JP2002511232A - Secreted proteins and polynucleotides encoding them - Google Patents

Abstract

  (57) [Summary] Disclosed are novel polynucleotides and the proteins encoded by them.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, as well as therapeutic, diagnostic and research utilities for these polynucleotides and proteins.

BACKGROUND proteinaceous factors invention (e.g., lymphokines, interferons, including cytokine such as CSFs and interleukins) has methods for the purpose of finding this 10
Matured rapidly over the years. Currently, conventional hybridization and expression cloning methods rely on information directly related to the discovered protein (ie,
In the case of hybridization cloning, depending on the partial DNA / amino acid sequence of the protein; in the case of expression cloning, the activity of the protein).
The new polypeptide is cloned "directly". Signal sequence cloning (
DNA sequences are isolated based on the presence of the currently well-recognized secretory leader sequence motifs), as well as more recent "indirect" cloning methods, such as various PCR or low stringency hybridization cloning methods. A large number of DNA / amino acid sequences for proteins known to have biological activity due to their secreted nature in the case of cloning of the leader sequence, or due to the cell or tissue source in the case of the PCR method. Has made progress by enabling. The present invention is directed to these proteins and the polynucleotides that encode them.

[0003] In one embodiment of the invention, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) comprising the nucleotide sequence of SEQ ID NO: 1 (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 24 to nucleotide 548; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 525 to nucleotide 548; (d) A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 115 to nucleotide 317; (e) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone B196 deposited under accession number ATCC 98021; (f) accession number ATCC9 cD of clone B196, which is deposited as 021
A polynucleotide encoding the full length protein encoded by the NA insert; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone B196 deposited under accession number ATCC 98021; CD of clone B196
A polynucleotide encoding the mature protein encoded by the NA insert; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2; (j) a biologically active SEQ ID NO: 2 (K) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); (1) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such a polynucleotide is a nucleotide sequence from nucleotide 24 to nucleotide 548 of SEQ ID NO: 1;
Nucleotide sequence from nucleotide 115 to nucleotide 317 of SEQ ID NO: 1; accession number ATCC
The nucleotide sequence of the full length protein coding sequence of clone B196 deposited as 98021; or the mature protein coding sequence of clone B196 deposited as accession number ATCC 98021. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone B196 deposited under accession number ATCC 98021. In yet another preferred embodiment, the invention provides a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 118 to amino acid 162 of SEQ ID NO: 2. Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In another embodiment, the present invention provides a composition comprising a protein comprising an amino acid sequence selected from the group consisting of: a protein substantially free of other mammalian proteins: (b) an amino acid sequence from amino acid 118 to amino acid 162 of SEQ ID NO: 2; (c) a fragment of the amino acid sequence of SEQ ID NO: 2; and (d) a deposit under accession number ATCC 98021. CD of clone B196 isolated
Amino acid sequence encoded by NA insert. Preferably, such a protein is the amino acid sequence of SEQ ID NO: 2 or amino acid 118 to amino acid 1 of SEQ ID NO: 2.
Includes up to 62 amino acid sequences.

[0004] In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also,
An organism having enhanced, reduced, or modified expression of a gene corresponding to a polynucleotide sequence disclosed herein is provided by the invention. Further, a method for producing a protein, comprising: (a) growing a culture of a host cell transformed with such a polynucleotide composition in a suitable medium; and (b) purifying the protein from the culture. There is also provided a method comprising: The protein produced by such a method is also provided by the present invention. Preferred specific examples include those in which the protein produced by such a method is a mature form of the protein. The protein composition of the present invention may further contain a pharmaceutically acceptable carrier. Compositions comprising antibodies specifically reactive with such proteins are also provided by the present invention. Also provided is a method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the invention and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION Isolated Proteins and Polynucleotides The nucleotide and amino acid sequences for each of the clones and proteins disclosed herein are reported below. By sequencing the deposited clones by known methods, the actual nucleotide sequence of each clone can be readily determined. The deduced amino acid sequence (both full length as well as mature) can then be determined from such nucleotide sequences. By expressing the clones in a suitable host cell, collecting the proteins, and then determining the sequence, the amino acid sequence of the protein encoded by each clone can also be determined. For each protein disclosed, Applicants have identified what has been determined to be the best identifiable reading frame using sequence information available at the time of filing. As used herein, the term "secreted" protein refers to that which is transported across a membrane when expressed in a suitable host cell, including transport as a result of a signal sequence in its amino acid sequence. “Secreted” proteins include, but are not limited to, proteins that are totally secreted (eg, soluble proteins) or partially secreted (eg, receptors) from the cell in which they are expressed. "Secreted" proteins also include, but are not limited to, those that are transported across the endoplasmic reticulum membrane. Clone "B196" The polynucleotide of the present invention has been identified as clone B196. B196
Describe a method selective for cDNAs encoding secreted proteins (US Pat. No. 5,366,663).
Adult blood (Phytohemagglutinin and phorbol meristate acetate and peripheral blood mononuclear cells treated with mixed lymphocyte reaction)
Based on computer analysis of the amino acid sequence of the protein isolated or encoded from the NA library, it was identified as encoding a secreted or transmembrane protein. B196 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "B196 protein"). The nucleotide sequence of B196 determined this time is shown in SEQ ID NO: 1. This time, what the applicant considers to be the correct reading frame for the coding region is shown in SEQ ID NO: 2. The deduced amino acid sequence of B196 protein corresponding to the above nucleotide sequence is shown in SEQ ID NO: 2. Amino acids 155 to 167 are predicted leaders /
The signal sequence, the predicted mature amino acid sequence starting at amino acid 168, or amino acids 155 to 167 is the transmembrane domain. The additional nucleotide sequence of B196 is shown in SEQ ID NO: 3. Applicant has determined that SEQ ID NO: 3 is an immature
Consider a cDNA molecule generated from an RNA transcript. This is because base pairs 205 to 352 of SEQ ID NO: 3 are considered to be intron sequences. SEQ ID NO: 1 was derived from SEQ ID NO: 3 by deleting this putative intron sequence. Clone B196 was purchased on April 4, 1996 under the Budapest Treaty
Deposited with the Type Culture Collection under the accession number ATCC 98021 as the original deposit. All restrictions on public use of the deposited material are set forth in 37 CFR. F. R
. Except for the provisions of § 1.808 (b), this will be terminated by a patent. The clone was digested with EcoRI / NotI (5 ′ site, EcoRI; 3 ′ site, No.
By performing tI), it can be removed from the deposited vector. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone B196 should be approximately 1800 bp. The deduced amino acid sequence disclosed herein for B196 was compared to BLASTN / BLASTX
And searched against GenBank and GenSeq nucleotide sequence databases using the FASTA search protocol. B196 is AA235452 (zt35c01.s1 Soares ovar
y tumor NbHOT Homo sapiens cDNA clone 724320 3 'similar to contains Alu
repetitive element), T09157 (EST07050 Homo sapiens cDNA clone HIBBP87 5 '
end), T34456 (EST68380 Homo sapiens cDNA 5 'end similar to None), T3503
9 (EST79238 Homo sapiens cDNA similar to None), and T70971 (yc49f08.r
1 Homo sapiens cDNA clone 84039 showed at least some similarity to the sequence identified as 5 '). Based on sequence similarity, the B196 protein and each similar protein or peptide may share at least some activity.

