies can be isolated using other techniques, such as phage display libraries (Houge).
nboom and Winter, J. et al . Mol. Biol. 227 :
381 (1991); Marks et al., J. Am . Mol. Biol. 2 22: 581 (1991)), or the like can be produced also by using a. Similarly, human antibodies
It can also be formed by introducing a human immunoglobulin locus into a transgenic animal, eg, a murine in which an endogenous immunoglobulin gene has been partially or completely inactivated. Observations of antibody production in humans have been made in response to challenge, but in all respects, including gene rearrangement, assembly, and antibody repertoire, it closely resembles what occurs in the human body. This is described, for example, in the following reports: US Patent Nos. 5,545,807; 5,545,806; 5,569,825.
5,625,126; 5,633,425; 5,661,016, Marks.
Et al. ( Bio / Technology 10 , 779-783 (992);
Lonberg et al. ( Nature 368, 856-859 (1994).
); Morrison ( Nature 368 , 812-13 (199).
4); Fishwild et al., ( Nature Biotechnology 14 , 845-51 (1996)); Neuberger ( Nature Biotechnology 14 , 826 (1996)); and Lonberg v. Inl. Hurzar .
unol. 13 65-93 (1995)).

Human antibodies can also be produced using transgenic animals that have been modified to produce fully human antibodies rather than their own endogenous antibodies (PCT Publication WO9.
4/02602). The animal's endogenous loci, which inactivate the animal's endogenous genes encoding the heavy and light chains of the immunoglobulin and instead encode the heavy and light chain immunoglobulins in the host genome, instead Has been inserted. Human genes are introduced, for example, using yeast artificial chromosomes containing the necessary portions of human DNA. Animals that provide all of the desired corrections are obtained as a progeny by cross-breeding intermediate transgenic animals that do not contain the corrections altogether. Among such animals, Mus musculus is preferred, and it is called Xenomouse, as shown in PCT publications WO 96/33735 and WO 96/34086. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from such animals after immunization with the immunizing antigen of interest, eg, in preparation of polyclonal antibodies. Alternatively, it can be obtained from immortalized B cells of animal origin, such as hybrid cells that produce monoclonal antibodies. It is also possible to directly obtain an antibody by restoring and expressing a gene encoding an immunoglobulin having a human variable site. Alternatively, further modifications can be made to obtain antibody analogs such as single chain Fv molecules.

Methods for producing non-human hosts deficient in endogenous immunoglobulin heavy chain expression, such as mice, are shown in US Pat. No. 5,939,598. It can be obtained in a manner that includes, but is not limited to, deleting the gene for the J portion from at least one of the endogenous heavy chain loci present in embryonic stem cells. It is to prevent rearrangement of that locus and to prevent the creation of copies of rearranged immunoglobulin heavy chain loci. Deletion is effected by the targeting vector containing the gene encoding the selectable marker. Then, from the embryonic stem cells, transgenic mice containing a gene encoding a selective marker in somatic cells and germ cells are produced.

Methods for producing sought antibodies, such as human antibodies, are described in US Pat. No. 5,916,771. It introduces an expression vector containing a nucleotide sequence encoding a heavy chain into a mammalian host cell in culture, and an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, It is concerned with the method of fusing two cells into a hybrid cell. Hybrid cells
It expresses antibodies containing heavy and light chains.

As a method of further improving this method, a method of identifying an epitope clinically related to an immune antigen, and a complementary method of selecting an antibody that immunospecifically binds to the epitope with high affinity are described. , PCT Publication WO 99/53049.

5.13.5 FAB Fragments and Single Chain Antibodies In the present invention, techniques for producing single chain antibodies specific for the antigenic proteins of the present invention include:
It is adjustable (see, eg, US Pat. No. 4,946,778). Furthermore, the method for constructing a Fab expression library may be adjusted in order to rapidly and effectively identify a monoclonal Fab fragment having a preferred specificity for a protein, or a derivative, fragment, analog, or homologue thereof. Possible (eg Huse et al., 1989)
Science 246: 1275-1281). An antibody fragment containing a genotype specific to a certain protein antigen can be produced using a technique well known in the art. For example, (i) an F (ab ′) 2 fragment produced by digesting an antibody molecule with pepsin; (ii) a Fab fragment produced by reducing the disulfide bridge of F (ab ′) 2; (iii) papain. Fab fragments produced by treating the antibody molecule with a reducing agent; and (iv) Fv fragments.

5.13.6 Biospecific Antibodies Bispecific antibodies are monoclonal antibodies (preferably human or humanized antibodies) that have binding specificities for at least two different antigens. . In the present case, one of the binding specificities is the antigenic protein of the invention. The second binding target is another antigen, which is preferably a cell surface protein, receptor, or receptor subunit.

Methods for making bispecific antibodies are well known in the art. Conventionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs. In this case, the two heavy chains have different specificities (Milstein and Cuello, Nature, 305.
: 537-539 (1983)). Since the immunoglobulin heavy and light chains are randomly combined, the hybrid cell (quadroma) produces a mixture of 10 different antibody molecules. Only one of them has the correct bispecific structure.
Purification of the correct molecule is usually done by affinity chromatography. A similar method is described in PCT Publication WO 93/08829 (May 1, 1993).
3 day) and Traunecker et al., EMBO J. , 10: 365
5-3659.

Variable regions of antibodies with the required binding specificities (antigen-antibody combining sites) can be fused to immunoglobulin constant region sequences. A fusion with a constant region of an immunoglobulin heavy chain composed of at least part of the hinge, CH2 and CH3 sites is preferred. It is preferred that the first heavy-chain constant region containing the site necessary for light chain binding, is present in at least one of the fusions. The DNA encoding the fusion of the immunoglobulin heavy chain (and possibly the immunoglobulin light chain) is inserted into a different expression vector and cotransfected into a suitable host organism. For further details on the method for producing a bispecific antibody, see, for example, Suresh et al., Enzymatic Chemistry,
121: 210 (1986).

According to another method described in WO 96/27011, it is also possible to devise the interface between a pair of antibody molecules so as to maximize the proportion of heterodermers recovered from the recombinant cell culture. Is. A preferred interface consists of at least a portion of the CH3 site that is in the antibody constant region. According to this method, small amino acid side chains obtained from the interface of the first antibody molecule can be replaced with large side chains such as tyrosine and tryptophan. By replacing the large amino acid side chain with a small amino acid side chain such as alanine or threonine, a compensating "cavity" of the same or similar size to the large side chain can be created on the interface of the second antibody molecule. Can be made on top. In this way it is possible to increase the number of heterodimers over unfavorable end products such as homodimers.

Bispecific antibodies can be full-length antibodies or fragments of antibodies (eg, F (ab ')).
2 bispecific antibody). Techniques for making bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985),
A method for proteolytic cleavage of intact antibodies to produce F (ab ') 2 fragments is described. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenate, thus stabilizing vicinal dithiols and inhibiting the formation of intermolecular disulfide bonds. The Fab ′ fragment thus generated is converted to a thionitrobenzoate (TNB) derivative. The Fab'-TNB derivative is then converted back to Fab'-thiol upon reduction with mercaptoethylamine,
Mixed with equimolar amounts of'-TNB derivative to form bispecific antibody. The produced bispecific antibody can be used as a selective immobilizing agent for an enzyme.

Alternatively, Fab ′ fragments are directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al. Exp. Med. 17
5: 217-225 (1992) is a fully humanized bispecific antibody F (
ab ') describes the production of two molecules. Each Fab ′ fragment is separately secreted from E. coli and chemically bound in vitro, resulting in the formation of bispecific antibodies. The bispecific antibody thus formed can bind to cells overexpressing the ErbB2 receptor and normal human T cells, while at the same time lysing cytotoxic lymphocytes against human breast cancer. Induce activity.

