JP2004041214A - SIALOADHESIN FAMILY 4 (SAF-4) cDNA - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new polypeptide, to provide a polynucleotide encoding the polypeptide, to provide a method of using the polypeptide and the polynucleotide in therapy or in identification of a compound which is an agonist, an antagonist, and/or a inhibitor useful in the therapy, and to provide a method for producing the polypeptide and the polynucleotide. <P>SOLUTION: A sialoadhesin family 4 (SAF-4) polypeptide, the polynucleotide, the method for producing the polypeptide by a recombination technique are disclosed in the specification, respectively. Further, the method for utilizing the SAF-4 polypeptide and the polynucleotide in the therapy and a method of diagnostic assay for utilizing the same are disclosed, respectively. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to newly identified polypeptides, polynucleotides encoding the polypeptides, and compounds that are agonists, antagonists and / or inhibitors that may be effective in or for treating the polypeptides and polynucleotides. The present invention relates to the use in identification, and to a method for producing the polypeptide and polynucleotide.

The drug discovery process is undergoing a radical change today. Because it extends to "functional genomics", high-throughput (high-efficiency) genomic or gene-based biology. This approach is rapidly replacing a relatively early approach based on “positional cloning”. The phenotype, ie, biological function or genetic disease, will be identified, and then the etiological gene will be located based on its genetic map location.

Functional genetic science relies heavily on various bioinformatics tools to identify gene sequences of interest from many currently available molecular biology databases. There is still a need to identify and characterize as yet unknown genes and their related polypeptides / proteins as targets for drug discovery.

SUMMARY OF THE INVENTION The present invention relates to SAF-4, particularly SAF-4 polypeptides and polynucleotides, recombinant materials, and methods for producing the same. In another embodiment, the invention relates to cancer, inflammation, autoimmune disease, allergy, asthma, rheumatoid arthritis, central nervous system inflammation, cerebellar degeneration, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic side Chordosclerosis, head injury damage, and other neurological abnormalities, septic shock, sepsis, stroke, osteoporosis, osteoarthritis, ischemic reperfusion injury, cardiovascular disease, kidney disease, liver disease, ischemic Injury, myocardial infarction, hypotension, hypertension, AIDS, myelodysplastic syndrome and other hematological abnormalities, aplastic anemia, male pattern baldness, and bacterial, fungal, protozoal and viral infections The present invention relates to methods for using the polypeptides and polynucleotides, including the treatment of diseases (hereinafter collectively referred to as "the diseases"). In another aspect, the present invention provides a method of identifying agonists and antagonists / inhibitors using the agents provided by the present invention, and treating a condition associated with SAF-4 imbalance using the identified compounds. About. In yet another embodiment, the invention relates to diagnostic assays for detecting diseases associated with inappropriate SAF-4 activity or levels.

DESCRIPTION OF THE INVENTION In one embodiment, the present invention relates to SAF-4 polypeptides. This kind of peptide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Comprises an isolated polypeptide comprising an amino acid sequence having at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include polypeptides comprising the amino acid sequence of SEQ ID NO: 2.

Another peptide of the invention has an amino acid sequence of at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Or more preferably at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include polypeptides consisting of the amino acid sequence of SEQ ID NO: 2.

更 Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

ポ リ Polypeptides of the invention are considered to be members of the sialoadhesin family. Therefore, they are interesting. This is because sialoadhesin, CD33, CD22 and myelin-binding glycoprotein (MAG), which are proteins of the sialoadhesin family, are used as cell-interacting molecules. These proteins specifically bind to carbohydrates on target cells in a sialic acid-dependent manner. Its extracellular domain consists of several immunoglobulin-like domains of V-like and C2-like subtypes, and its homology to any signaling motif is not known for its intracellular portion. Sialoadhesin expression is restricted to macrophages, has 17 Ig-like domains, and the specific recognition sequence on target cells is Neu5Aca2,3Galb13GalNAc. Known target cells are developing myeloid cells in the bone marrow and lymphocytes in the spleen and lymph nodes (Crocker, P.R., et al., EMBO J 1994, 13: 4490-4503). CD22 is expressed only on B cells and has a and b isoforms (with 5 and 7 Ig-like domains, respectively). CD22 is known to bind T cells, B cells, monocytes, granulocytes and erythrocytes by recognizing Neu5Aca2,6Galb1,4Glc (NAc) in N-linked glycans (Crocker, PR et al., EMBO J 1994, 13: 4490-4503; Stamenkovic, I. and Seed, B. Nature, 1990, 345: 74-77; Wilson, GL et al., J Exp Med, 1991, 173: 137-146). Since myelin-binding glycoprotein (MAG) is expressed by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, MAG is thought to be involved in cell adhesion to axons. MAG has two alternatively spliced variants, a large MAG (L-MAG) and a small MAG (S-MAG), each of which is either during embryonic development or adult. Is expressed in any of This alternative splicing results in expression of the same extracellular domain but different intracellular domains (Pedraza, L. et al., JCB, 1990, 111: 2651-2661).