[0006] Fragments of the protein of the present invention that can exhibit biological activity are also included in the present invention. Protein fragments may be linear or, for example, HUSaragovi
, et al., Bio / Technology 10, 773-778 (1992) and RSMcDowell, et al., J
Amer. Chem. Soc. 114, 9245-9253 (1992) (both references are deemed to be described herein by reference) may be cyclized using known methods. . For many purposes, including increasing the number of valencies at the protein binding site,
Such a fragment may be fused to a carrier molecule along with the immunoglobulin.
For example, a protein fragment may be fused to the Fc portion of an immunoglobulin via a "linker". For the bivalent form of the protein, such a fusion is made to the F molecule of the IgG molecule.
This can be done for part c. Such fusions may be obtained using other immunoglobulin isotypes. For example, a protein-IgM fusion will yield a ten-valent form of a protein of the invention. The invention also provides the full length and mature forms of the disclosed proteins. The full length form of such a protein has been identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. Mature forms of such proteins may be obtained by expressing the disclosed full-length polynucleotides (preferably those deposited with the ATCC) in appropriate mammalian cells or other host cells. The sequence of the mature form of the protein may be determined from the amino acid sequence of the full-length form.

[0007] The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. "
A “corresponding gene” is a region of the genome that is transcribed to produce mRNA,
CDNA is derived from the RNA and the flanking regions of the genome (including coding sequences, 5 'and 3' untranslated regions) or spliced exons, introns, promoters, enhancers, and Also includes silencer or suppressor elements. Corresponding genes can be isolated using the sequence information disclosed herein. Corresponding genes can be isolated using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information to identify and / or amplify genes in a suitable genomic library or other source of genomic material. An "isolated gene" is a gene that has been separated from flanking coding sequences, if present, present in the genome of the organism from which the gene was isolated.

[0008] Provided are organisms having enhanced, reduced, or altered expression of a gene corresponding to a polynucleotide sequence disclosed herein. Desirable changes in gene expression are achieved by using antisense polynucleotides or by using ribozymes that bind and / or cleave mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15 (7): 250-2
54; Lavarosky et al., 1997, Biochem.Mol. Med. 62 (1): 11-22; and Hampel,
1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are herein incorporated by reference). A transgenic animal having a multicopy gene corresponding to a polynucleotide disclosed herein, preferably obtained by transforming cells with a genetic construct that is stably maintained in transformed cells and their progeny. A transgenic animal is provided. Also provided are transgenic animals having altered genetic control regions that increase or decrease gene expression levels or alter the temporal or spatial pattern of gene expression (see European Patent No. 0 649 464 B1; All of which are considered to be described herein by reference). In addition, an organism is provided wherein the gene corresponding to the polynucleotide sequence disclosed herein has been incompletely or completely inactivated, either by insertion of an external sequence or by deletion of all or part of the corresponding gene. By insertion, preferably by insertion after incorrect resection of the transposable element (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 19).
93, Proc. Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Pr.
USA 91 (2): 719-722; all of which are incorporated herein by reference), or by homologous recombination, preferably by positive / negative genetic selection Homologous recombination (Mansour et a
l., 1988, Nature 336: 348-352; US Patent Nos. 5,464,764; 5,487,992; 5,
627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are deemed herein to be incorporated by reference), incomplete or complete gene inactivation can be performed. These organisms with altered gene expression
Preferably they are eukaryotes, and more preferably mammals. Such organisms are useful for developing non-human models for studying diseases associated with the corresponding gene, and for developing assay systems for identifying molecules that interact with protein products from the corresponding gene.

If the protein of the invention is membrane-bound (eg, a receptor), the invention also provides a soluble form of such protein. In such a form, some or all of the intracellular and transmembrane domains of the protein are removed so that the protein is sufficiently secreted from the host in which it was expressed. The intracellular and transmembrane domains of the protein of the present invention can be identified by known methods for determining such domains from sequence information. The proteins and protein fragments of the present invention comprise at least 25% (more preferably at least 50%, most preferably at least 75%) of the amino acid sequence of the disclosed protein.
And has at least 60% sequence identity (more preferably at least 75% identity, most preferably at least 90%) to the disclosed proteins.
Or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. It has at least 75% sequence identity (more preferably at least 85% identity, most preferably at least 95% identity) to any of the disclosed protein segments, preferably 8 or more ( Proteins or protein fragments containing segments comprising more preferably 20 or more, most preferably 30 or more) contiguous amino acids are also encompassed by the present invention. Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. The term "species homolog" herein is a protein or polynucleotide that relates to a species different from the source of the protein or polynucleotide, but as determined by one of skill in the art,
Those having significant sequence similarity. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein, and then screening for an appropriate nucleic acid source from the desired species. The present invention also relates to allelic variants of the disclosed polynucleotides, i.e., naturally occurring alternative forms of isolated polynucleotides that encode a protein identical, homologous, or related to the protein encoded by the polynucleotide of the present invention. Include. The present invention also encompasses polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein. The invention also encompasses polynucleotides that are capable of hybridizing to the polynucleotides described herein under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions. I do. The following table shows examples of strictness conditions. The very strict conditions are, for example, at least as strict as the conditions A to F, and the strict conditions are, for example, at least as strict as the conditions GL, and the reduced conditions are, for example, For example, it is at least as strict as the conditions M to R.

[0010] a): The hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. b): SSPE (1XS) in hybridization and wash buffer
SPE was 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1.25 mM E
DTA, pH 7.4) can be replaced by SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate). After completion of hybridization, washing is performed for 15 minutes. * T _B -T _R : The hybridization temperature for hybrids that are expected to be less than 50 base pairs in length is 5-10 times greater than the melting temperature (T _m ) of the hybrid.
C should be lowered. T _m is determined by the following equation. For hybrids less than 18 base pairs in length, T _m (−C) = 2 (# of A + T bases) +4 (# of G + C base
s). For hybrids 18 to 49 base pairs in length, T _m (−C) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N), where N is the hybrid And the sodium ion concentration in the hybridization buffer ([Na ⁺ ] for 1 × SSC = 0.165 M).

Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF Fritsch, and T. Maniatis, 1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Ha
rbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,
1995, FM Ausubel et al., Eds., John Wiley & Sons, Inc., sections 2.10
and 6.3-6.4 and are deemed to be incorporated herein by reference. Preferably, each such hybridizing polynucleotide is at least 25% of the polynucleotide of the invention to be hybridized.
(More preferably, at least 50%, most preferably at least 75%) and at least 60% sequence identity (more preferably at least 75%) to the polynucleotide of the invention to be hybridized. , Most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps.