Various methods for forming and isolating bispecific antibody fragments directly from transgenic cell culture have also been reported. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al. Immunol. 148 (
5): 1547-1553 (1992). The leucine zipper peptides obtained from the Fos and Jun proteins are F-linked to two different antibodies by gene fusion.
It was linked to the ab 'part. The antibody homodimer was reduced at the hinge to make a monomer, which was reoxidized to form an antibody heterodimer. This method can also be used for the production of antibody homodimers. Hollinger et al . , Proc. Nati. Acad. Sci. USA , 90: 6444-6448 (1993).
The “diabody” technology described in US Pat. The fragment consists of a heavy chain variable region (VH) and a light chain variable region (VL) linked together by a linker. Since the linker is short,
It does not pair between two regions on the same chain. Thus, the VH and VL regions of one fragment are forced to pair with the complementary VL and VH regions, resulting in the formation of two antigen binding sites. A method for producing a bispecific antibody fragment using a single chain Fv (sFv) dimer has also been reported. Gruber et al., J
． Immunol. 152: 5368 (1994).

Antibodies with three or more valencies are also contemplated. For example, avian-specific (trispecific) antibodies can be prepared. Tutt et al. Immunol. 147: 50
(991). A typical bispecific antibody can bind two different epitopes. At least one of them is derived from the protein antigen of the present invention. The arm for the anti-antigen of the immunoglobulin molecule is provided with a trigger molecule (for example, CD2, CD3, C
T cell receptor molecules such as D28 and B7), or F of IgG (FcγR)
c receptor (eg, FcγRI (CD64), FcγRII (CD32), Fc
It is also possible to link with the arm bound to γRIII (CD16)). This is to concentrate the defense mechanism of cells on cells expressing a specific antigen. Bispecific antibodies can be used to direct cytotoxic agents to cells expressing a particular antigen. Such an antibody possesses an arm that binds to an antigen and an arm that binds to a cytotoxic substance or a radionuclide chelating agent (EOTUBE, DPATA, DOTA, TETA, etc.). Another bispecific antibody also binds to the protein antigens described in this document and also to tissue factor (TF).

5.13.7 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. As a method of using such an antibody, for example, a method of attacking undesired cells by immune tissue cells (US Pat. No. 4,676,980)
And treatment of HIV infection (WO 91/00360; WO 92/200373
; EP 03089) has been proposed. It is considered that such an antibody can be prepared in vitro using a method well known in synthetic protein chemistry (for example, a method using a cross-linking substance etc.). For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Suitable reagents for this purpose are, for example, iminothiolate, methyl-4-mercaptobutyrimidate,
Examples include reagents listed in US Pat. No. 4,676,980.

5.13.8 Effector Function Engineering The antibody of the present invention is preferably adjusted in terms of effector function. This is to enhance the effect of the antibody in the treatment of cancer, for example. For example, a cystine residue is introduced at the Fc site and intramolecular SS bond formation is allowed to occur at that site. The homodimeric antibody thus produced may have enhanced internalization capabilities and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity. Caron et al. Exp. Me
d. 176: 1191-1195 (1992); Shopes, J.
． Immunol. , 148: 2918-292 (1992). Homodimeric antibodies with enhanced anti-cancer activity are described in Wolff et al., Cancer Research,
53: 2560-2565 (1993).
It is also possible to prepare using a dual-acting crosslinker. It is also possible to produce antibodies with Fc dual sites to enhance complement lysis and ADCC capabilities. Stevens
See et al., Anticancer drug design, 3: 219-230 (1980).

5.13.9 Immunoconjugate The present invention provides chemotherapeutic agents, toxins (eg, enzymatically active toxins from bacteria, molds, plants, animals, or fragments thereof), radioactive isotopes (ie, radioactive conjugates). )
It is also related to immunoconjugation in which a cytotoxic substance such as is bound to an antibody.

The chemotherapeutic agents used to generate such immunoconjugates are as described above. Enzymatically active toxins and fragments thereof include, for example, diphtheria A chain, non-binding active fragments of diphtheria venom, and exotoxin A chain (Pseudomonas aerugino).
sa origin), ricin A chain, abrin A chain, modesin A chain, alpha-sarcin,
Aleurites fordii protein, dianthin protein, Phytol
aca Americana proteins (PAPI, PAPII, and PAP-
S), momordica charantia inhibitor, curcin, crotin,
sapaonaaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, trichothecin and the like.
For the production of radioactive conjugated antibody, 212Bi, 131I, 131In, 90Y
, 186 Re, and many radionuclides are available.

Various dual-acting protein binding agents are used for binding the antibody to the cytotoxic substance. Bifunctional protein binders include N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), and bifunctional derivatives of imide esters (dimethyl adipimidate HCL, etc.), Active ester (disuccinimidyl / suberate, etc.), aldehyde (glutaraldehyde, etc.), bisazide compound (bis (P-azidobenzoyl) hexanediamine, etc.), bis-diazonium derivative (bis- (P-diazoniumbenzoyl)) Ethylenediamine etc.), diisocyanate (triene 2,6-diisocyanate etc.), bis-active fluorine compound (1,5-difluoro-2,4-dinitrobenzene etc.) and the like. For example, ricin immunotoxins are described in Vitetta et al., Science, 238: 1.
098 (1987). Carbon-14 labeled 1-
Isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (M
X-DTPA) is a typical chelating agent for the binding of radionuclides to antibodies.
See WO94 / 11026.

It is also possible to conjugate the antibody to a "receptor" such as streptavidin, in preparation for tumor attack. In that case, the antibody / receptor conjugate is administered to the patient, and the conjugate that is not bound thereafter uses a clarifying agent, and then a “ligand” (which binds to the cytotoxin) such as avidin is used. By administration, it is cleared from the circulatory system.

5.14 Computer Readable Sequences In one application, the nucleotide sequences of the invention can be recorded on computer readable media. As used in this document, "computer-readable medium" means any medium that can be read and accessed by a computer. For example, a magnetic storage medium such as a floppy (registered trademark) disk, a hard disk storage medium, a magnetic tape, an optical storage medium such as a CD-ROM, RAM or R.
It is a hybrid of an electronic storage medium such as OM, a magnetic storage medium, an optical storage medium, or the like. A skilled technician should readily know which of the currently known computer-readable media can be used to make a computer-readable media product recording the nucleotide sequences of the present invention. Is. The phrase "recorded" as used in this document refers to the process of storing information on a computer-readable medium. A skilled technician should readily be able to employ any method of recording information on a computer readable medium to produce a product containing the nucleotide sequence information of the present invention.

A skilled technician can make computer readable media recording nucleotide sequences of the invention using a variety of data storage structures. Which data storage structure to select is usually determined by the means by which the recorded information is accessed. Furthermore,
A variety of data processing programs and formats can be used to record the nucleotide sequence information of the present invention on a computer readable medium. The sequence information can be represented as a word processing text file formatted by a commercial software such as WordPerfect or Microsoft Word, or can be represented as ASCII in the form of a database such as DB2, Sybase or Oracle. Is. A skilled technician can readily use a data processing structure format (text file or database) to obtain a computer readable medium having the nucleotide sequence information of the present invention recorded therein.

SEQ ID NO: 1-3, 5, or 12 representative fragments thereof, or SE
Q ID NO: 1-3, 5, or 12 nucleotide sequences and at least 9
By providing nucleotide sequences that are 5% identical in computer readable form, a competent technician can access that sequence information at any time and for a variety of purposes. Any number of computer software is available commercially that enables access to the sequence information provided in the form of a computer readable medium. The following example shows BLAST (Altschul) on the SYBASE system.
Et al., J. Mol. Biol. 215: 403-410 (1990).
) And BLAZE (Brutlag et al., Comp. Chem. 17:
203-207 (1993)) using software that executes a search algorithm,
1 shows a method for confirming an open reading frame (ORF) in a nucleic acid sequence. Such an ORF may be a protein-encoding fragment, and is used in the production of commercially important proteins such as enzymes used in fermentation reactions and enzymes used in the production of commercially useful metabolites. It may be.

As used herein, the term "computer-based system" refers to the hardware, software, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware required for the computer utilizing system of the present invention is a central processing unit (CPU), an input device, an output device,
And a data storage device. A skilled technician can readily discern which of the currently available computer-based systems are suitable for use with the present invention. As described above, the computer utilizing system of the present invention comprises a data storage device recording the nucleotide sequence of the present invention, and hardware and software devices necessary for supporting and executing the searching means. As used in this document, the term "data storage device" refers to a storage device (memory) capable of recording the nucleotide sequence information of the present invention, or a memory access device for accessing a product recording the nucleotide sequence information of the present invention. Means either.