  CD33 is most related to SAF-4. Because they are the most closely related of all members of the family. Normally, CD33 is expressed on a differentiating myeloid monocytic lineage. Although CD33 is not present on early stem cells, granulocyte, erythroid, monocyte, and megakaryocyte colony forming units (CFU-GEMM) and granulocyte and mononuclear phagocyte progenitor cell colony forming units (CFU-GEMM). GM). CD33 is down-regulated by mature granulocytes, but retained by mature monocytes and macrophages (Andrews, R.G. et al., Blood, 1983, 62: 124; Griffin, J.D. et al., Leuk Res 1984, 8: 521). Because CD33 has two Ig-like domains, it more readily binds targets expressing NeuAca2,3GAl in N-linked and O-linked glycans. CD33 maps to chromosome 19q13.1-13.3 in the genome that is closely associated with MAG and CD22 (Freeman, S.D., et al., Blood, 1995, 85: 2005-2012).

  In addition, CD33 has been found to be expressed on leukemic myeloblasts in approximately 85% of patients with acute myeloid leukemia (AML), and AML and acute lymphoblastic leukemia (ALL ) Is often used to distinguish In the treatment of AML, monoclonal antibodies against CD33 have been used therapeutically ex vivo to clear residual myeloblasts from autologous bone marrow transplants (Robertson, MJ et al., Blood, 1992 79: 2229- 2236). More recently, humanized monoclonal antibodies to CD33 have been evaluated in the in vivo treatment of AML (Caron, PC. Et al., Blood, 1994, 83: 1760-1768). These properties are hereinafter referred to as “SAF-4 activity” or “SAF-4 polypeptide activity” or “biological activity of SAF-4”. These activities include the antigenic activity and the immunological activity of the SAF-4 polypeptide, particularly the antigenic activity and the immunological activity of the polypeptide of SEQ ID NO: 2. Preferably, the polypeptide of the present invention exhibits at least one biological activity of SAF-4.

ポ リ The polypeptides of the present invention may be in the form of the “mature” protein or may be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or stable during recombinant production. There are additional arrangements to ensure performance.

Also included in the invention are variants of the polypeptide, ie, polypeptides that differ from a reference polypeptide by conservative amino acid substitutions (where one residue is replaced by another residue of similar properties). . Typical such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; between the basic residues Lys and Arg; or aromatic. Occurs between group residues Phe and Tyr. In particular, a mutant in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acids are substituted, deleted, or added in any combination is preferable.

ポ リ The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. It is. Means for producing such polypeptides are well understood in the art.

に お い て In a further aspect, the present invention relates to a SAF-4 polynucleotide. Such a polynucleotide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, Preferably, an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 95% identity is included. In this regard, polypeptides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. . Such polynucleotides include polynucleotides comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 2.

A further polynucleotide of the invention has at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity, over its entire coding region, to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2. An isolated polynucleotide comprising a nucleotide sequence having at least 90% identity, more preferably at least 95% identity, is included. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. .

Additional polynucleotides of the present invention include at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the polynucleotide of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1. And more preferably an isolated polynucleotide comprising a nucleotide sequence having at least 95% identity. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. . Such polynucleotides include polynucleotides comprising the polynucleotide of SEQ ID NO: 1 and polynucleotides of SEQ ID NO: 1.

The present invention also provides a polynucleotide complementary to any of the above polynucleotides.

ヌ ク レ オ チ ド The nucleotide sequence of SEQ ID NO: 1 shows homology to CD33 (Simmons, D. and Seed, B., JI 141: 2797-2800, 1988). The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and includes a polypeptide coding sequence (nucleotide numbers 51 to 1970) encoding a polypeptide of 639 amino acids which is a polypeptide of SEQ ID NO: 2. Even though the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is identical to the polypeptide coding sequence contained in SEQ ID NO: 1, the polypeptide of SEQ ID NO: 2 is still duplicated due to the redundancy (degeneracy) of the genetic code. It may be a sequence other than the sequence contained in SEQ ID NO: 1 that encodes. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins in the sialoadhesin family and has homology to CD33 (Simmons, D. and Seed, B., JI 141: 2797-2800, 1988) and And / or have structural similarity.

好 適 Suitable polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions / properties as the polypeptides and polynucleotides homologous thereto. Further, preferred polypeptides and polynucleotides of the present invention have at least one SAF-4 activity.

The present invention also relates to partial or other polynucleotides and polypeptides that were initially identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Thus, in a further aspect, the present invention provides:
(a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 A nucleotide sequence having an identity of, most preferably 97-99%,
(b) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 A nucleotide sequence having an identity of, most preferably 97-99%,
(c) the polynucleotide of SEQ ID NO: 3, or (d) the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity. A nucleotide sequence that encodes a polypeptide consisting of an amino acid sequence having a percent identity, more preferably at least 95% identity, and most preferably 97-99% identity;
And a polynucleotide of SEQ ID NO: 3.

Further, the present invention provides
(a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 A polypeptide comprising an amino acid sequence having an identity of, most preferably 97-99%,
(b) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 A polypeptide having an amino acid sequence having the identity of, most preferably 97-99%,
(c) a polypeptide comprising the amino acid of SEQ ID NO: 4, and
(d) a polypeptide that is the polypeptide of SEQ ID NO: 4,
And a polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 3,
I will provide a.

ヌ ク レ オ チ ド The nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are derived from an Expressed Sequence Tag (EST) sequence. One skilled in the art will appreciate that there will necessarily be some nucleotide sequence reading errors in the EST sequence (see Adams, M.D. et al., Nature 377 (supp) 3, 1995). Thus, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are subject to the same inherent limitations in sequence accuracy. In addition, the peptide sequence encoded by SEQ ID NO: 3 may contain regions identical to the protein with the highest homology or structural similarity, or regions of high homology and / or structural similarity (eg, conservative amino acid differences). Contains.