[0012] The isolated polynucleotide encoding the protein of the present invention is described in Kaufman et al., Nucl.
The protein may be recombinantly produced by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed in eic Acids Res. 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. Many suitable expression control sequences are known in the art. General methods for recombinant protein expression are also known and are described in R. Kaufman, Methods in Enzymology 185, 537-566.
(1990) is a typical example. "Operably linked," as defined herein, means that the isolated polynucleotide of the present invention and the expression control sequence are placed in a vector or cell and transformed (transfected) with the ligated polynucleotide / expression control sequence. It is meant to be expressed by a host cell. Many types of cells can act as suitable host cells for expressing the proteins of the present invention.
Mammalian cells include, for example, monkey COS cells, Chinese hamster ovary (CH
O) obtained from in vitro culture of cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, primary tissues Cell line, primary explant, H
Includes eLa cells, mouse cells, BHK, HL-60, U937HaK or Jurkat cells. Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. A potentially suitable yeast strain is Saccharomyces ce
revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or yeast strains capable of expressing heterologous proteins. A potentially suitable bacterial strain is Escherichia
coli, Bacillus subtilis, Salmonella typhimurium, or bacterial strains capable of expressing heterologous proteins. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced therein, for example, by phosphorylation or glycosylation at appropriate sites to obtain a functional protein. Such covalent linkages may be made using known chemical or enzymatic methods. The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors and the protein obtained by using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are described, for example, in In
Commercially available in kit form from Vitrogen, San Diego, California, USA (MaxBac ^R kit), such methods are well known in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
) (Assumed to be described herein by reference). As used herein, insect cells capable of expressing a polynucleotide of the present invention have been "transformed." The protein of the present invention may be produced by culturing the transformed host cell under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein may then be purified from such culture (ie, medium or cell extract) using known purification processes, such as gel filtration and ion exchange chromatography. Purification of the protein may also be accomplished by affinity columns containing an agent that will bind to the protein; one or more column steps with an affinity column such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibacrom blue 3GA Sepharose ^R ;
One or more steps using hydrophobic interaction chromatography with a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may be expressed in an easily purified form. For example, maltose binding protein (MBP), glutathione-S-tonsferase (GST)
Alternatively, it may be expressed as a fusion protein such as a fusion protein with thioredoxin (TRX). Kits for expression and purification of such fusion proteins are available from New En
Commercially available from glan BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen. The protein can also be tagged with an epitope and then purified by using a specific antibody directed against such epitope. One such epitope ("flag") is commercially available from Kodak (New Haeven, CT). Finally, the protein is further purified using one or more reverse phase high quality liquid chromatography (RP-PLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. can do. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as "isolated protein".

The protein of the present invention may be used as a product of a transgenic animal, for example, a component of milk of a transgenic cow, goat, pig, or sheep characterized by a somatic cell or a germ cell containing a nucleotide sequence encoding the protein. May be expressed as The protein may be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed protein sequences may have common biological properties, including protein activity, by sharing primary, secondary or tertiary structure and / or conformational properties. Thus, they may be used as biological activities or immunological substitutes for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and generating antibodies.

The proteins described herein are characterized by an amino acid sequence similar to that of the purified protein, but may be modified therein, either spontaneously or by derivatization. For example, modifications of the peptide or DNA sequence can be made by those skilled in the art using known methods. Modifications of interest in the protein sequence include changes, substitutions, exchanges, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Methods for such changes, substitutions, exchanges, insertions or deletions are well known to those skilled in the art (eg, US Pat. No. 415).
No. 8584). Preferably, such changes, substitutions, exchanges, insertions or deletions retain the desired activity of the protein. Other fragments and derivatives of the protein sequence that are expected to retain protein activity in whole or in part, and thus may be useful in screening or other immunological methods, can also be made by those skilled in the art from the disclosure herein. . Such modifications are believed to be encompassed by the present invention.

Uses and Biological Activities The polynucleotides and proteins of the present invention are believed to exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. Can be The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, such as a vector suitable for gene therapy or DNA transfer). Can be provided.

Research Uses and Utility The polynucleotides provided by the present invention can be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes "Gene chips" or other probes as probes for subtraction To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization techniques, and to generate anti-DNA antibodies or to generate additional immunizations. Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Polynucleotides can be used in an interaction trap assay (eg, as described in Gyuris et al., Cell 75: 791-803 (1993)) to identify The proteins provided by the present invention may be used in assays to determine biological activity, including a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses; As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. A literature disclosing such a method can be found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Sp.
ring Harbor Laboratory Press, Sambrook, J., EF Fritsch and T. Maniatis
eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Tec
hniques ", Academic Press, Berger, SL and AR Kimmel eds., 1987.

Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate individual or liquid formulation, such as in the form of a powder, pill, solution, suspension or capsule. it can. In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Cytokines and Cell Proliferation / Differentiation Activity The protein of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or produce other cytokines in certain cell populations. Can be induced. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention is determined in cell lines (32D, DA
2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (pr
eB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTL
(Including, but not limited to, L2, TF-1, Mo7e and CMK) are confirmed by any of a number of conventional factor-dependent cell proliferation assays. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for T cell or thymocyte proliferation are described in Current Protocols in Immunology, Ed by JE Coligan, AM Kruisbeek, D .;
H. Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et
al., J. Immunol. 137: 34943500, 1986; Bertagnolli et al., J. Immunol. 145
: 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133: 327-341,
1991; Bertagnolli, et al., J. Immunol. 149: 3778-3783, 1992; Bowman et al.
., J. Immunol. 152: 1756-1761, 1994, but are not limited thereto. Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM and Shevach, EM In Cur
rent Protocols in Immunology. JEea Coligan eds. Vol 1 pp. 3.12.1-3.1
2.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and h
uman Interferon g, Schreiber, RD In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toront
o. Includes, but is not limited to, those described in 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottoml
y, K., Davis, LS and Lipsky, PE In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 1991; deVries et al., J. Exp.Med. 173: 1205-1211, 1991; Moreau et al.
., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
USA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6
-Nordan, R. In Current Protocols in Immunology. JEea Coligan eds. V
ol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al.,
Proc. Natl. Acad. Sci. USA 83: 1857-1861, 1986; Measurement of human I
nterleukin 11-Bennett, F., Giannotti, J., Clark, SC and Turner, K. J
. In Current Protocols in Immunology. JEea Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and hum
an Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC and Turner,
KJ In Current Protocols in Immunology. JEea Coligan eds. Vol 1 p
p. 6.13.1, including, but not limited to, those described in John Wiley and Sons, Toronto. 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions and direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Ed by JE. Coligan, AM Kruisbeek, D.
H. Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7,
Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sc
i. USA 77: 6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11: 405-411.
, 1981; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J.
Immunol. 140: 508-512, 1988, including, but not limited to.

Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including, but not limited to, the activity in the assays described herein. Protein is
It may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), for example, in up-regulating or down-regulating T and / or B lymphocyte proliferation, and in N
It may be useful in activating the cytolytic activity of K cells and other cell populations. These immunodeficiencies can be genetic and can include viral (
For example, it may be caused by HIV) and bacterial or fungal infections, or may be caused by an autoimmune disease. More specifically, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, for example, various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated using the protein of the present invention. Of course, in this regard, the proteins of the present invention may be used where a boost to the immune system is indicated, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis,
Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory Eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions in which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems, may be treated with the proteins of the invention. Other conditions for which immunosuppression is desired (including, for example, organ transplantation)
May be treated using the protein of the present invention. The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response already in progress, or may involve interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped.
Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (eg, such as B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the graft is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Mixed with peptides,
Alternatively, pre-transplant administration of a soluble, monomeric form of a peptide with B7-2 activity alone, induces binding of the molecule to its native ligand on immune cells without transmitting a responsive costimulatory signal. there is a possibility. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Lensch
ow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad.
Sci. USA, 89: 11102-11105 (1992)). Furthermore, a rat model of GVHD (Paul ed., Fundamental Immunology, Ravan Press, New York, 1
989, pp. 846-847) can be used to determine the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo. Also, blocking antigen function can be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic deep erythematosus in MRL lpr / lpr or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and experimental murine myasthenia gravis. (Paul ed., Fundamental Immunology, Raven Pres
s, New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful.
Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection.
In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of stimulatory forms of B lymphocyte antigen.

Alternatively, the T cells are taken from a patient and co-stimulated in vitro with a viral antigen to which ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of a soluble peptide of the invention, The in vitro activated T cells may then be reintroduced into the patient to enhance the antiviral immune response in the infected patient. Another method of promoting an antiviral immune response is
The infected cells are removed from the patient and transfected with them a nucleic acid encoding a protein of the invention as described herein so that the cells express all or part of the protein on their surface. Will be re-introduced. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with a peptide having B7-2-like activity alone or with a peptide having B7-1-like activity and / or B7-3-like activity, It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, tumor cells can be targeted for transfection in vivo using gene therapy. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, MHC
Tumor cells lacking class I or MHC class II molecules, or sufficient MHC
Class I or tumor cells can not be re-expressed MHC class II molecules, MHC class Iα chain protein and beta ₂ microglobulin protein or an MHC class IIα chain and an MHC class IIβ chain all or part of the protein (e.g., cytoplasmic truncations protein Domain) can be transfected, whereby the class I or class II MHC protein can be expressed on the cell surface. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is a T cell-mediated transfected tumor Induces an immune response against the cells. If desired, a gene encoding an antisense construct that blocks expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays suitable for thymocyte or spleen cell cytotoxicity are described in Current Protocols in Immunology, Ed by JE Coligan, AM Kruisbeek, D .;
H. Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann
Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., Proc.
J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1
572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al.,
J. Immunol. 140: 508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci.
USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1
982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Im.
munol. 137: 3494-3500, 1986; Bowmanet al., J. Virology 61: 1992-1998; Ta
kai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular.
Immunology 133: 327-341, 1991; Brown et al., J. Immunol. 153: 3079-3092
, 1994, including, but not limited to. Assays for T cell-dependent immunoglobulin responses and isotype switching, specifically to modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described in Maliazewski, J. Immunol. 144: 3028-3033, 1990, including but not limited to assays for B cell function as described in In vitro antibody production, Mond, JJ and Brunswick, M. In Current.
Protocols in Immunology JEColigan eds.Val 1 pp.3.8.1-3.8.16, John Wile
y and Sons, Tronto 1994. The mixed lymphocyte reaction (MLR) assay, which identifies proteins that primarily produce Th1 and CTL responses, is described in Current Protocols in Immunology, Ed by JE Coligan, AM Kruisbeek, D .;
H. Margulies, EM Shevach, W Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et
al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 50
8-512, 1988; including but not limited to the assays described in Bertagnolli et al., J. Immunol. 149: 3778-3783, 1992. Dendritic cell-dependent assays, specifically identifying proteins expressed by dendritic cells that activate intact T cells, are described by Guery et al., J. Immunol. 134: 536-544, 1995; Inaba. et al., Journal of E
xperimental Medicine 173: 549-559, 1991; Macatonia et al., Journal of Im
munology 154: 5071-5079, 1995; Porgador et al., Journal of Experimental.
Medicine 182: 255-260, 1995; Nair et al., Journal of Virology 67: 4062-4
069, 1993; Huang et al., Science 264: 961-965, 1994; Macatonia et al.,
Journal of Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al.,
Journal of Clinical Investigation 94: 797-807, 1994; and Inaba et al., J
Includes, but is not limited to, the assays described in ournal of Experimental Medicine 172: 631-640, 1990. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1
951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of I
mmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 199
3; including but not limited to the assays described in Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Early stage T cell commitment and development assays
Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 15
5: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Pr
USA 88: 7548-7551, 1991, including but not limited to oc. Natl. Acad. Sci.

Hematopoietic modulatory activity The proteins of the present invention are useful in the regulation of hematopoiesis and therefore in the treatment of bone marrow and lymphocyte deficiencies. Even minimal biological activity supporting colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis.
For example, supporting the proliferation of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing usefulness in the treatment of various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all of hematopoietic cells, therefore, a variety of stem cell disease (
It is usually a disease that is treated by transplantation, and is useful in supporting the growth of, for example, aplastic anemia and paroxysmal hemoglobinuria), and is also useful in vivo or ex vivo. It is also useful in repopulating the stem cell compartment as normal cells after radiation / chemotherapy (ie, in combination with bone marrow transplantation) or after being engineered for gene therapy. In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the proliferation and differentiation of various hematopoietic cell lines have already been cited. Assays for embryonic stem cell differentiation, particularly those that identify proteins that affect embryonic hematopoiesis, are described in Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al.
, Molecular and Cellular Biology 13: 473-486, 1993; McClanahan et al., B
lood 81: 2903-2915, 1993, including, but not limited to. Assays for stem cell survival and differentiation, particularly identifying proteins that modulate lympho-hematopoiesis, are described in Methylcellulose colony forming assays, Freshney, MG In Culture of Hema.
topoietic Cells.RI Freshney, et al. eds.Vol pp. 265-268, Wiley-Liss
, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA
89: 5907-5911, 1992; Primitive hematopoietic colony forming cells with h
igh proliferative potential, McNiece, IK and Briddell, RA In Culture
of Hematopoietic Cells.RI Freshney, et al. eds.Vol pp. 23-39, Wile
y-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology.
22: 353-359, 1994; Cobblestone area forming cell assay, Ploemacher, RE
In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol pp.
1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow cultu
res in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen
, T. In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol.
pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture ini
tiating cell assay, Sutherland, HJ In Culture of Hematopoietic Cells.
RI Freshney, et al. Eds.Vol pp. 139-162, Wiley-Liss, Inc., New York,
Includes, but is not limited to, the assays described in NY. 1994.