The term “probing tool” as used in this document refers to one or more implemented on a computer-based system to compare a target sequence or target structural motif with sequence information recorded in a data storage device. Means multiple programs. Searching means are used to identify fragments or known sequence sites that match a particular target sequence or target motif. Various algorithms have already been commercialized, and a large number of search means execution software that can be used in the computer-using system of the present invention are commercially available. Examples of such software include Smith-Waterman and MacP.
attern (EMBL), BLASTN, BLASTA (NPOLYPE
PTIDEIA) etc. A competent technician should readily be able to determine which of the various algorithms or execution software units for homology search are available for the computer-based system of the present invention. As used herein, the term "target sequence" refers to any nucleic acid, an amino acid sequence of 6 or more nucleotides, or an amino acid sequence of 2 or more amino acids. A competent technician should immediately recognize that the longer the target sequence, the less likely it will occur randomly in the database. The most preferred length of the target sequence is about 10 to 100 amino acids, or about 30 to 300 nucleotide residues. However, as is well known, the search for commercially important fragments such as sequence fragments related to gene expression, protein processing, etc. may be performed in a considerably short length.

The term “target structural motif” or “target motif” as used in this document refers to a rationally selected sequence or combination of sequences that forms on the folds of the target motif. Selected based on the three-dimensional structure Various types of target motifs are known in the art. The target motif of a protein is, for example, the active site of an enzyme or a signal sequence. Target motifs of nucleic acids include promoter (promoter) sequences, hairpin structures, inducible expression elements (protein binding sequences), and the like.

Triple Helix Formation Furthermore, the fragments of the invention can be used in a broad sense to control gene expression through triple helix formation or antisense DNA / RNA. Both methods are based on DNA or RNA binding of polynucleotide sequences. Polynucleotides suitable for such use usually have a base length of 20 to 40 and have a gene site related to transcription (triple helix-Lee et al., Nucl.
cids Res. 6: 3073 (1979); Cooney et al., S.
Science, 15241: 456 (1988); Dervan et al.,
Science, 251: 1360 (1991)), or m
RNA itself (antisense Olmno, J. Neurochem. 56:
560 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Vocarayton, Florida (1988)) and are designed with complementarity. Triple helix formation blocks transcription of DNA to RNA under optimal conditions, and antisense RNA hybridization method
Block the translation of the A molecule into a polypeptide. Both techniques have proven effective in model systems. The information contained in the sequences of the present invention is necessary for the design of antisense or triple helix oligonucleotides.

5.16 Diagnostic Assays and Tools The present invention employs nucleic acid probes or the antibodies of the present invention (which can be appropriately labeled) to determine the presence or expression of one ORF of the present invention or its homologues. Also provides a method of identifying in a test sample.

A method of detecting a polynucleotide of the present invention, which is typically one in which a compound that binds to the polynucleotide and forms a complex is contacted with a sample for the time required to form the complex, and then the complex is formed. To detect. As a result, the polynucleotide of the invention will also be detected in the sample if the complex is present. The method further comprises subjecting the sample, under stringent conditions of hybridization, to a nucleic acid primer that anneals the polynucleotide of the invention under such conditions, and then amplifying the annealed polynucleotide. If the polynucleotide is amplified, the polynucleotide of the invention will be detected in the sample.

A method of detecting a polypeptide of the invention, which is typically one in which a compound that binds to the polypeptide and forms a complex is contacted with a sample for the time required to form the complex, and then the complex is formed. To detect. As a result, the polypeptide of the invention will also be detected in the sample if the complex is present.

These methods are described in more detail by incubating a test sample with one or more of the antibodies of the invention, or one or more of the nucleic acid probes, followed by testing of the components of the test sample and nucleic acid The binding state with the antibody is analyzed.

Conditions for incubating the nucleic acid probe or antibody with the test sample are various. The culture conditions depend on the format used for the analysis, the detection method, and the type and nature of the nucleic acid probe or antibody used for the analysis. Those who are trained in the technology of the industry,
One can readily determine which of the commonly available hybridization (hybridization), amplification, or immunoassay formats are suitable for use with the nucleic acid probes or antibodies of the invention. Such an assay is described by Chard, T .; , Introduction to Radioimmunoassay and Related Technologies, Elsevier Science Publisher, Amsterdam, The Netherlands (1986); Bullock, G .; R. Etc, immunocytochemistry technology,
Academic Press, Orlando, Florida, Volume 1 (1982), Volume 2 (
1983), Volume 3 (1985); Tijssen, P .; , Practice and theory of immunoassays: experimental techniques in biochemistry and molecular biology, Elsevier Science Publisher, Amsterdam, Netherlands (1985), etc. The test sample of the present invention includes cells, cell proteins or membrane extracts, biological fluids (sputum, blood, serum, plasma, urine, etc.). The test sample used in the above method differs depending on the assay format, the nature of the detection method, the nature of the cell tissue, cells, or extracts used as the assay sample. The method for preparing a protein extract or membrane extract of cells is well known in the art and can be easily adjusted, so that a sample suitable for the system to be used can be immediately obtained.

In another application of the invention, there is provided a set of instruments containing the reagents necessary to carry out the assays of the invention. In particular, the present invention provides a compartmental tool for containing one or more containers. It is composed of: (1) a first container containing one of the probes or antibodies of the invention; (2) another one or more containers containing wash reagents, bound probes. Alternatively, it contains one of the reagents capable of detecting the presence of the antibody.

To describe in more detail, compartmentalization tools include all tools that contain reagents in different containers. Such containers include small glass containers, plastic containers,
Or, there is a piece of plastic or paper. If you use such a container, 1 reagent
It can be effectively transferred from one compartment to another. The reagent and the sample do not mix, and it is also possible to quantitatively add the substance or solution contained in each container from one compartment tool to another compartment tool. Examples of such containers include containers for test samples, containers for antibodies used in assays, containers for washing reagents (phosphate buffered saline, Tris buffer, etc.), and detection of bound antibodies and probes. There are containers that contain reagents. The detection reagent includes a labeled nucleic acid probe, a labeled secondary antibody, or when the primary antibody is labeled, an enzyme or antibody binding reagent capable of reacting with the labeled antibody. One of ordinary skill in the art will readily recognize which of the probe formats or antibodies disclosed in the present invention are suitable for any of the device formats well known in the art.

5.17 Medical Imaging The new polypeptides and binders of the invention are useful for medical imaging of sites expressing the molecules of the invention (eg where the polypeptides of the invention are involved in immune responses). , Images of inflammation or infected parts, etc.). See, for example, Kunkel et al., US Pat. No. 5,413,778. Such methods involve chemical attachment of labeling agents or imaging agents, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging of the labeled polypeptide at a target site in vivo. is doing.

5.18 Screening Analysis Method The present invention uses the isolated proteins and polynucleotides to provide ORF (
SEQ ID NO: corresponding to any of the nucleotide sequences set forth in 1-3, 5, or 12), the agent bound to the polypeptide encoded by the polypeptide, or the specificity of the polypeptide encoded by the nucleic acid. Also provided is a method for obtaining and identifying an agent bound to a specific domain. The details of the above method consist of the following steps. The isolated protein encoded by the ORF of the present invention is contacted with a substance. Determining whether the substance binds to the protein or the nucleic acid. Therefore, normally, to identify a compound that binds to a polynucleotide of the invention, the compound of the invention is contacted with the compound for the time necessary to form a polynucleotide / compound complex, and then the complex is detected. . By doing so, if the polynucleotide / compound complex is detected, the compound that binds to the polynucleotide of the invention is identified.

[0349] Thus, also commonly, to identify compounds that bind to a polypeptide of the invention,
The polypeptide of the invention and the compound are contacted for the time required to form the polypeptide / compound complex, and then the complex is detected. By doing so, if the polypeptide / compound complex is detected, the compound that binds to the polynucleotide (mom) of the present invention is identified.

