WO1998011217A2 - HUMAN PROTEINS HAVING SECRETORY SIGNAL SEQUENCES AND DNAs ENCODING THESE PROTEINS - Google Patents
HUMAN PROTEINS HAVING SECRETORY SIGNAL SEQUENCES AND DNAs ENCODING THESE PROTEINS Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- the present invention relates to human proteins having secretory signal sequences and DNAs encoding these proteins .
- the proteins of the present invention can be used as pharmaceuticals or as antigens for preparing antibodies against said proteins .
- the cDNAs of the present invention can be used as probes for the gene diagnosis and gene sources for the gene therapy. Furthermore, the cDNAs can be used as gene sources for large-scale production of the proteins encoded by said cDNAs .
- such a secretory protein has been obtained by a method comprising the isolation and purification of the target protein from a large amount of the blood or a cell culture supernatant by using the biological activity as an indicator, determination of its primary structure followed by cloning of the corresponding cDNA on the basis of the information on the thus-obtained amino acid sequence, and production of the reco binant protein using said cDNA.
- the contents of the secretory proteins are generally so low that the isolation and purification are difficult in many cases.
- secretory proteins and type-I membrane proteins possess hydrophobic sequences, defined as the secretory signal sequences, consisting of about 20 amino acid residues at the amino acid termini (the N-termini). Therefore, the cloning of genes encoding the secretory proteins or type-I membrane proteins is expected to be performed by using the presence or the absence of these secretory signal sequences as indicators.
- the object of the present invention is to provide novel human proteins having secretory signal sequences and DNAs encoding said proteins .
- the present inventors were successful in cloning of cDNAs having secretory signal sequences from a human full-length cDNA bank, thereby completing the present invention. That is to say, the present invention provides proteins containing any of the amino acid sequences represented by Sequence No. 1 to Sequence No. 9 that are human proteins having secretory signal sequences. The present invention, also, provides DNAs encoding said proteins exemplified as cDNAs containing any of the base sequences represented by Sequence No. 10 to sequence No. 18.
- Each of the proteins of the present invention can be obtained, for example, by a method for isolation from human organs, cell lines, etc, a method for preparation of the peptide by the chemical synthesis on the basis of the amino acid sequence of the present invention, or a method for production with the recombinant DNA technology using the DNA encoding the human secretory protein of the present invention, wherein the method for obtainment by the recombinant DNA technology is employed preferably.
- an in vitro expression can be achieved by preparation of an RNA by the in vitro transcription from a vector having a cDNA of the present invention, followed by the in vitro translation using this RNA as a template.
- the recombination of the translation domain to a suitable expression vector by the method known in the art leads to the expression of a large amount of the encoded protein by using Escherichia coll , Bacillus subtilis, yeasts, animal cells, and so on.
- a protein of the present invention is expressed by a microorganism such as Escherichia coli
- the translation region of a cDNA of the present invention is constructed in an expression vector having an origin, a promoter, ribosome-binding site(s), cDNA-cloning site(s), a terminator, etc.
- a maturation protein can be obtained by performing the expression with inserting an initiation codon in the translation region where the secretary signal sequence is removed.
- a fusion protein with another protein can be expressed. Only a protein portion encoding said cDNA can be obtained by cleavage of said fusion protein with an appropriate protease.
- the protein of the present invention can be secretory-produced as a maturation protein outside the cells, when the translation region of said cDNA is subjected to recombination to an expression vector for animal cells that has a promoter for the animal cells, a splicing domain, a poly(A) addition site, etc., followed by transfection into the animal cells.
- the proteins of the present invention include peptide fragments (more than 5 amino acid residues) containing any partial amino acid sequence of the amino acid sequences represented by Sequence No. 1 to Sequence No. 9. These fragments can be used as antigens for preparation of the antibodies.
- the proteins of the present invention are secreted in the form of maturation proteins outside the cells, after the signal sequences are removed. Therefore, these maturation proteins shall come within the scope of the present invention.
- the N-terminal amino acid sequences of the maturation proteins can be easily identified by using the method for the cleavage-site determination in a signal sequence [Japanese Patent Kokai Publication No. 1996-187100]. Furthermore, many secretory proteins are subjected to the processing after the secretion to be converted to the active forms .
- activated proteins or peptides shall come within the scope of the present invention.
- glycosylation sites are present in the amino acid sequences, expression in appropriate animal cells affords glycosylated proteins. Therefore, these glycosylated proteins or peptides also shall come within the scope of the present invention.
- the DNAs of the present invention include all DNAs encoding the above-mentioned proteins . Said DNAs can be obtained using the method by chemical synthesis, the method by cDNA cloning, and so on.
- Each of the cDNAs of the present invention can be cloned from, for example, a cDNA library of the human cell origin.
- the cDNA is synthesized using as a template a poly(A) RNA extracted from human cells.