Tissue Proliferation Activity The protein of the present invention is useful in compositions used for the growth or regeneration of bone, cartilage, key, ligament and / or nervous tissue, as well as for wound healing and tissue repair, and in the treatment of burns, lacerations and ulcers. Has utility. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, finds use in healing fractures and cartilage damage or defects in humans and other animals. Such formulations using the protein of the present invention are useful for reducing closed and open fractures and for improving fixation of artificial joints. De novo osteogenesis induced by osteogenic agents contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects and is also useful in cosmetic surgery. The protein of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such agents provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells,
Alternatively, it can induce differentiation of osteogenic cell precursors. The protein of the present invention may be used to stimulate osteoporosis or osteoarthritis, for example, by stimulating bone and / or cartilage repair or by blocking the process of tissue destruction mediated by inflammation or inflammatory processes (collagenase activity, osteoclast activity, etc.). May also be useful in the treatment of Another category of tissue development activity that may be due to the proteins of the present invention is key / ligament formation. Proteins of the invention that induce the formation of key / ligament-like or other tissues in an environment in which such tissues are not normally formed can be used to prevent key or ligament laceration, deformation and other key or ligament defects in humans and other animals. Applied to healing, Such formulations using the key / ligament-like tissue inducing protein may be used to prevent damage to the key or ligament tissue as well as to improve the fixation of the key or ligament to bone or other tissue. The de novo key / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of congenital, traumatic, or oncological resection-induced craniofacial defects, and furthermore the attachment of keys or ligaments Or it is also useful in cosmetic surgery for repair. The compositions of the present invention provide an environment for attracting key- or ligament-forming cells, stimulate the growth of key- or ligament-forming cells, and induce the differentiation of key- or ligament-forming cell precursors. Or induce the growth of key / ligament cells or progenitors ex vivo so as to effect tissue repair when returned to vivo.
The compositions of the present invention are also useful for keratitis, carpal tunnel syndrome and other key or ligament defects. The compositions of the present invention may include a suitable matrix and / or sequestering agent, such as a carrier well known in the art. The protein of the present invention is useful for the proliferation of nerve cells and the regeneration of nerve and brain tissues, namely, central and peripheral nervous system diseases and neuropathies and mechanical diseases and traumatic diseases (including degeneration, death or trauma to nerve cells or nerve tissues). ) May also be useful. More particularly, diseases of the peripheral nervous system such as peripheral nerve injury, peripheral and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager
The protein may be used in the treatment of diseases of the central nervous system, such as the syndrome. Additional conditions that may be treated according to the present invention include mechanical and traumatic diseases such as spinal cord diseases, cerebrovascular diseases such as head trauma and stroke. Peripheral neuropathy caused by chemotherapy or other medical therapies can be treated with the proteins of the present invention. The protein of the present invention is also useful for promoting more preferable and prompt closure of non-healing wounds including (but not limited to) pressure ulcers, ulcers related to vascular dysfunction, surgical and trauma wounds and the like. It is possible. The protein of the present invention may also be used in other tissues such as organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), and vascular (including vascular endothelium) tissue. Or an activity for promoting the growth of cells constituting such a tissue. Part of the desired effect may be the regeneration of normal tissue by suppressing fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity. The proteins of the present invention may also be useful in protecting or regenerating intestines and treating conditions caused by lung or liver fibrosis, reperfusion injury of various tissues, and systemic cytokine damage. The protein of the present invention promotes or suppresses differentiation of the above-mentioned tissue from precursor tissue or cells;
Alternatively, it may be useful for suppressing the growth of the tissue. The activity of the protein of the invention may be measured, inter alia, by the following method: Tissue regeneration activity assay is described in WO 95/16035 (bone, cartilage, key).
International Publication WO95 / 05846 (nerves, neurons); International Publication WO91 / 05
7491 (skin, endothelium), but is not limited thereto. Wound healing activity assays are described in Winter, Epidermal Wound Healing, pps. 71-112.
(Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc.,
Including, but not limited to, those described in Chicago (modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978)).

Activin / Inhibin Activity The protein of the present invention may also exhibit activin- or inhibin-related activity. Inhibins are characterized by their ability to inhibit follicle stimulating hormone (FSH) release,
Activins are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces the fertility of female mammals and reduces the spermatogenesis of male mammals. Can be useful as Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the protein of the present invention, as a homodimer or as a heterodimer with subunits of other proteins of the inhibin-β group, can be used to stimulate fertility based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells. May be useful as therapeutic agents that induce See, for example, U.S. Pat. No. 4,798,885. The proteins of the invention may also be useful for improving reproduction in sexually immature mammals, resulting in increased fertility during the life of livestock such as cattle, sheep and pigs. The activity of the protein of the invention may be measured, inter alia, by the following method: Activin / inhibin activity assays are described in Vale et al., Endocrinology 91:
562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Natu
re 321: 776-779, 1986; Mason et al., Nature 318: 659-663,1985; Forage et
Natl. Acad. Sci. USA 83: 3091-3095, 1986, including, but not limited to.

Chemotaxis / chemokinetic activity The protein of the present invention can be used for the chemotactic or chemokinetic activity of mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells (for example, (Acting as a chemokine). Chemotactic and chemokinetic proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for treating injuries and other trauma to tissues and for treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent. Certain proteins or peptides have chemotactic activity on a particular cell population and, when stimulable, direct or indirectly direct the movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce adhesion of one cell population to another cell population as well as the ability of the protein to adhere to another cell population. Suitable assays for migration and adhesion are described in Current Protocols in Immunology, Ed by JE Coligan, AM Kruisbeek, DH
Margulies, EM Shevach, W. Strober, Pub. Greene Publishing Associates
and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chem
okines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95: 1370-1376, 1995;
Lind et al. APMIS 103: 140-146, 1995; Muller et al Eur. J. Immunol. 25:
1744-1748; Gruber et al. J. of Immunol. 152: 5860-5867, 1994; Johnston
et al. J. of Immunol. 153: 1762-1768, 1994, including but not limited to the assays described.

Hemostatic and thrombolytic activity The protein of the present invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various clotting disorders, including hereditary disorders such as hemophilia, or in the treatment of injuries caused by trauma, surgery, or other causes, as well as clotting and other hemostasis. Can be promoted. The protein of the present invention may be useful for the treatment and prevention of thrombolysis or inhibition of thrombus formation and conditions resulting therefrom (eg, myocardial infarction and infarction of central nervous system blood vessels (eg, stroke)). The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for haemostatic and thrombolytic activities are described in Linet et al., J. Clin. Pharmacol. 26: 131-140,1986; Burdick et al. , Thro
mbosis Res. 45: 413-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1
991); including but not limited to the assay described in Schaub, Prostaglandins 35: 467-474, 1988.