As a method of identifying a compound that binds to the polypeptide of the present invention, a method of contacting the polypeptide of the present invention with a compound in a cell for a time necessary to form a polypeptide / compound complex is also provided. is there. In that case, since the complex promotes the expression of the gene sequence of the receptor of the cell, the complex can be detected by detecting the phenotypic expression of the gene sequence of the receptor. As a result, if a polypeptide / compound complex is detected, a compound that binds to the polypeptide of the invention is identified.

Compounds identified in this manner also include compounds that modulate the activity of the polypeptides of the invention (ie, compounds that increase or decrease the activity of the polypeptide relative to its activity in the absence of the compound). Can be included. Included among the compounds identified in this way are compounds that modulate the activity of the polynucleotides of the invention (ie, compounds that increase or decrease phenoexpression relative to the level of expression in the absence of the compound). Is also possible. Compounds as identified using the methods of the invention can be tested for their ability to regulate activity or expression of phenotypes using standard assay methods already known in the art.

The substances to be screened by the above method include peptides, carbohydrates, vitamin derivatives, and other pharmaceutical substances. Such substances can be randomly selected or screened. Alternatively, protein modeling technology can be used to intelligently select and design.

For random screening, peptides, carbohydrates, pharmaceutical agents, etc. are randomly selected and assayed for their ability to bind to the protein encoded by the ORF of the invention. The substance can be intelligently selected or designed. In this document, when a substance is selected based on the structure of a particular protein, that substance is "
It is reasonably selected or designed ”. For example, a person trained in medical technology
The existing method is applied to easily produce a peptide or a pharmaceutical substance having an ability to bind to a specific peptide sequence, and thus an rationally designed anti-peptide (mom) peptide (for example, Hurby etc., Applications of synthetic peptides: antisense peptides, synthetic peptides, user guides, WH Freeman, NY
(1992) 289-307, and Kaspczak et al., Biochemistry 28:92.
30-8 (1989)), or pharmaceutical substances can be made.

In addition to the foregoing, in a broad sense, a class of substances of the invention are compounds of the invention O
It can be used to control gene expression through binding to one of RF or EMF. As mentioned above, such substances may be randomly screened,
You may reasonably design and select. One of skill in the art can regulate the expression of a single ORF or multiple ORFs (both of which depend on the same EMF in terms of expression control) by targeting the ORF or EMF to achieve sequence specificity or element specificity. Can design sexual substances. One class of DNA binding agents contains residues of bases that bind to DNA or RNA to form a triple helix. Such materials may be based on classical phosphodiester, ribonucleic acid backbones, or various sulfhydryl or polymerized derivatives with base attachment ability.

The materials used in these methods usually contain 20 to 40 bases, so that they are complementary to the gene sites involved in transcription (triple helix-Lee et al.,
Nuel. Acids. Res. 6: 3073 (1979); Coo
ney et al., Science, 241: 456 (1988); and Der.
van et al., Science 251: 1360 (1991)), or as complementary to the mRNA itself (antisense Okano,
J. Neurochem. 56: 560 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Boca Leighton, FL (1988)). Triple helix formation yields the best results in terms of inhibiting transcription of DNA to RNA, and antisense RNA hybridization inhibits the process of translating mRNA into a polypeptide. Both techniques have proven effective in model systems. The information contained in the sequences of the present invention is necessary for the design of antisense, triple helix oligonucleotides, and other DNA binding agents.

Agents that bind to the protein encoded by one of the ORFs of the invention can be used as diagnostic agents. Agents that bind to the protein encoded by one of the ORFs of the present invention can be formulated according to known techniques used to produce pharmaceutical compounds.

5.19 Use of Nucleic Acids as Probes The present invention also provides nucleic acid hybridization probes specific for polypeptides that can be hybridized using naturally occurring nucleotide sequences. The hybridisation probe of the present invention is obtained from any of the nucleotide sequences SEQ ID: 1-3, 5, or 12. Hybridization probes obtained from any of the nucleotide sequences SEQ ID: 1-3, 5, or 12 are useful for expressing such tissue cells in a sample because the corresponding gene is expressed only in a limited number of tissues. Type of RN
It can be used as an index indicating the presence of A.

[0358] For example, an appropriate hybridization technique such as in situ hybridization can be used. The PCR described in US Pat. Nos. 4,683,195 and 4,965,188 also provide another use for oligonucleotides based on nucleotide sequences. Such probes used in PCR are produced recombinantly, chemically synthesized, or mixed. The probe is composed of different nucleotide sequences for the detection of identical sequences or a degenerate pool of possible sequences for the identification of closely related genomic sequences.

Other methods of generating nucleic acid-specific hybridization probes include cloning nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known to those of skill in the art and are commercially available,
It can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase or SP6 RNA polymerase as T7 and an appropriately radiolabeled nucleotide.

Nucleotide sequences can be used to construct hybridizing probes to map each genomic sequence. The nucleotide sequences provided by the present invention can be mapped to certain chromosomes or specific regions of chromosomes by commonly known genes and / or chromosome mapping techniques. These techniques include in situ hybridization, linkage analysis for known chromosomal markers, and hybridization screening using chromosomal preparations or libraries with known flow classification specific to known chromosomes. A method for in situ hybridization of fluorescent distribution of chromosomes is described by Bama et al. (1988) Human Chromosomes: A Manu.
al of Basic Technologies, Pergamon Pre
ss, New York NY. (Human chromosome: Manual of basic methods,
(Pergamon Press, New York City, NY).

Fluorescent in situ hybridization of chromosome preparations and other physical staining map generation techniques can be matched by additional gene map data. An example of genetic map data is
1994 Genome Issue of Science (Genome Special Issue Science) (265: 1981f). The correlation between the location of a nucleic acid on a physical chromosomal map and a particular disease (or constitution for a particular disease) helps to define the region of DNA associated with that particular disease. The nucleotide sequences of the invention can be used to detect differences in gene sequences between normals, carriers or patients.

5.20 Preparation of Oligonucleotides Immobilized on a Support Oligonucleotides, ie small segments of nucleic acids, can be prepared, for example, using an automated oligonucleotide synthesizer, that is to say the direct chemistry of oligonucleotides that is commonly performed. It can be easily created by composition.

Support-immobilized oligonucleotides can be prepared by methods using a suitable support, such as glass, polystyrene or Teflon®, well known to those skilled in the art. One method is to accurately identify the synthesized oligonucleotides using standard synthesizers. Immobilization methods include passive adsorption (Inoue and Honda, 1990 J. Clin M
icrobiol 28 (6) 1462-72); UV) (Nagata et al., 1985; Dahlen et al., 1987, Morrissey and Col.
Lins, Mol. Cell Probes 1989 3 (2) 189-
207) or a covalent bond of base-modified DNA (Keller et al., 1988).
1989), etc., and the references cited here are incorporated in the present application. Another method is to use the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl.
Acad. Sci USA 91 (8) 3072-6 describes the use of biotinylated probes, which are immobilized with double probes, streptavidin-coated magnetic beads.

Streptavidin-coated beads can be purchased from Dinard of Oslo. Of course, the same binding chemistry can be applied for coating surfaces with streptavidin. Biotinylated probes are available from various sources, such as, for example, Operon Technologies, Inc. (Alameda, Calif.).

Suitable materials can also be obtained from Nunk Laboratories (Naperville, IL). Nunk Laboratories can covalently bond to a microwell surface called Cobalink NH. Cobalt-Link NH is a polystyrene surface onto which the second stage amino group (> HN) has been grafted to provide a bridgehead for further covalent bonding. The Kovalink module was purchased from Nunk Laboratories. The DNA molecule is 5'of Kobalink due to the host amide bond.
It can be bound only at the ends and allows the immobilization of more than 1 pmol of DNA (Rasmussen et al. (1991) Anal Biochern 19).
8 (l) 138-42).