  Polynucleotides of the present invention can be analyzed by EST analysis (Adams, MD et al., Science (1991)) from a cDNA library derived from mRNA in cells of human primary dendritic cells by standard cloning and screening techniques. 252: 1651-1656; Adams, MD et al., Nature (1992) 355: 632-634; Adams, MD et al., Nature (1995) 377 Supp: 3-174). The polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, and can also be synthesized using a commercially available known technique.

When a polynucleotide of the present invention is used for recombinant production of a polypeptide of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide alone or other coding sequences (eg, a leader or secretory sequence, a pre- -, Coding for a pro- or pre-pro-protein sequence, or other fusion peptide moiety). For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is provided by the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 86: 821-824. Such as a hexa-histidine peptide, or an HA tag. The polynucleotide may also include 5 'and 3' non-coding sequences, for example, transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

As further specific examples of the present invention, several, for example, 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid residues may be substituted or deleted in any combination. Alternatively, there is an added polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2.

  Polynucleotides that are identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 can be used to isolate full-length cDNA and genomic clones encoding a polypeptide of the present invention, and to SEQ ID NO: 1. To isolate cDNA and genomic clones of other genes with high sequence similarity (including homologues from non-human species and genes encoding orthologs), It can be used as a hybridization probe or as a primer for a nucleic acid amplification (PCR) reaction. Generally, these nucleotide sequences are 70%, preferably 80%, more preferably 90%, and most preferably 95% identical to the reference nucleotide sequence. Probes or primers usually contain 15 or more nucleotides, preferably 30 or more, and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30 to 50 nucleotides.

Polynucleotides encoding the polypeptides of the present invention (including homologues and orthologs derived from non-human species) can be prepared using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof under stringent hybridization conditions. The method comprises the steps of screening an appropriate library below and isolating a full-length cDNA and a genomic clone containing the polynucleotide sequence. Such hybridization techniques are known to those skilled in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt solution, 10% dextran sulfate and 20 μg / ml Incubation at 42 ° C. overnight in a solution containing denatured and sheared salmon sperm DNA, and then washing the filters at about 65 ° C. in 0.1 × SSC. Thus, the present invention also includes a polynucleotide obtained by screening an appropriate library under stringent hybridization conditions using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.

よ う As will be appreciated by those skilled in the art, the isolated cDNA sequence will often be incomplete, since in many cases the region encoding the polypeptide will be truncated at the 5 'end of the cDNA. It is due to reverse transcriptase, which originally has a low "processivity" (a measure of the enzyme's ability to remain attached to the template during the polymerization reaction), and the mRNA template during first-strand cDNA synthesis. DNA copy cannot be completed.

There are several methods known and available to those skilled in the art for obtaining full-length cDNAs or extending short-chain cDNAs, such as those based on the rapid cDNA end amplification method (RACE) (eg, See Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent improvements in the above techniques, as demonstrated, for example, by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly simplified the search for longer cDNAs. In the Marathon ^™ technology, cDNA is prepared from mRNA extracted from a given tissue, and an “adapter” sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deleted" 5 'end of the cDNA. Next, a “nested” primer, a primer designed to anneal inside the amplification product (typically an adapter-specific primer that anneals further 3 ′ to the adapter sequence and an additional 5 ′ to the known gene sequence) The PCR reaction is repeated using a gene-specific primer that anneals to the side. The product of this reaction is then analyzed by DNA sequencing and the product is directly linked to the existing cDNA to complete the sequence, or another full-length sequence using new sequence information for 5 ′ primer design. By performing PCR, a full-length cDNA can be constructed.

組 換 え The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing the expression system using methods known in the art. Thus, in a further aspect, the invention relates to an expression system containing one or more polynucleotides of the invention, a host cell engineered with said expression system, and the production of a polypeptide of the invention by recombinant methods. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention.

For recombinant production, host cells are genetically engineered to incorporate the polynucleotide expression system of the present invention or a portion thereof. Introduction of polynucleotides into host cells is described in Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). And can be performed by methods described in many standard laboratory manuals. Suitable such methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, bullet loading. There is a ballistic introduction or infection.

  Representative examples of suitable hosts include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2, Spodoptera Sf9). ), Animal cells (eg, CHO, COS, HeLa, C 127, 3T3, BHK, HEK 293, Bowes melanoma cells) and plant cells.

  A wide variety of expression systems can be used. Such expression systems include, for example, systems derived from chromosomes, episomes and viruses, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion factors, yeast chromosome elements, viruses (eg, baculovirus, SV40 Such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus), and vectors derived from combinations thereof, such as plasmids such as cosmids and phagemids and bacteriophage genetics. Some come from elements. These expression systems may contain control regions that regulate as well as cause expression. Generally, any system or vector that can maintain, augment, and express a polynucleotide for production of the polypeptide in the host can be used. The appropriate nucleotide sequence can be inserted into the expression system by any of the commonly used and known techniques, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the polypeptide of interest to secrete the translated protein into the endoplasmic reticulum lumen, into the periplasmic space, or into the extracellular environment. These signals may be endogenous to the polypeptide of interest or heterologous signals.

場合 When trying to express a polypeptide of the present invention for use in screening assays, it is generally preferred that the polypeptide be produced on the surface of cells. If so, the cells are harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium is recovered to recover and purify the polypeptide. If produced intracellularly, it is necessary to first lyse the cell and then recover the polypeptide.