Receptor / Ligand Activity The protein of the present invention may also exhibit activity as a receptor, receptor ligand or inhibitor or agonist of receptor / ligand interaction. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cells-
Receptors involved in cell interactions and their ligands (such as cell adhesion molecules (selectins, integrins and their ligands)) and receptors / ligands involved in antigen presentation, antigen recognition, development of cellular and humoral immune responses But not limited thereto. Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors for related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interaction. The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for receptor-ligand activity are described in Current Protocols in Immunology, Ed by JE Coligan, AM Kruisbeek, D.
H. Margulies, EM Shevach, W. Strober, Pub. Greene Publishing Associ
ates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhes
ion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. A
cad. Sci. USA 84: 6864-6868, 1987; Bierer et al., J. Exp. Med. 168: 1145.
-1156, 1988; Rosenstein et al., J. Exp.Med. 169: 149-160 1989; Stolte
nborg et al., J. Immunol.Methods 175: 59-68, 1994; Stitt et al., Cell 8
0: 661-670, 1995, including, but not limited to.

Anti-inflammatory activity The protein of the present invention can exhibit anti-inflammatory activity. Anti-inflammatory activity involves stimulating cells that are involved in the stimulus response, resulting in cell-cell interactions (eg, attachment).
By inhibiting or promoting, thereby inhibiting or promoting the chemotaxis of cells involved in the inflammatory process, thereby inhibiting or promoting cell extravasation,
Alternatively, it is exerted by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Symptoms of inflammation (
Includes chronic or acute symptoms, infection-related inflammation (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, lethal endotoxin injury, arthritis, complement mediated Super-acute rejection,
Nephritis, lung injury induced by cytokines or chemokines, inflammatory bowel disease, Crohn's disease or diseases resulting from overproduction of cytokines such as TNF or IL-1). . The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials.

Cadherin / Tumor Inhibition Inhibitory Activity Cadherin is a calcium-dependent adhesion molecule and appears to play a major role during development, particularly in determining specific cell types. Loss or alteration of normal cadherin expression can induce changes to cell adhesion properties associated with tumor growth and metastasis. Cadherin dysfunction has also been implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (autoimmune cutaneous bullous disease), Crohn's disease, and some developmental abnormalities. The cadherin superfamily contains over 40 members, each with a different expression pattern. All members of the superfamily share common conserved extracellular repeats (cadherin domains), but show structural differences in other parts of the molecule. Calcium is necessary for their attachment, since the cadherin domains bind to calcium to form their tertiary structure. Since the small number of amino acids in the first cadherin domain provides the basis for homophilic attachment, modification of this recognition site can alter the specificity of cadherin, thereby recognizing only itself Instead, the mutant molecule will be able to bind to different cadherins. In addition, some cadherins are involved in heterophilic attachment with other cadherins. E-cadherin, a member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in the tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of a polynucleotide expressing E-cadhedrin into a cancer cell line can reverse the altered cell morphology, restore cell-to-cell and adherence to other substrates, reduce cell growth rates, By dramatically reducing the growth of dependent cells,
The cancer-related changes were reversed. Thus, reintroducing E-cadhedrin expression returns the carcinoma to a less advanced stage. Other cadherins may have the same inhibitory role in carcinomas from other tissue types. Therefore, cancer can be treated using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Introducing such proteins or polynucleotides into cancer cells may reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells are also known to express cadherins of different tissue types than originally, so these cancer cells can invade different tissues and metastasize. The protein of the present invention having cadherin activity, and the polynucleotide of the present invention encoding such a protein replaces cadherin inappropriately expressed in these cells, restores normal cell adhesion properties, and reduces the tendency of cells to metastasize. Or can be removed. Furthermore, an antibody that recognizes and binds to cadherin can be obtained using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Such antibodies can also be used to block the attachment of inappropriately expressed tumor cells cadherin, so that the cells do not form tumors elsewhere. Such anti-cadhedrin antibodies can be used as markers for cancer grade, pathological type, and prognosis. That is, as the cancer progresses, the expression of cadherin decreases, and by using a cadherin-binding antibody, the decrease of cadherin expression can be detected. Using a fragment of a protein of the present invention having cadherin activity, preferably a decapeptide at the cadherin recognition site, and a polynucleotide of the present invention encoding such a protein fragment, to bind to cadherin to prevent cadherin binding that produces undesirable effects. Can also block cadherin function. Further, using a fragment of the protein of the present invention having cadherin activity, preferably a truncated soluble cadherin fragment known to be stable in the circulatory system of cancer patients, and a polynucleotide encoding such a protein fragment, Correct cell-cell attachment can also be disrupted. Assays for cadherin adhesion and entry inhibitory activity are described in Hortsch et al. J Bio
l Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552
, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.

Tumor Inhibitory Activity In addition to the activities described above for the immunological treatment or prevention of tumors, the proteins of the present invention may exhibit other antitumor activities. Certain proteins directly or indirectly affect tumor growth (eg,
(Via ADCC). Certain proteins act on tumor tissue or progenitor tissue to inhibit the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) and to inhibit tumor growth. Tumor inhibitory activity may be exhibited by causing the production of a factor, agent or cell type, or by removing or inhibiting a factor, agent or cell type that promotes tumor growth.

Other Activities The proteins of the present invention may exhibit one or more of the following additional activities or effects: infectious agents, including but not limited to bacteria, viruses, fungi, and other parasites. Killing; height, weight, hair color, eye color, skin or other tissue pigmentation, or the size or form of an organ or body part (eg, breast augmentation or vice versa)
Effects on body characteristics (suppression or promotion); effects on digestion of consumed fat, protein or carbohydrates; appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) Effects on behavioral characteristics, including but not limited to, violent behavior; providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormones or endocrine Activity; for enzymes,
Correcting enzyme deficiency and treating related disorders; treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement);
And the ability to act as an antigen in a vaccine composition to generate an immune response to such proteins or other substances or entities that cross-react with such proteins.