The use of Cobalink NH stop for covalent attachment of DNA to the 5'end has been published (Rasmussen et al., 1991). Phosphoramidate linkages are used in this technique. (Chiu et al., 1983 Nucleic A
cids 11 (18) 6513-29) This has the advantage of using only one covalent bond for immobilization. The phosphoramidate linkage binds the DNA via a 2 nm long spacer arm to the Covalink NH secondary amino group located at the end of the spacer arm covalently grafted onto the polystyrene surface. In order to link an oligonucleotide to Cobalink NH via a phosphoramidate bond, the oligonucleotide terminal must have a 5'end phosphate group. It is possible to covalently attach biotin to cobalink and use streptavidin for probe attachment.

More specifically, this linking method involves the steps of dissolving DNA in water (7.5 ng / ul), denaturing for 10 minutes at 95 ° C. and chilling on ice for 10 minutes. Next, ice-cooled 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm ⁷ ).
To give a final concentration of 10 mM I-MeIm ⁷ . And ss DNA
The solution is then fed to an ice-cooled Cobalink NH strip (75 ul / well).

Carbodiimide 0.2 M ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) dissolved in 10 mM 1-MeIm ⁷ was newly made and 25 ul was added per well. The strips were incubated at 50 ° C for 5 hours. After culturing, the strips were washed using, for example, Nunc-ImmunoWash.
The wells were first washed 3 times, then immersed in wash solution for 5 minutes, and finally washed 3 times (wherein the wash solution was warmed to 50 ° C. with 0.4 N NaOH, 0.25).
% SDS).

A further suitable method for the present invention is PCT patent application WO 90/03382.
(Southern & Muskos), which is hereby incorporated by reference. In this method of making oligonucleotides immobilized on a support,
Also included is a method of attaching a nucleoside 3'-reagent via a phosphate to a support-supported aliphatic hydroxyl group by a covalent phosphodiester link.

The oligonucleotide is then synthesized on the supported nucleoside and removed from the synthesized oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidites and nucleoside hydrogen phosphorates.

DNA probes can be made on a chip for making a DNA probe array. For example, performing directly-addressable laser-activated photodeprotection on a glass plate is described by Forder et al. (1991) Scie.
nce 251 (4995) 767-73,
Reference here is made part of the present application. Immobilizing the probe on a nylon support is described by Fannes et al. (1991) Nucleic Acid.
s Res. 19 (12) 3345-50.
The probe is also Duncan and Cavalier, (1988) Anal 10Bi.
Ochem 169 (l) 104-8 using Teflon®
Can also be linked to; all these references are incorporated herein by reference.

To link oligonuclotides to nylon supports, see Fanness et al. (199).
As described in 1), it is necessary to activate the nylon surface by alkylation and selectively activate the 5'-amine of the oligonucleotide with cyanuric chloride.

Another method for making support-immobilized oligonucleotides is described by Peas et al. (1994) Proc. Natl. Acad. Sci USA 9
1 (11) 5022-6. These authors have used recent photolithographic methods to create arrays of immobilized oligonucleotide probes (DNA chips). These methods of synthesizing oligonucleotide probes using light in a dense and miniaturized array have been shown to be photolabile 5'-
It utilizes protected N-acyl-deoxynucleoside phosphoramidite, surface linker chemistry and various combinatorial synthetic approaches. With this method, 25
An oligonucleotide probe matrix divided into 6 can be prepared.

5.21 Generation of Nucleic Acid Fragments Nucleic acids are RNs that include cDNAs, genomic DNA, chromosomal DNA, microdissected chromosomal bands, cosmid or YAC inserts, and mRNA without amplification steps.
Obtained from various sources such as A. For example, Sambrook et al. (1989)
Describe three protocols for separating high molecular weight DNA from mammalian cells (p. 9.14-9.23).

DNA fragments can be cloned into M13, plasmids or lambda vectors and / or directly from genomic DNA or cDNA by PCR or other amplification methods. Samples can be made and sorted on multiwell plates. About 100-1000 ng of DNA sample is produced in a final volume of 2-500 ml.

Nucleic acids may be sonicated and Na-treated using any method known to those of skill in the art, for example using the restriction enzymes described in 9.24-9.28 of Sambrook et al. (1989).
It is fragmented by the method of shearing by OH treatment.

The low pressure shear method is also described by Schlierfer et al. (1990) Nucleic
Acids Res. 18 (24) 7455-6. In this method, a small French pressure cell is subjected to various pressures from low to medium to pass a DNA sample. Low to medium pressure regulation of cells is controlled by a lever device. The results of these studies have revealed that low pressure shear can be an alternative to sonication and enzymatic DNA fragmentation methods.

A particularly suitable method for fragmenting DNA is Fitzgerald et al. (1
992) Nucleic Acids Res. 20 (14) 3753-6
A method of using the two-base discriminating endonuclease CviJI disclosed by No. 2 is considered. These authors described a method for performing rapid fragmentation and fractionating DNA to the size of properties they considered reasonable for shotgun cloning and sequencing.

The restriction endonuclease CviJI normally splits the discriminating sequence PuGCPy between G and C, leaving a blunt end. The special reaction conditions that change the specificity of this enzyme (CviIL **) are that quasi-randomly distributed DNA fragments are bound to the small molecule pUC 1
9 (2688 base pairs). Fitzgerald et al. (192) described the randomness of this fragmentation method by size fractionation by rapid gel filtration.
Using the CviJI ** digest of pUC19 and ligating directly to the rack Z-M13 cloning vector without end repair,
It was evaluated quantitatively. Sequence analysis of 76 clones revealed that CviJI ** in addition to PuGCPy restricted pyGCPy and PuGCPu, and new sequence data accumulated at a rate consistent with random fragmentation.

As reported in the literature, the advantages of this method are: compared to the sonication and agarose gel fragmentation methods: lower DNA requirements (0-5 instead of 2-5 ug).
． 2-0.5 ug); Fewer processing steps (no need for pre-ligation, edge repair, chemical extraction, or agarose gel electrophoresis and elution etc.).

Regardless of the method of obtaining and making the nucleic acid fragments, it is important to denature the DNA to provide a single strand for hybrid formation. This is a DNA solution 80-
This is achieved by incubating at 90 ° C for 2-5 minutes. The solution is then rapidly cooled to 2 ° C. to prevent the DNA fragments from refolding by the time they contact the chip.
The phosphate group must be removed from the genomic DNA by methods known to those of skill in the art.

5.22 Preparation of DNA Array An array can be prepared by spotting a DNA sample on a support such as a nylon membrane. Spotting is an array of metal pins (the location of which corresponds to an array of wells in a microtiter plate)
Can be carried out by repeatedly transferring about 20 nl of the DNA solution to a nylon membrane. Offset printing provides a dot density that is greater than or equal to the density of the wells. There can be 1 to 25 dots per mm ² depending on the label used. By avoiding spotting on specific preselected rows and columns, another subset (subarray) can be formed. The samples in one subarray can be the same genomic segment (or the same gene) of DNA from different individuals, or different overlapping genomic clones. Each sub-array may represent replica spotting of the same sample. In one example, the selected gene segment was amplified from 64 patients. For each patient, the amplified gene segment may be on one 96-well plate (all 96 wells have the same sample). One plate is prepared for each of the 64 patients. Using the 96 pin device, all samples are spotted on a single 8x12 cm membrane. The sub-array can also be provided with a total of 64 samples, one from each patient. When 96 sub-arrays are identical, the dot span is 1 mm ² and there may be 1 mm spacing between the sub-arrays.

Another method is to use a membrane or plate (Nunc Inc., Naperville, Ill.) Separated by a physical spacer, in which case the spacer is made of plastic molded over the membrane. With a grid, the grid may be similar to a multiwell plate, or a membrane provided on the bottom of a hydrophobic strip.
Fixed physical spacers are not desirable when imaging by exposing a flat phosphorus storage screen, or X-ray film.

The invention is illustrated in the following examples. It will be apparent to those skilled in the art from this disclosure that various modifications and changes can be made within the scope of the present invention. Therefore,
The broad possibilities of the present invention should not be limited by the following examples. The present invention should not be limited by the examples which are intended to illustrate only one aspect of the invention, and functionally equivalent compositions and methods are within the scope of the invention. In fact, many modifications and variations are possible to those skilled in the art in practicing the invention, given the preferred embodiments presented herein. Accordingly, the only limitations within the scope of the invention are the claims provided.