To recover and purify the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxylapatite chromatography and lectin chromatography can be used. Most preferably, high performance liquid chromatography is used for purification. When a polypeptide is denatured during intracellular synthesis, isolation and / or purification, known techniques for regenerating the protein can be used to restore the active conformation.

  The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic. Detection of a variant of a gene characterized by the polynucleotide of SEQ ID NO: 1 associated with a dysfunction is useful for diagnosing a disease caused by under-expression, over-expression or altered expression of the gene or susceptibility to the disease. It will provide a diagnostic tool that can be added or diagnosed. Individuals with mutations in the gene can be found at the DNA level by various techniques.

核酸 Nucleic acid for diagnosis can be obtained from cells of a subject, for example, cells from blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection, or may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can be used in a similar manner. Deletions and insertions can be detected by a change in size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to a labeled SAF-4 nucleotide sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments on gels, with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985)). 230: 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or by chemical cleavage (Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. See). In another embodiment, an array of oligonucleotide probes comprising a SAF-4 nucleotide sequence or a fragment thereof can be constructed, for example, to perform efficient screening for gene mutations. Array techniques are known and have general applicability and have been used to solve various molecular genetics problems, including gene expression, genetic linkage and genetic variability (eg, M Chee et al., Science, Vol. 274, pp. 610-613 (1996)).

(4) The diagnostic assay provides a method for diagnosing or determining the susceptibility to the disease by detecting a mutation in the SAF-4 gene by the method described above. In addition, the disease can be diagnosed by a method of measuring an abnormal decrease or increase in polypeptide or mRNA levels from a sample obtained from a subject. The decrease or increase in expression can be determined at the RNA level by any of the polynucleotide quantification methods known in the art, such as nucleic acid amplification (eg, PCR, RT-PCR), RNase protection, Northern blotting, or other hybridization methods. Can be measured. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample obtained from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays and flow cytometry analysis.

Thus, in another embodiment, the present invention provides
(a) the polynucleotide of the present invention (preferably, the nucleotide sequence of SEQ ID NO: 1) or a fragment thereof,
(b) a nucleotide sequence complementary to the nucleotide sequence of (a),
(c) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2) or a fragment thereof, or (d) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2),
And a diagnostic kit comprising:

  It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component. Such a kit may be a disease or susceptibility to a disease, especially cancer, inflammation, autoimmune disease, allergy, asthma, rheumatoid arthritis, central nervous system inflammation, cerebellar degeneration, Alzheimer's disease, Parkinson's disease, multiple sclerosis, muscle Amyotrophic lateral sclerosis, head injury damage, and other neurological abnormalities, septic shock, sepsis, stroke, osteoporosis, osteoarthritis, ischemic reperfusion injury, cardiovascular disease, kidney disease, liver disease , Ischemic injury, myocardial infarction, hypotension, hypertension, AIDS, myelodysplastic syndrome and other hematological abnormalities, aplastic anemia, male pattern baldness, and bacterial, fungal, and protozoal infections And for diagnosing viral infections or susceptibility to these.

  The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can specifically target a particular location on an individual human chromosome and hybridize to that particular location. Creating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available online at Johns Hopkins University Welch Medical Library). Then, the relationship between the gene mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

差異 Differences in cDNA or genomic sequence between affected and unaffected individuals can also be examined. If the mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation may be responsible for the disease.

ポ リ The polypeptide of the present invention, a fragment or an analog thereof, or a cell expressing the same can also be used as an immunogen for producing an antibody immunospecific for the polypeptide of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for the polypeptides of the present invention than its affinity for other related polypeptides in the prior art.

抗体 Antibodies to the polypeptide of the present invention can be obtained by administering a fragment, analog or cell containing the polypeptide or epitope to an animal (preferably an animal other than a human) using a conventional protocol. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma method (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), the trioma method, the human B cell hybridoma method (Kozbor et al., Immunology Today (1983) 4:72). And the EBV-hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

  To make single-chain antibodies to a polypeptide of the invention, methods for preparing single-chain antibodies as described in US Patent No. 4,946,778 can be adapted. Also, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies.

ク ロ ー ン Using the above-described antibody, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

  Antibodies to the polypeptides of the present invention may find use, inter alia, in the treatment of the aforementioned diseases. In addition, antibodies to SAF-4 polypeptide, to sub-characterize the cell population that is differentiating into the hematopoietic system, cancer expressing SAF-4 as a diagnostic marker to distinguish different forms of cancer As a tool to promote ex vivo expansion (proliferation and / or differentiation) of hematopoietic progenitor cells expressing SAF-4 to clear cells from bone marrow ex vivo, It can be used as a stimulus in vivo and as a chemoprotectant in vivo.

  In a further aspect of the present invention, the present invention provides a polypeptide of the invention or a fragment thereof and various parts of the constant regions of the heavy or light chains of the immunoglobulins of the various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising: The immunoglobulin is preferably the constant region of the heavy chain of human IgG, especially IgG1, in which case the fusion takes place in the hinge region. In certain instances, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods for the genetic engineering of these fusion proteins and their use in drug screening, diagnosis and therapy. In another approach, a soluble form of the SAF-4 polypeptide that is still capable of binding the ligand in competition with endogenous SAF-4 may be administered. A typical embodiment of such a competitor comprises a fragment of a SAF-4 polypeptide. In one embodiment, the extracellular domain of SAF-4 fused to the human immunoglobulin Fc region is used, and this domain can be used to treat cancer, inflammation, autoimmune diseases, allergies, etc. . In addition, SAF-4 / Fc polypeptides are used as a tool to promote ex vivo expansion (proliferation and / or differentiation) of hematopoietic progenitor cells expressing SAF-4 ligand, for recruiting stem cells to the periphery. It can be used as an in vivo stimulus and as an in vivo chemoprotectant to wipe out cancer cells expressing SAF-4 ligand from bone marrow ex vivo. A further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology can be found in International Patent Applications WO94 / 29458 and WO94 / 22914.