Administration and Dose The protein of the present invention (which may be from any source, including but not limited to recombinant and non-recombinant methods) is mixed with a pharmaceutically acceptable carrier. May be used in pharmaceutical compositions. Such compositions may contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable"
A non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the present invention comprises M-CSF, GM-CSF, T
NF, 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, TNF1, TNF2, G-CSF
, Meg-CSF, thrombopoietin, stem cell factor, and cytokines such as erythropoietin, lymphokines, or other hematopoietic factors. The pharmaceutical composition may further contain other agents that enhance protein activity or supplement its activity or utility in therapy. Such additional factors and / or agents may be included in a pharmaceutical composition to exert a synergistic effect with the protein of the invention or to minimize side effects. Conversely, specific cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or antithrombotic factors, or by incorporating the protein of the present invention into the formulation of anti-inflammatory agents, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or Side effects of antithrombotic factors or anti-inflammatory agents may be minimized. A protein of the invention may be active in multimers (eg, heterodimers or homodimers) or in complexes with itself or other proteins. Consequently, the pharmaceutical composition of the present invention may contain such a multimeric or complexed form of the protein of the present invention. The pharmaceutical composition of the present invention may be in the form of a complex of the protein of the present invention and a protein or peptide antigen. Protein and / or peptide antigens can be
It will deliver a stimulus signal to the lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins.
MHC and structurally related proteins, including those encoded by the host cell's class I and class II MHC genes, will serve to present peptide antigens to T lymphocytes. The antigen component is supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, antibodies capable of binding to surface immunoglobulins and other molecules on B cells can be mixed with the pharmaceutical compositions of the present invention. The pharmaceutical composition of the present invention may be in the form of a liposome, in which the protein of the present invention further comprises another pharmaceutically acceptable carrier, an amphipathic agent such as a lipid (in the form of an aggregate such as micelles in an aqueous solution). , Present as an insoluble monolayer, liquid crystal or lamellar layer). Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatide, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposome formulations is within the level of ordinary skill in the art and is described, for example, in US Pat.
And 4737323, all of which are herein incorporated by reference. As used herein, the term "therapeutically effective amount" refers to the amount of each active ingredient of a pharmaceutical composition or method that is sufficient to show significant patient benefit, e.g., amelioration of symptoms of such symptoms, healing, increased rate of healing. Means the total amount. When used for an individual active ingredient administered alone, the term refers only to that ingredient. When used in a combination of active ingredients, it refers to the total amount of active ingredients that produces a therapeutic effect regardless of sequential or simultaneous administration. In practicing the therapeutic methods of the invention, a therapeutically effective amount of a protein of the invention is administered to a mammal having the condition to be treated. According to the method of the invention, the protein of the invention may be administered alone or with other therapeutic agents, such as cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the proteins of the invention may be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors. If administered sequentially, the attending physician will determine the appropriate order of administration for combinations of the proteins of the present invention with cytokines, lymphokines, other hematopoietic, thrombolytic or antithrombotic factors. The administration of the protein of the present invention in the pharmaceutical composition of the present invention or used in the practice of the present invention can be performed by various conventional methods, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. Intravenous injection into a patient is preferred. When a therapeutically effective amount of a protein of the invention is administered orally, the protein of the invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5 to 95% of a protein of the present invention;
It preferably contains about 25-90% of the protein of the invention. When administered in liquid form, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. Liquid form pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of the protein of the present invention, preferably about 1 to 50% of the protein of the present invention. When administering a therapeutically effective amount of a protein of the invention intravenously, intradermally or subcutaneously,
The protein of the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection include, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride for injection, and Ringer's solution containing lactic acid for injection, Or it should contain other carriers known in the art. The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of the protein of the invention in the pharmaceutical composition of the invention depends on the nature and severity of the condition to be treated,
As well as the nature of the treatment the patient has already received. Ultimately, your doctor
The amount of the protein of the invention to be treated for each patient will be determined. First, the attending physician administers a low dose of the protein of the invention and observes the patient's response. Higher doses of the protein of the invention may be administered until the optimal therapeutic effect is obtained, with no further increase in the dose at the time the optimal therapeutic effect is obtained. Various pharmaceutical compositions for carrying out the method of the present invention contain about 0.01 μg to about 100 mg of the protein of the present invention (preferably,
It should contain from about 0.1 μg to about 10 mg per kg body weight, more preferably from 0.1 μg to about 1 mg per kg body weight. The duration of treatment by intravenous administration using the compositions of the present invention will vary with the severity of the disease to be treated and the condition and response of each patient. It is believed that the duration of each application of the protein of the invention ranges from 12 to 24 hours for continuous intravenous administration. Ultimately, the attending physician will decide the duration of intravenous treatment using the pharmaceutical composition of the present invention.

An animal may be immunized using the protein of the present invention to obtain polyclonal and monoclonal antibodies that specifically react with the protein of the present invention. Such antibodies may be obtained using whole proteins or fragments thereof as immunogens. The peptide immunogen may further include a cysteine residue at the carboxy terminus and binds to haptens such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, RP Merrifield, J. Amer. Che.
m. Soc. 85, 2149-2154 (1963); JL Krstenansky, et. al., FEBS Lett. 211
, 10 (1987). A monoclonal antibody that binds to the protein of the present invention can be a useful diagnostic agent for immunodetection of the protein of the present invention. Neutralizing antibodies that bind to the protein of the present invention can be useful as therapeutics for conditions related to the protein of the present invention,
Further, it may be useful in the treatment of certain tumors in the treatment of some forms of cancer in which aberrant expression of the protein of the invention is involved. For cancer cells or white blood cells,
Neutralizing monoclonal antibodies against the protein of the present invention may be useful for detecting and preventing metastatic spread of cancer cells mediated by the protein of the present invention. For compositions of the invention useful for regenerating bone, cartilage, tendons or ligaments, the method of treatment comprises:
It involves administering the composition locally, systemically, or locally, such as an implant or device. When administered, the therapeutic composition used in the present invention is, of course, in a pyrogen-free, physiologically acceptable form. In addition, if desired, the composition may be encapsulated or injected in a viscous form and delivered to the site of bone, cartilage or tissue damage. Topical administration is suitable for wound healing and tissue repair. Therapeutically useful agents other than the protein of the present invention, which may be contained in the above composition, may be alternatively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Preferably, for bone and / or cartilage formation, the composition delivers the protein-containing composition to the site of bone and / or cartilage damage, provides a structure for bone and cartilage development, and more optimally. Will contain a matrix that can be absorbed into the body. Such matrices may be made of materials currently used for other implanted medical devices. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The appropriate formulation will be determined by the particular application of the composition. Possible matrices for the composition may be biodegradable and chemically known calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydride. Other usable materials are biodegradable and biologically well known, such as bone or skin collagen. Further, the matrix may contain pure proteins or extracellular matrix components.
Other possible matrices are not biodegradable, such as sintered hydroxyapatite, bioglass, aluminate, or other ceramics, but are chemically known. The matrix may comprise a combination of materials of the above type, for example polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be modified in the composition, for example in calcium-aluminate-phosphate, and processed to pore size, particle size,
The particle shape and biodegradability may be varied. A 50:50 (molar weight) copolymer of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150 to 800 microns is presently preferred. In some applications, it may be useful to use a sequestering agent, such as carboxymethylcellulose or an autologous clot, to prevent dissociation of the protein composition from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose; Most preferred are the cationic salts of (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestering agent useful in the present invention is from 0.5 to 20% by weight, preferably from 1 to 10% by weight, based on the total formulation weight, and this amount prevents desorption of proteins from the polymer matrix. An amount that allows for proper handling of the composition, but not too much so that the progenitor cells infiltrate the matrix, thereby conferring the protein with the opportunity to promote the osteogenic activity of the progenitor cells. Not to be. In a further composition, the protein of the present invention may be mixed with other agents that are beneficial in treating bone and / or cartilage defects, wounds, or tissues. These agents include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). At present, the therapeutic compositions are also valuable for veterinary use. For details,
In addition to humans, livestock and thoroughbred horses are desirable patients for such treatments using the proteins of the present invention. The rules of administration of the protein-containing pharmaceutical composition used for tissue regeneration may include various factors that alter the action of the protein, such as the tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of the wound, The physician will decide on the type of tissue required (eg, bone), patient age, gender, and diet, severity of infection, duration of administration, and other clinical factors. Dosages may vary depending on the type of matrix used for reconstitution and the inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin-like growth factor I), to the final composition can also affect the dose. Progress can be monitored by periodically assessing tissue / bone growth and / or repair, for example, by X-ray, histomorphological examination and tetracycline labeling. The polynucleotide of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells in vivo or ex vivo and expressed in a mammalian subject. The polynucleotides of the present invention may be administered by other known methods for introducing nucleic acids into cells or organisms, including but not limited to viral vectors or in the form of naked DNA. ). The cells may be cultured and grown ex vivo in the presence of the protein of the present invention, or may have a desired effect on such cells, or may have a desired activity in such cells. The treated cells can then be introduced in vivo for therapeutic purposes. The patents and publications cited herein are considered to be entirely incorporated herein by reference.