All references mentioned in this specification are hereby incorporated by reference in their entireties.

6.0 Example Example 1 Isolation of SEQ ID NO 1 from a Testis cDNA Library A cDNA library obtained from testis was sequenced using hybridisation sequence signature analysis and Sanger sequencing techniques using standard PCR. As a result of the determination, a plurality of novel nucleic acids (Hyseq clone recognition number 2924342 (SEQ ID NO:
1)) was obtained. The insert of the library was amplified by PCR using primers specific for the vector sequences flanking the insert. This sample was spotted on a nylon membrane and assayed for sequence signature using an oligonucleotide probe. These clones were sorted by similar or identical sequences and a single representative clone from each group was selected for gel sequencing. The 5'sequence of the amplified insert was deduced a priori using the reverse M13 sequence primer in a typical Sanger sequence protocol. The PCR products were purified and then subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was performed using a 377 Applied Biosystems (ABI) sequencer. It was confirmed that the insert is a new sequence that has never been obtained from this library, and that it is not in the public database. This sequence is SEQ
It was named ID NO: 1.

Example 2 Assembly of SEQ ID NO: 2 The nucleic acid named SEQ ID NO: 2 according to the present invention has SEQ ID NO: 2.
Collected using NO: 1 as a seed. Then, the cyclic algorithm is applied to the seed and expanded to obtain the expanded set. In that case, a different database belonging to this set (ie a database containing the Hyseq EST sequences, dbE
Additional sequences were derived from ST version 114, gb pri 114, and UniGene version 101). The algorithm stopped when no additional sequences to expand the set were available from the above database. Incorporation of component sequences into the set was based on BLASTN hits for the expanding set with BLAST scores greater than 300 and greater than 95% identity.

The nearest neighbor result of the set contig uses the FASTXY algorithm and G
Obtained by FASTA version 3 search against empept release 114. FASTXY is an improved version of FASTA alignment that allows frameshifts within codons. The closest flanking results showed each set of homologues closest to Genpept (and also containing the translated amino acid sequence encoded by the set). The closest neighbor results are shown below.

[0389]

[Table 1] The polypeptide was predicted to be encoded by SEQ ID NO: 2, as shown below. The polypeptide is FASTY (http://fasta.bioch.virginia.com) which selects the polypeptide by comparing the translated novel polynucleotide with a known polypeptide.
(available from edu) (WR Pearson, Methods in Enzymology, 18).
3: 63-98 (1990), which is hereby incorporated by reference).

[0390]

[Table 2] Example 3 Set of SEQ ID NOs: 3, 4, and 12 The set of novel nucleotides SEQ ID NO: 3 and 12 is ES
Achieved using T-array SEQ ID NO: 1 as seed. The seed was extended by gel sequencing (377 Applied Biosystems (ABI) sequencer) using primers to extend the 3'end (primer extension). Marathon-Ready ^™ cDNA User Manual (PT
1156-1) (Clontech), the 5'end of which was extended using RACE as disclosed in (herein incorporated by reference). At position 230 of the nucleotide sequence, guanine (SEQ ID N
It was confirmed to be either O: 3) or thymine (SEQ ID NO: 12).

The polypeptide was predicted to be encoded by SEQ ID NO: 3 or 12, as shown below. The polypeptide was predicted using a software program called BLASTX that selects polypeptides by comparing translated novel polynucleotides to known polynucleotides. The first methionine begins at position 237 of SEQ ID NO: 3 and the putative stop codon TGA begins at position 567 of the nucleotide sequence.
The first methionine also begins at position 237 of SEQ ID NO: 12 and the putative stop codon TGA also begins at position 567.

FIG. 1 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) A protein encoded by the CUB domain polypeptide and Afri
Bone morphogenetic protein of can Clawed Frog (Xenopus laevis)
1 shows a BLASTX amino acid sequence alignment with the 1 (BMP-1) precursor SEQ ID NO: 9, both sequences of SEQ ID NO: 4 11
It shows 57% similarity for two amino acid residues and 40% identity for the same 112 amino acid residues of SEQ ID NO: 4.

FIG. 2 shows that SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) BLASTX between the protein encoded by the CUB domain polypeptide and human BMP-1 CUB2 domain SEQ ID NO: 10
Amino acid sequence alignment is shown, both sequences being SEQ ID NO: 4
With 54% similarity to 108 amino acid residues of SEQ ID NO:
It shows that 43% of the same 108 amino acid residues of 4 are identical.

FIG. 3 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) shows the MSI GeneAtlas amino acid sequence alignment between the protein encoded by the CUB domain polypeptide and bovine spermadhesin SEQ ID NO: 11, both sequences being identical to SEQ ID NO: 4. 40.4% similarity for residues, 19
． It means having 2% identity. E value of PSI-BLAST is 4
． 2e-17, protein database ID number entry = 1sfp (Resea
rch Collaboratory for Structural Bio
informations. http: // www. rcsb. org / pbd)
, Confirmation score = 0.42, present at residues 1-106 of SEQ ID NO: 4.

GeneAt, Molecular Simulations, Inc.
las software (Molecular Simulations, Inc.,
(San Diego, Calif.), The CUB domain polypeptide SEQ ID NO: 4 has a region at residues 1-103 having a motif characteristic of the major seminal plasma glycoprotein of sus scrofa molecule. Was confirmed. PSI-BLAST has an e value of 1.7e-15, protein database ID number entry = 1 sppB (Research Collaborative fo
r Structural Bioinformatics. http: ///
ww. rcsb. org / pbd), confirmation score = 0.24, SEQ ID
NO: present in residues 1-103 of 4.

GeneAt, Molecular Simulations, Inc.
las software (Molecular Simulations, Inc.,
It was confirmed that the CUB domain polypeptide SEQ ID NO: 4 has a region at residues 2-107 having a motif characteristic of sus scrofa seminal plasma protein by using San Diego, CA). It was PSI
-E value of BLAST is 1.7e-15, protein database ID number entry = 1spp (Research Collaboratory for Str)
Structural Bioinformatics. http: // www. rc
sb. org / pbd), P value = 4.7e-02, SEQ ID NO: 4
Present in residues 2-107 of.

The predicted about 15 residue signal peptide has SEQ ID NO: 4 (
It is encoded by residues 1 to 15 of SEQ ID NO: 6). Extracellular potions are effective by themselves. This can be confirmed by mammalian cell expression and degradation product sequencing. Signal peptide site is Kyte-Do
little hydrophobicity prediction algorithm (J. Mol Biol. 155,
pp. 105-31 (982), incorporated herein by reference). Those skilled in the art will appreciate that the actual split site may be different than that predicted by the computer program.