  A further aspect of the present invention relates to a method of eliciting an immunological response in a mammal, said method comprising an antibody and / or a T-cell immune response sufficient to protect the animal from the disease, in particular the invention. Inoculating a mammal with a polypeptide of the formula (I). Yet another aspect of the invention directs the expression of a polynucleotide encoding a polypeptide of the invention in vivo to elicit an immunological response that produces an antibody that protects the mammal from the disease. A method for eliciting an immunological response in a mammal, comprising providing the polypeptide via a vector.

  A further aspect of the present invention relates to immunological / vaccine formulations (compositions) that, when introduced into a mammalian host, elicit an immunological response against the polypeptide of the invention in the mammal. Polypeptide or polynucleotide. The vaccine formulation may further include a suitable carrier. Since the polypeptide may be degraded in the stomach, it is preferably administered parenterally (eg, by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injections which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may contain agents or thickeners. Such formulations may be presented in single or multi-dose containers (eg, sealed ampules and vials) and may be stored in a lyophilized state which requires only the addition of a sterile liquid carrier immediately before use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or other adjuvant systems known in the art. The dose varies with the specific activity of the vaccine, but can be easily determined by routine experimentation.

  The polypeptides of the present invention have been implicated in a variety of biological functions, including many pathological conditions, particularly those diseases. Therefore, it is desirable to develop screening methods that identify compounds that stimulate or suppress the function of this polypeptide. Thus, in a further aspect, the present invention provides a method of screening a compound to identify a compound that stimulates or suppresses the function of the polypeptide. Generally, agonists or antagonists are used for the purpose of treating and preventing the above-mentioned diseases. Compounds can be identified from a variety of sources, such as a mixture of cells, cell-free preparations, chemical libraries and natural products. The agonist, antagonist or inhibitor so identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide, and its structural or functional mimetic. (See Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)).

  In the screening method, the binding of the candidate compound to the polypeptide of the present invention, or a cell or membrane carrying the polypeptide, or a fusion protein thereof is easily performed using a label directly or indirectly bound to the candidate compound. Can be measured. Alternatively, the screening method may use competition with a labeled competitor. In addition, such screening methods can test whether a candidate compound results in a signal resulting from activation or suppression of the polypeptide, using a detection system suitable for cells bearing the polypeptide. . Generally, inhibitors of activation are assayed in the presence of a known agonist to determine the effect of the presence of the candidate compound on activation by the agonist. Constitutively active polypeptides are used in screening methods for inverse agonists or inhibitors by examining whether a candidate compound inhibits activation of the polypeptide in the absence of the agonist or inhibitor. Further, in these screening methods, a candidate compound and a solution containing the polypeptide of the present invention are mixed to form a mixture, the SAF-4 activity in the mixture is measured, and the SAF-4 activity of the mixture is determined as a standard. Simply include each step of comparing with. In high-throughput screening assays to identify antagonists of the polypeptides of the present invention, fusion proteins such as those made from the Fc portion and SAF-4 polypeptides described above can also be used (D. Bennett et al., J. Mol. Recognition, 8: 52-58 (1995) and K. Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

ス ク リ ー ニ ン グ In addition, a screening method for detecting the effect of an additional compound on the production of mRNA or polypeptide in cells can be assembled using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention. For example, using monoclonal or polyclonal antibodies by standard methods known in the art, an ELISA assay can be constructed to measure the level of secretion or cell binding of the polypeptide, which can be performed on properly engineered cells or It can be used for searching for a substance that suppresses or enhances the production of a polypeptide from a tissue (also referred to as an antagonist or an agonist, respectively).

If membrane bound or soluble receptors are present, the polypeptides of the invention can be used to identify such receptors by standard receptor binding methods known in the art. it can. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays, in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg, ¹²⁵ I). (Eg, biotinylation) or fused to a peptide sequence suitable for detection or purification and incubated with a putative receptor source (cells, cell membranes, cell supernatants, tissue extracts, body fluids, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can also be used to identify agonists or antagonists of the polypeptide that compete for binding to the polypeptide or its receptor, if present. Standard methods for performing screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, and in some cases, oligonucleotides or proteins (eg, ligands, ligands, etc.) that are closely related to the ligands, substrates, receptors, enzymes, etc., of the polypeptide. Fragments of a substrate, receptor, enzyme, etc.) or small molecules that bind to a polypeptide of the invention but do not elicit a response (and thus prevent the activity of the polypeptide).

Thus, in another aspect, the invention is directed to identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc., of the polypeptides of the invention, or compounds that decrease or increase the production of such polypeptides. Regarding the screening kit, this kit
(a) a polypeptide of the present invention,
(b) a recombinant cell expressing the polypeptide of the present invention,
(c) a cell membrane expressing the polypeptide of the present invention, or (d) an antibody against the polypeptide of the present invention,
Wherein the polypeptide is preferably the polypeptide of SEQ ID NO: 2. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component.