[0033]

[Sequence list]

[Brief description of the drawings]

FIG. 1A is a schematic representation of the pED6 vector used for depositing the clones disclosed herein.

FIG. 1B shows the pNOTs used to deposit the clones disclosed herein.
1 schematically shows a vector.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 7/04 A61P 9/10 4H045 9/10 17/00 17/00 19/00 19/00 25/00 25/00 25/06 25/06 25/28 25/28 29/00 29/00 35/00 35/00 37/04 37/04 37/06 37/06 43/00 105 43/00 105 C07K 14 / 47 C07K 14/47 A61K 37/02 C12N 5/10 C12N 5/00 B 15/09 ZNA 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD) , TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, Z ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU , CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, AZ, VN, YU, ZW (72) John M. McCoy, Inventor, United States 01867 Massachusetts, Reading, 56 Howard Street 113 Harvard, Ann Lee Road (72) Inventor Lisa A Collins-Lacy School Street 124, Acton, MA 01720 Massachusetts USA (72) Inventor David Marberg Orchard Drive 2, Acton, MA 01720 Massachusetts, USA No. (72) Inventor Cheryl Evans Bent Willow Circle 18801 F-term (Reference) 4B024 AA01 BA21 BA80 CA04 DA03 EA04 FA17 GA11 4B064 AG01 AG02 CA10 CA19 CC24 DA01 4B065 AA93X AA93Y CA24 CA44 4C084 AA02 AA06 AA07 AA13 BA01 BA02 BA08 BA19 BA20 BA22 BA23 CA53 CA56 DA01 NA14 ZA012 ZA512 ZA532 ZA542 ZA862 ZA942 ZA962 ZA972 ZB072 ZB112 ZB212 ZB262 ZB312 ZC612 4C086 AA01 ZA01 ZA12 ZA12 ZA12 ZB31 ZC61 4H045 AA10 AA30 BA10 CA42 DA01 EA20 FA74

Claims (24)

[Claims] 1. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 24 to nucleotide 548 of SEQ ID NO: 1; A polynucleotide comprising the nucleotide sequence from nucleotide 525 to nucleotide 548; (d) a polynucleotide comprising the nucleotide sequence from nucleotide 115 to nucleotide 317 of SEQ ID NO: 1; A polynucleotide comprising the nucleotide sequence of a protein coding sequence; (f) the cD of clone B196 deposited under accession number ATCC 98021
A polynucleotide encoding the full length protein encoded by the NA insert; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone B196 deposited under accession number ATCC 98021; CD of clone B196
A polynucleotide encoding the mature protein encoded by the NA insert; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2; (j) a biologically active SEQ ID NO: 2 (K) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); (1) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); And (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (j). A composition comprising an isolated polynucleotide of choice. 2. The composition of claim 1, wherein said polynucleotide is operably linked to at least one expression control sequence. 3. A host cell transformed with the composition of claim 2. 4. The host cell according to claim 3, wherein said cell is a mammalian cell. 5. The encoded by the composition of claim 2, comprising: (a) growing the culture of the host cell of claim 3 in a suitable medium; and (b) purifying the protein from the culture. Method for producing protein. 6. A protein produced by the method of claim 5. 7. The protein of claim 6, comprising a mature protein. 8. A composition comprising a protein comprising an amino acid sequence selected from the group consisting of: a protein substantially free of other mammalian proteins: (a) the amino acid of SEQ ID NO: 2 (B) the amino acid sequence from amino acid 118 to amino acid 162 of SEQ ID NO: 2; (c) a fragment of the amino acid sequence of SEQ ID NO: 2; and (d) the cD of clone B196 deposited under accession number ATCC 98021.
Amino acid sequence encoded by NA insert. 9. The composition according to claim 8, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2. 10. The protein according to claim 1, wherein the protein is from amino acid 118 to amino acid 16 of SEQ ID NO: 2.
9. The composition of claim 8, comprising up to two amino acid sequences. 11. The composition of claim 8, further comprising a pharmaceutically acceptable carrier. 12. A method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the composition of claim 11. 13. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 14. The composition of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1. 15. The composition of claim 1, wherein said polynucleotide comprises a nucleotide sequence from nucleotide 24 to nucleotide 548 of SEQ ID NO: 1. 16. The composition of claim 1, wherein the polynucleotide comprises the nucleotide sequence of the full-length protein coding sequence of clone B196 deposited under accession number ATCC 98021. 17. The composition of claim 1, wherein the polynucleotide encodes a full-length protein encoded by the cDNA insert of clone B196 deposited under accession number ATCC 98021. 18. The composition of claim 1, wherein the polynucleotide comprises the nucleotide sequence of the mature protein coding sequence of clone B196 deposited under accession number ATCC 98021. 19. The composition of claim 1, wherein the polynucleotide encodes a mature protein encoded by the cDNA insert of clone B196 deposited under accession number ATCC 98021. 20. The composition of claim 1, wherein the polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 2. 21. The polynucleotide, wherein the polynucleotide has biological activity.
2. The composition of claim 1, which encodes a protein comprising a fragment of the amino acid sequence of 22. The composition of claim 8, wherein the protein comprises a biologically active fragment of the amino acid sequence of SEQ ID NO: 2. 23. Clone B wherein the protein was deposited under accession number 98021
9. The composition of claim 8, comprising an amino acid sequence encoded by the 196 cDNA insert. 24. The polynucleotide comprises nucleotide 525 of SEQ ID NO: 1.
2. The composition of claim 1, comprising the nucleotide sequence of from 1 to 548.