SEQ ID NOs: 3 and 12 were confirmed to be present in the following tissues: Adult brain (GIMCO) (Hyseq library name AB3001 and A.
BD003) and (Clontech) (Hyseq library name ABR00
1, ABR006, ABR008) and (Invitrogen) (Hyse
q library name ABT004), cultured preadipocytes (Stratage)
ne) (Hyseq library name ADP001), adrenal gland (Clontech
) (Hyseq library name ADR002), adult heart (GIBCO) (
Hyseq library name AHR001), adult kidney (GIBCO) (H
yseq library name AKD001) and (Invitrogen) (Hy
seq library name AKT002), adult lung (GIBCO) (Hyseq library name ALG001), lymph node (Clontech) (Hyseq library name ALN001), young liver (GIBCO) (Hyseq library name ALG001), adult liver (Invitrogen) (Hyseq library name ALV002) and (Clontech) (Hyseq library name A
LV003), adult ovary (Invitrogen) (Hyseq library name AOV001), adult placenta (Clontech) (Hyseq library name APL001), adult spleen (GIBCO) (Hyseq library name ASP001), testis (GIBCO) ( Hyseq library name ATS00
1), adult bladder (Invitrogen) (Hyseq library name BLD
001), bone marrow (Clontech) (Hyseq library name BMD00
1 and BMD002), adult colon (Invitrogen) (Hyseq
Library name CLN001), adult cervix (BioChain) (Hyse
q Library name CVX001), Endothelial cells (Stratagene) (Hy
seq library name EDT001), fetal brain (Clontech) (Hy
seq library names FBR001, FBR004, and FBR006) and (Invitrogen) (Hyseq library name FBT002) and (G
IBCO) (HFB001), fetal heart (Invitrogen) (Hyse
q Library name FHR001), fetal kidney (Clontech) (Hyse
q Library names FKD001 and FKD002), fetal lung (Clonte
ch) (FLG001) and (Invitrogen) (Hyseq library name FLG003), fetal liver / spleen (Columbia University) (Hyseq library names FLS001, FLS002, and FLS003), fetal liver (ch.
Invitrogen) (Hyseq library name FLV001) and (Cl
ontech) (Hyseq library names FLV002 and FLV004)
, Fetal muscle (Invitrogen) (Hyseq library name FMS0
01 and FMS002), fetal skin (Invitrogen) (Hyseq
Library names FSK001 and FSK002), Umbilical cord (BioChain)
) (Hyseq library name FUC001), macrophage (Invitr
gen) (Hyseq library name HMP001), infant brain (Columbia University) (Hyseq library names IB2002, IB2003, and IB)
S001) Lung fibroblasts (Stratagene) (Hyseq library name FLB001), Lung tumor (Invitrogen) (Hyseq library name LGT002), Lymphocytes (ATCC) (Hyseq library name LP)
C001), white blood cells (GIBCO) (Hyseq library name LUC001)
And (Clontech) (Hyseq library name LUC003), melanoma (ATCC) obtained from cell line ATCC # CRL1424 (Hyseq library name MEL004), mammary gland (Invitrogen) (Hyseq library name MMG001), induced nerve cell ( Stratagene) (Hyseq library name NTD001), retinoic acid-induced nerve cells (Stratagen)
e) (Hyseq library name NTR001), nerve cells (Stratege)
ne) (Hyseq library name NTU001), pituitary gland (Clonte
ch) (Hyseq library name PIT004), placenta (Clontech)
(Hyseq library name PLA003), prostate (Clontech) (H
yseq library name PRT001), rectum (Invitrogen) (Hy
seq library name REC001), salivary gland (Clontech) (Hyse
q library name SAL001), small intestine (Clontech) (Hyseq library name SIN001), skeletal muscle (Clontech) (Hyseq library name) SKM001), spinal cord Clontech) (Hyseq library name SPC001), adult spleen (Clontech) (Hyseq library name SPLc01), stomach (Clontech) (Hyseq library name STO0
01), thalamus (Clontech) (Hyseq library name THA002)
, Thymus (Clontech) (Hyseq library names THM001 and T
HMc02), thyroid (Clontech) (Hyseq library name THR001), trachea (Clontech) (Hy
seq library name TRC001), and uterus (Clontech) (Hys
eq library name UTR001). Tissue expression information is SEQ ID NO:
It was determined using an EST tissue source consisting of 3 and 12 and other EST tissue sources of the cluster to which they belong. Clusters were created based on the sequence signature of each sequence as described in Example 1.

Example 4 A. Expression of SEQ ID NO: 4 in cells Chinese hamster ovary (CHO) cells or other suitable cell types were DMEM (ATCC) and 10% fetal bovine serum (FBS) (Gibco).
Grow until 70% confluence. Prior to transfection, the medium was DMEM and 0.
Changed to 5% FCS. The cells have SEQ ID NO: 3, 5, or 12
The cDNA is transfected with or the pBGal vector is transfected with FuGENE-6 transfection reagent (Boehringer).

Briefly, 4 μl of FuGENE-6 was diluted in 100 μl of DMEM and incubated for 5 minutes. Thereafter, 1 μg of DNA was added to this, and 1
After culturing for 5 minutes, CHO cells are added to a 35 mm dish. CHO
Cells are cultured at 37 ° C. with the addition of 5% CO ² . After 24 hours, media and cell lysates were collected, centrifuged and dialyzed against assay buffer (15 mM.
Tris pH 7.6, 134 mM NaCl, 5 Mm glucose, 3
mM CaCl ₂ and MgCl ₂ ).

B. Expression studies using SEQ ID NO: 1-3, 5, or 12 Expression of SEQ ID NO: 1-3, 5, or 12 in various tissues was performed using a semi-quantitative polymerase chain reaction-based approach. Was analyzed. Target tissues (adult bladder, adult brain, adult heart, adult kidney, adult lymph node, adult liver, adult lung, adult ovary, adult placenta, adult rectum, adult spleen, Human testis, bone marrow, thymus, thyroid, fetal kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocytes and macrophages) A cDNA library was used. From the sample, using a gene-specific primer, SEQ ID NO: 1-3
Amplification of 5, or 12 parts was performed. The amplified product was separated on an agarose gel, transferred and chemically linked to a nylon filter. The filter is then SEQ ID NO: 1-3,5, or radioactivity produced from 12 by the label ⁽³³ P-dCTP) are double-stranded probe, using Klenow polymerase, hybrids with random primed method Been formed. The filter was washed (high stringency wash) and exposed to the phosphoimaging screen for several hours.
The resulting stripes indicate the presence of cDNA containing SEQ ID NO: 1-3, 5, or 12 sequences within a particular library, and thus the presence of mRNA expression in the corresponding cell type or tissue.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows that SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) A protein encoded by the CUB domain polypeptide and Afri
Bone morphogenetic protein of can Clawed Frog
1 shows a BLASTX amino acid sequence alignment with the 1 (BMP-1) precursor SEQ ID NO: 9, both sequences of SEQ ID NO: 4 11
It shows 57% similarity for two amino acid residues and 40% identity for the same 112 amino acid residues of SEQ ID NO: 4. A = in the figure
Alanine, C = Cysteine, D = Aspartic acid, E = Glutamic acid, F = Phenylalanine, G = Glycine, H = Histidine, I = Isoleucine, K = Lysine, L = Leucine, M = Methionine, N = Asparagine, P = Proline , Q = glutamine, R = arginine, S = serine, T = threonine, V = valine, W = tryptophan, Y = tyrosine. Gaps are indicated by horizontal lines.

FIG. 2 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) BLASTX between the protein encoded by the CUB domain polypeptide and human BMP-1 CUB2 domain SEQ ID NO: 10
Amino acid sequence alignment is shown, both sequences being SEQ ID NO: 4
With 54% similarity to 108 amino acid residues of SEQ ID NO:
It shows that 43% of the same 108 amino acid residues of 4 are identical. In the figure, A = alanine, C = cysteine, D = aspartic acid, E = glutamic acid, F
= Phenylalanine, G = glycine, H = histidine, I = isoleucine, K
= Lysine, L = leucine, M = methionine, N = asparagine, P = proline,
Q = glutamine, R = arginine, S = serine, T = threonine, V = valine,
W = tryptophan, Y = tyrosine. Gaps are indicated by horizontal lines.