Those skilled in the art will readily appreciate that the polypeptides of the present invention can also be used in methods for designing agonists, antagonists or inhibitors of the polypeptides based on their structure. This method
(a) first analyzing the three-dimensional structure of the polypeptide,
(b) assuming the three-dimensional structure of the likely reactive or binding site of the agonist, antagonist or inhibitor;
(c) synthesizing a candidate compound which is expected to bind or react with the putative reaction site or binding site, and
(d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor,
Comprising. It will be further appreciated that this is usually an interactive process.

  In a further embodiment, the present invention relates to any of an excess or a deficiency of SAF-4 polypeptide activity, such as cancer, inflammation, autoimmune disease, allergy, asthma, rheumatoid arthritis, central nervous system inflammation. Cerebellar degeneration, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, head injury damage, and other neurological abnormalities, septic shock, sepsis, stroke, osteoporosis, osteoarthritis Ischemic reperfusion injury, cardiovascular disease, kidney disease, liver disease, ischemic injury, myocardial infarction, hypotension, hypertension, AIDS, myelodysplastic syndrome and other hematological abnormalities, aplastic anemia, male pattern Provided are treatments for baldness and abnormal conditions such as bacterial, fungal, protozoal and viral infections.

ア プ ロ ー チ If the activity of the polypeptide is in excess, several approaches are available. One approach is to inhibit the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. Administering an inhibitor compound (antagonist) as described above to a patient in an amount effective to do so with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding ligands, substrates, enzymes, receptors, etc., in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of a SAF-4 polypeptide.

  In yet another approach, expression of the gene encoding endogenous SAF-4 polypeptide can be suppressed using expression blocking techniques. These known techniques require the use of antisense sequences produced in the body or administered separately (eg, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (oligodeoxynucleotides as antisense inhibitors of gene expression), CRC Press, Boca Raton, FL (1988), O'Connor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides that form triple helices with this gene can be provided (eg, Lee et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 1360). These oligomers can be administered as such, or the related oligomers can be expressed in vivo.

  For treating abnormal conditions related to an under-expression of SAF-4 and its activity, several approaches are also available. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It becomes. Alternatively, gene therapy can be used to produce SAF-4 endogenously in the relevant cells of the patient. For example, a polynucleotide of the invention may be engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. These producer cells are administered to a patient for in vivo cell manipulation and in vivo polypeptide expression. For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches (and references therein) in Human Molecular Genetics, T Strachan and AP Read, BIOS Scientific Publishers Ltd (1996). thing. Another approach is to administer a therapeutic amount of a polypeptide of the present invention together with a suitable pharmaceutical carrier.

  In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or excipient. And a pharmaceutical composition containing the same. Such carriers include, but are not limited to, saline, saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention as described above. The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds, for example, therapeutic compounds.

O The pharmaceutical composition can be adapted to the route of administration, for example, systemic or oral. A suitable form for systemic administration is infusion (injection), typically intravenous. Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Alternative means of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts, fusidic acid, and other surfactants. In addition, oral administration is possible provided the polypeptide or other compound of the present invention can be formulated as an enteric coating or capsule. These compounds may be administered topically and / or localized in the form of an ointment, paste, gel or the like.

The required dosage range depends on the choice of peptide or other compound of the invention, the route of administration, the nature of the formulation, the condition of the patient, and the judgment of the physician. However, suitable dosages range from 0.1 to 100 μg / kg of patient weight. Given the variety of compounds available and the different efficiencies of the routes of administration, the required dosage is expected to vary widely. For example, oral administration would be expected to require higher dosages than intravenous administration. Such dosage level variations can be adjusted using standard empirical optimization procedures, as is well understood in the art.

  The polypeptide used for the treatment can also be produced in the body of the patient in the above-mentioned treatment called “gene therapy”. For example, cells from a patient are engineered ex vivo with a polynucleotide, such as a DNA or RNA, encoding the polypeptide, for example, using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

  Polynucleotide and polypeptide sequences provide a valuable source of information in identifying alternative sequences having similar homology. This is facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as GCG. Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and / or a polypeptide encoded thereby.

定義 The following definitions are intended to help understand terms that are often used in the above description.

抗体 As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, as well as Fab fragments, including Fab or other products of an immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or separated from its original environment, or both. For example, a polynucleotide or polypeptide naturally occurring in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-localized material is described herein. As used herein, "isolated".

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA in which single- and double-stranded regions are mixed, single- and double-stranded RNA, single-stranded RNA with mixed region and double-stranded region, hybrid molecule containing DNA and RNA (can be single-stranded or more typically double-stranded, where single-stranded and double-stranded regions are mixed May be included). In addition, "polynucleotide" means a triple-stranded region consisting of RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases, and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and special bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides commonly occurring in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to a peptide or protein comprising two or more amino acids linked by a peptide bond or a modified peptide bond (ie, a peptide isostere). "Polypeptide" refers to both short chains (commonly referred to as peptides, oligopeptides or oligomers) and long chains (commonly referred to as proteins). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences that have been modified by natural processes, such as post-translational processing, or by any of the chemical modification methods known in the art. Such modifications are elaborated on basic textbooks, more detailed scholarly articles and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. The same type of modification may be present at the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides can be branched for ubiquitination or cyclic, with or without branching. Cyclic, branched or branched cyclic polypeptides can result from post-translation natural processes and can also be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond. Crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination Transfer RNA-mediated addition of amino acids to proteins such as, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, arginylation, ubiquitination and the like (eg. , Proteins-Structure and Molecular Properties 2nd Ed., TE Creighton, WH Freeman and Company, New York, 1993; Posttranslational Covalent Modification of Proteins, edited by BC Johnson, Academic Press, New York, Wold, F. in 1983, Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12; Seifter et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182: 626-646; and Rattan et al., “Protein Synthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci. (1992) 663: 48-62).

  As used herein, "variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. Changes in the nucleotide sequence of this variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, deletions, additions, fusions and truncations of the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. In general, the sequences of the reference polypeptide and the variant are generally closely similar and limited to differences that are the same in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, or additions in any combination. The amino acid residues to be substituted or added may or may not be those encoded by the genetic code. The variant of the polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis methods or by direct synthesis.

  "Identity," as known in the art, is the relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strands of such sequences. "Identity" can be calculated without difficulty by known methods. Examples of such methods include Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW. , Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). , But is not limited thereto. Methods for determining identity are designed to give the largest match between the sequences considered. In addition, methods for determining identity have been compiled into publicly available computer programs. Computer program methods for determining the identity between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403-410 (1990)). The BLAST X program is generally available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990)). The known Smith Waterman algorithm can also be used to determine identity.

Parameters for comparing polypeptide sequences include the following:
1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA, 89: 10915-10919 (1992)
Gap penalty: 12
Gap length penalty: 4
A program useful with these parameters is generally available as the "gap" program from the Genetics Computer Group (Madison WI). The above parameters are default parameters for peptide comparison (no terminal gap penalty).

Parameters for comparing polynucleotide sequences include the following:
1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: match = + 10, mismatch = 0.
Gap penalty: 50
Gap length penalty: 3
A useful program with these parameters is available from the Genetics Computer Group (Madison WI) as the "gap" program. These are the default parameters for nucleic acid comparison.

Suitable meanings for "identity" of polynucleotides and polypeptides are optionally provided below in (1) and (2).
(1) Specific examples of the polynucleotide comprise a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1. It further includes an isolated polynucleotide. The polynucleotide sequence may be the same as the reference sequence of SEQ ID NO: 1, or may include up to an integer number of nucleotide variations with respect to the reference sequence. The mutation is selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being at the 5 ′ or 3 ′ terminal position of the reference nucleotide sequence, or at these terminal positions. Between the nucleotides in the reference sequence, individually or as one or more contiguous groups within the reference sequence. The number of nucleotide variations is determined by multiplying the total number of nucleotides of SEQ ID NO: 1 by the respective percent identity (divided by 100) and subtracting the product from the total number of nucleotides of SEQ ID NO: 1, ie,
_{n n ≦ x n - (x} n · y)
Can be obtained by Wherein, n _n is the number of nucleotide alterations, x _n is the total number of nucleotides in SEQ ID NO: 1, y is about 0.70,80% for 0.60,70% for 0.60 for 60% for 50% for 0.90,95% for 0.85,90% for 0.80,85% 1.00 for 0.97 or 100% for 0.95,97%, • is the symbol for the multiplication operator, the x _n and y Non-integer products are rounded down to the nearest integer before subtracting the product from _xn . The alteration of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 causes a nonsense, missense or frameshift mutation in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutation. be able to.

By way of example, a polynucleotide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2, ie, has 100% identity to the reference sequence or has a% identity less than 100%. The sequence can include up to an integer number of amino acid variations. The mutation is selected from the group consisting of deletion, substitution (including transition and transversion) or insertion of at least one nucleic acid, wherein such mutation is at the 5 'or 3' end position of the reference polynucleotide sequence, or at these ends. It may be anywhere between the positions and may be interposed between the nucleic acids in the reference sequence individually or as one or more contiguous groups within the reference sequence. The number of nucleic acid mutations is determined by multiplying the total number of amino acids of SEQ ID NO: 2 by the respective percent identity (divided by 100) and subtracting the product from the total number of amino acids of SEQ ID NO: 2, ie:
_{n n ≦ x n - (x} n · y)
Can be obtained by Wherein, n _n is the number of amino acid alterations, x _n is the total number of amino acids in SEQ ID NO: 2, y is about 70% for example and the like 0.85 for 0.80,85% for 0.70,80% ,. Are symbols representing the multiplication operator, and the non-integer product of _xn and y is rounded down to the closest integer before subtracting the product from _xn .
(2) A specific example of a polypeptide is a simple polypeptide comprising a polypeptide having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 2. Further comprising an isolated polypeptide. The polypeptide sequence may be the same as the reference sequence of SEQ ID NO: 2, or may include up to an integer number of amino acid mutations with respect to the reference sequence. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence. , Or anywhere between these terminal positions, and may be interspersed individually between amino acids in the reference sequence or as one or more contiguous groups within the reference sequence. The number of amino acid mutations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the value of each percent identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2, Required by

n _a ≦ x _a − (x _a · y)
Where n _a is the number of amino acid mutations, x _a is the total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.70 for 80% Is 0.80, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and are symbols representing a multiplicative operator, x _a and y the product of the non-integer of prior to subtracting it from x _a, rounded down to the nearest integer.