FIG. 3 shows SEQ ID NO: 3 or 12 (ie SEQ ID NO:
4) MSI GeneAtla between the protein encoded by the CUB domain polypeptide and bovine spermadhesin SEQ ID NO: 11.
s amino acid sequence alignments, both sequences having SEQ ID NO:
It has 40.4% similarity and 19.2% identity for 4 identical residues. In the figure, A = alanine, C = cysteine, D = aspartic acid,
E = glutamic acid, F = phenylalanine, G = glycine, H = histidine,
I = isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine, P = proline, Q = glutamine, R = arginine, S = serine, T = threonine, V = valine, W = tryptophan, Y = Means tyrosine. Gaps are indicated by horizontal lines.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/00 A61P 11/02 4C084 7/10 11/06 4H045 11/00 17/00 11/02 17 / 02 11/06 17/04 17/00 19/00 17/02 19/02 17/04 19/04 19/00 19/10 19/02 25/00 19/04 25/16 19/10 25/28 25 / 00 27/02 25/16 29/00 101 25/28 31/00 27/02 31/04 29/00 101 31/12 31/00 31/18 31/04 33/06 31/12 35/00 31 / 18 37/00 33/06 37/02 35/00 37/08 37/00 41/00 37/02 43/00 101 37/08 105 41/00 107 43/00 101 111 111 105 C07K 14/47 107 16 / 18 111 C12N 1/15 C07K 14/47 1/19 16/18 1/21 C12N 1/15 C12P 21/02 C 1/19 C12Q 1/02 1/21 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/02 33/53 M 1/68 37/00 102 G0 N 33/15 A61K 48/00 33/50 C12N 15/00 ZNAA 33/53 F 37/00 102 5/00 A // A61K 48/00 A61K 37/02 (31) Priority claim number 09 / 678,216 (32) Priority date September 29, 2000 (September 29, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, U, CZ, DE, DK, DM, DZ, 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, MZ, 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 Arterburn, Matthew Sea. United States California 94566, Pleasanton, Via Del Cielo 5910 (72) Inventor Lin, Haishan United States California 94552, Castro Valley, Sherman Drive 20942 (72) Inventor Tan, Wy. Tom United States California 95118, San Jose, Lanwick Court 4230 (72) Inventor Liu, Chen Hoa United States California 95117, San Jose, Ranchero Way 1125 No. 14 (72) Inventor Jamanak, Radoetie. United States California 94303, Palo Alto, East Greenwich Place 850 F Term (Reference) 2G045 AA25 AA35 AA40 BA13 BB20 CB01 CB21 DA12 DA13 DA14 DA20 DA36 DA37 DA77 FB02 FB03 FB04 FB07 FB16 CA11 CA02 DA11 CA02 CA11 EA03 EA04 FA02 GA01 GA11 HA01 HA03 HA12 4B063 QA01 QA08 QA18 QA19 QQ01 QQ13 QQ42 QQ52 QQ79 QR08 QR33 QR42 QR55 QR59 QR62 QR74 QR80 QR82 QS05 QS25 QS34 QS36 QA02 CA01 A27A01 A27A01 A27A01 A27B01 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA19 CA01 CA20 CA24 CA25 CA44 CA46 4C084 AA02 AA07 AA13 AA17 AA20 AA24 BA01 BA08 BA21 BA22 BA23 CA18 CA20 CA30 CA51 DA01 DA40 DC50 MA01 MA02 NA14 ZA011 ZA021 ZA151 ZA161 ZA221 ZA331 ZA341 ZA361 ZA511 ZA671 ZA681 ZA751 ZA891 ZA941 ZA961 ZA971 ZB011 ZB021 ZB051 ZB071 ZB131 ZB151 ZB211 ZB212 ZB221 ZB261 ZB331 ZB351 ZB381 ZC061 ZC351 ZC551 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA00 DA76 EA20 EA50 FA72 FA74

Claims (30)

[Claims] 1. A nucleotide selected from the group consisting of SEQ ID NOs: 2-3, 5 and 12, a translated protein coding portion thereof, a mature protein coding portion thereof, an extracellular portion thereof, or an active domain thereof. An isolated polynucleotide comprising a sequence. 2. An isolated polynucleotide encoding a biologically active polypeptide, the polynucleotide comprising stringent hybridization (
Hybridization (hybridization) is performed under the conditions of hybridization (hybridization) with the complement of the polynucleotide of claim 1. 3. An isolated polynucleotide encoding a biologically active polypeptide, said polynucleotide comprising about 9 nucleotides of the polynucleotide of claim 1.
It has a sequence identity of 0% or more. 4. The polynucleotide of claim 1, which is a DNA sequence. 5. An isolated polynucleotide comprising the complement of the nucleotide of claim 1. 6. A vector comprising the polynucleotide of claim 1. 7. An expression vector comprising the polynucleotide of claim 1. 8. A host cell genetically engineered to express the polynucleotide of claim 1. 9. The host cell according to claim 8, wherein the polynucleotide is functionally associated with a regulatory sequence that controls the expression of the polynucleotide in the host cell. 10. SEQ ID NO: 4, 6, 7, and 8, selected from the group consisting of a translated protein coding portion thereof, a mature protein coding portion thereof, an extracellular portion thereof, or an active domain thereof. An isolated polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence. 11. A composition comprising the polypeptide of claim 10 and a carrier. 12. Having CUB domain-like activity, SEQ ID NO: 4
A polypeptide comprising at least 10 contiguous amino acids of a polypeptide sequence selected from the group consisting of :, 6, 7, and 8. 13. The polypeptide of claim 12, which has SEQ ID NO: 4
Containing at least 5 contiguous amino acids of a polypeptide sequence selected from the group consisting of, 6, 7, and 8. 14. A polynucleotide encoding the polypeptide of claim 12. 15. A polynucleotide encoding the polypeptide of claim 13. 16. A polynucleotide encoding the polypeptide of claim 10. 17. An antibody specific for the polypeptide of claim 10. 18. A method of detecting the polynucleotide of claim 1 in a sample, which comprises: a) forming a complex with a compound that binds to the polynucleotide of claim 1 to form a complex. The sample is contacted for a period of time necessary to do so, and b) the complex is detected, and if the complex is detected, the polynucleotide of claim 1 is detected. 19. A method of detecting the polynucleotide of claim 1 in a sample, which comprises: a) a nucleic acid primer that anneals to the polynucleotide of claim 1 under stringent (hybridization) conditions. Contacting the sample under hybridization conditions such as; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting the product, thereby detecting the product in the sample. Is detected. 20. The method of claim 19, wherein the polynucleotide is R
A NA molecule is included, and the method further includes reverse transcribing the annealed RNA molecule into a cDNA polynucleotide. 21. A method of detecting the polynucleotide of claim 1 in a sample, which comprises: a) forming a complex with a compound that binds to the polypeptide of claim 1 to form a complex. The sample is contacted for a period of time and under the conditions necessary to do so; b) the complex is detected, and if the complex is detected, the polynucleotide of claim 10 is detected. 22. A method of identifying a compound that binds to the polypeptide of claim 10, comprising: a) a period of time required to form a polypeptide / compound complex of the polypeptide of claim 10. B) detecting the complex and contacting the compound under conditions; and the detection of the polypeptide / compound complex shall identify the compound that binds to the polypeptide of claim 10. 23. A method of identifying a compound that binds to the polypeptide of claim 10, comprising: a) the polypeptide of claim 10 and the compound in a cell in a polypeptide / compound complex. To allow the complex to express a reporter gene sequence in the cell and b) detect the presence of the complex by detecting reporter gene sequence expression. If a complex of compounds is detected, then a compound that binds to the polypeptide of claim 10 has been identified. 24. A method for producing a CUB domain polypeptide comprising: a) culturing the host cell of claim 8 in a state necessary to express the polypeptide in said cell; b) a cell culture or step. The polypeptide is separated from the cells of (a). 25. A kit comprising the polypeptide of claim 10. 26. A nucleic acid array comprising the polynucleotide of claim 1 or a unique segment of the polynucleotide of claim 1 attached to a surface. 27. The array of claim 26, which detects a full match to the polynucleotide of claim 1 or a unique segment of a polynucleotide. 28. The array of claim 26, which detects a mismatch to the polynucleotide of claim 1 or a unique segment of a polynucleotide. 29. A method for treating a subject in need of enhancing the expression or activity of the CUB domain polypeptide according to claim 10, wherein a composition selected from the group consisting of the following items is administered to the subject: Including: (a) a therapeutic amount of an agonist of the polypeptide; (b) a therapeutic amount of the polypeptide; and (c) a polypeptide under conditions and conditions such that the polypeptide is produced. Therapeutic amount of a polynucleotide that encodes, and a pharmaceutically acceptable carrier. 30. A method of treating a subject in need of suppressing the expression or activity of the CUB domain polypeptide of claim 18, wherein a composition selected from the group consisting of the following items is administered to the subject. Including: (a) a therapeutic amount of an antagonist of the polypeptide; (b) a therapeutic amount of a polynucleotide that suppresses the expression of a nucleotide sequence encoding the peptide; and (c) a CUB domain polypeptide and the same. A therapeutic amount of polypeptide competing for ligand, and a pharmaceutically acceptable carrier.