By way of example, a polypeptide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2, ie, has 100% identity to the reference sequence, or has a% identity less than 100%. The sequence can include up to an integer number of amino acid variations. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence. , Or anywhere between these terminal positions, and may be interspersed individually between amino acids in the reference sequence or as one or more contiguous groups within the reference sequence. The number of amino acid mutations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the value of each percent identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2, Required by

n _a ≦ x _a − (x _a · y)
Wherein, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y is 0.85 mag for 0.80,85% for 0.70,80% for 70% e.g. ,. Are symbols representing the multiplication operator, and the non-integer product of x _a and y is rounded down to the nearest integer before subtracting the product from x _a .

  "Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A-0 464 describes a fusion protein comprising various parts of the constant region of an immunoglobulin molecule and another human protein or a part thereof. In many cases, for use in therapy and diagnosis, it will be advantageous to use the immunoglobulin Fc region as part of a fusion protein, for example, to improve pharmacokinetic properties (eg, EP-A-0232). 262). On the other hand, for some uses, it may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

配列番号２

配列番号３

配列番号４

All publications, including patents and patent application specifications, cited herein are hereby incorporated by reference in their entirety, as if each publication was clearly and individually indicated. Shall be.
Sequence information SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4

Claims (14)

An isolated polypeptide selected from the group consisting of the following (i) to (iii):
(i) at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2;
(b) 80% identity,
(c) 90% identity, or (d) 95% identity,
An isolated polypeptide comprising an amino acid sequence having
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or
(iii) an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. An isolated polynucleotide selected from the group consisting of the following (i) to (vi), or a nucleotide sequence complementary to the isolated polynucleotide:
(i) at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2;
(b) 80% identity,
(c) 90% identity, or (d) 95% identity,
An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having
(ii) at least (a) 70% identity to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over its entire length;
(b) 80% identity,
(c) 90% identity, or (d) 95% identity,
An isolated polynucleotide comprising a nucleotide sequence having
(iii) at least (a) 70% identity to the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1;
(b) 80% identity,
(c) 90% identity, or (d) 95% identity,
An isolated polynucleotide comprising a nucleotide sequence having
(iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2,
(v) an isolated polynucleotide consisting of the polynucleotide sequence of SEQ ID NO: 1,
and
(vi) Appropriate libraries are prepared under stringent hybridization conditions.
An isolated polynucleotide obtained by screening with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof. 抗体 An antibody immunospecific for the polypeptide of claim 1. A method of treating a subject, comprising:
When the subject needs to suppress the activity or expression of the polypeptide according to claim 1,
(a) administering to the subject a therapeutically effective amount of the antagonist to the polypeptide; and / or
(b) administering to the subject a nucleic acid molecule that suppresses the expression of a nucleotide sequence encoding the polypeptide; and / or
(c) administering to the subject a therapeutically effective amount of the polypeptide that competes with the polypeptide for its ligand, substrate, or receptor;
A method for treating the subject, comprising: A method for diagnosing a disease or a susceptibility of a subject related to the expression or activity of the polypeptide according to claim 1 in the subject,
(a) determining whether there is a mutation in the nucleotide sequence encoding the polypeptide in the subject's genome; and / or (b) expressing the polypeptide in a sample obtained from the subject. Analyzing the presence or amount of
A method comprising: A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1,
(a) measuring the binding between a candidate compound and the polypeptide (or a cell carrying the polypeptide or its membrane) or a fusion protein thereof by using a label directly or indirectly bound to the candidate compound; To do,
(b) measuring the binding between a candidate compound and the polypeptide (or a cell carrying the polypeptide or its membrane) or a fusion protein thereof in the presence of a labeled competitor;
(c) examining whether the candidate compound produces a signal resulting from activation or suppression of the polypeptide, using a detection system suitable for cells or cell membranes carrying the polypeptide;
(d) preparing a mixture by combining the candidate compound and a solution containing the polypeptide, measuring the activity of the polypeptide in the mixture, and comparing the activity of the mixture with a standard; and
(e) detecting the effect of the candidate compound on mRNA encoding the polypeptide in cells and production of the polypeptide, for example, using an ELISA assay;
A screening method comprising a method selected from the group consisting of: ア ン タ ゴ ニ ス ト An antagonist of the polypeptide according to claim 1. 発 現 An expression system containing a polynucleotide capable of producing the polypeptide of claim 1 when the following expression system is present in a compatible host cell. Claim 9 wherein the host cell produces, under suitable culture conditions, a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. A method for producing a recombinant host cell, comprising transforming or transfecting a cell with the described expression system. 組 換 え A recombinant host cell obtained by the method according to claim 9. 11. The membrane of a recombinant host cell according to claim 10, which expresses a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. A method for producing the polypeptide, comprising culturing the host cell according to claim 10 under conditions sufficient to produce the polypeptide, and recovering the polypeptide from the culture medium. An isolated polynucleotide selected from the group consisting of the following (a) to (d):
(a) an isolated polynucleotide comprising a nucleotide sequence having at least 70%, 80%, 90%, 95%, 97% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 ,
(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3,
(c) the polynucleotide of SEQ ID NO: 3, or
(d) comprising a nucleotide sequence encoding a polypeptide having at least 70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. An isolated polynucleotide. A polypeptide selected from the group consisting of the following (a) to (e):
(a) a polypeptide comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4,
(b) a polypeptide having an amino acid sequence having at least 70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4,
(c) a polypeptide comprising the amino acid of SEQ ID NO: 4,
(D) a polypeptide consisting of the polypeptide of SEQ ID NO: 4,
(e) a polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3.