- the human cells may be cells delivered from the human body, for example, by the operation or may be the culture cells.
- the cDNA can be synthesized by using any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol . Cell. Biol . 2: 161-170 (1982)], the Gubler-Hoffman method [Gubler, U. and Hoffman, J.
- the primary selection of a cDNA encoding a human protein having a secretory signal sequence is performed by the sequencing of a partial base sequence of the cDNA clone selected at random from the cDNA library, sequencing of the amino acid sequence encoded by the base sequence, and recognition of the presence or absence of hydrophobic site(s) in the resulting N-terminal amino acid sequence region.
- the secondary selection is carried out by determination of the whole base sequence by the sequencing and the protein expression by the in vitro translation.
- the ascertainment of the cDNA of the present invention for encoding the protein having the secretory signal sequence is performed by using the signal sequence detection method fYokoya a-Kobayashi , M.
- the ascertainment for the coding portion of the inserted cDNA fragment to function as a signal sequence is provided by fusing a cDNA fragment encoding the N-terminus of the target protein with a cDNA encoding the protease domain of urokinase and then expressing the resulting cDNA in COS7 cells to detect the urokinase activity in the cell culture medium.
- the cDNAs of the present invention are characterized by containing any of the base sequences represented by Sequence No. 10 to Sequence No. 18 or any of the base sequences represented by Sequence No. 19 to Sequence No. 27.
- Table 1 summarizes the clone number (HP number), the cells affording the cDNA, the total base number of the cDNA, and the number of the amino acid residues of the encoded protein, for each of the cDNAs .
- the same clone as any of the cDNAs of the present invention can be easily obtained by screening of the cDNA library constructed from the cell line or the human tissue employed in the present invention, by the use of an oligonucleotide probe synthesized on the basis of the corresponding cDNA base sequence depicted in Sequence No. 19 to Sequence No. 27.
- any cDNA that is subjected to insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides in Sequence No. 10 to Sequence No. 27 shall come within the scope of the present invention.
- any protein that is produced by these modifications comprising insertion or deletion of one or plural nucleotides and/or substitution with other nucleotides shall come within the scope of the present invention, as far as said protein possesses the activity of the corresponding protein having the amino acid sequence represented by Sequence No. 1 to Sequence No. 9.
- the cDNAs of the present invention include cDNA fragments (more than 10 bp) containing any partial base sequence of the base sequence represented by Sequence No. 10 to No. 18 or of the base sequence represented by Sequence No. 19 to No. 27.
- the portion encoding the secretory signal sequence can be employed as means to secrete an optionally selected protein outside the cells by fusing with a cDNA encoding another protein.
- DNA fragments consisting of a sense chain and an anti-sense chain shall come within this scope. These DNA fragments can be used as the probes for the gene diagnosis.
- Figure 1 A figure depicting the structure of the secretory signal sequence detection vector pSSD3.
- Figure 2 A figure depicting the construction of the secretory signal sequence - the urokinase fusion gene.
- Figure 3 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP00685.
- Figure 4 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP00714.
- Figure 5 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP00876.
- Figure 6 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP01134.
- Figure 7 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10029.
- Figure 8 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10189.
- Figure 9 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10269.
- Figure 10 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10298.
- Figure 11 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10368.
- the present invention is embodied in more detail by the following examples, but this embodiment is not intended to restrict the present invention.
- the basic operations and the enzyme reactions with regard to the DNA recombination are carried out according to the literature ["Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, 1989]. Unless otherwise stated, restrictive enzymes and a variety of modification enzymes to be used were those available from Takara Shuzo Co., Ltd. The manufacturer's instructions were used for the buffer compositions as well as for the reaction conditions, in each of the enzyme reactions.
- the cDNA synthesis was carried out according to the literature [Kato, S. et al., Gene 150: 243-250 (1994)].
- the fibrosarcoma cell line HT-1080 (ATCC CCL 121), the epidermoid carcinoma cell line KB (ATCC CRL 17), the histiocyte ly phoma cell line U937 (ATCC CRL 1593) stimulated by phorbol esters, tissues of stomach cancer delivered by the operation, and liver were used for human cells to extract mRNAs.
- Each of the cell lines was cultured by a conventional procedure .
- the product was dissolved in a mixed solution of 50 mM Tris-hydrochloric acid buffer solution (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol , and 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP), mixed with 200 units of a reverse transferase (GIBCO-BRL) , and the resulting solution at a total volume of 20 ⁇ l was allowed to react at 42°C for one hour.
- the thus- obtained pellets were dissolved in a mixed solution of 50 mM Tris-hydrochloric acid buffer solution (pH 7.5), 100 mM NaCl, 10 mM MgCl 2 , and 1 mM dithiothreitol. Thereto were added 100 units of EcoRI and the resulting solution at a total volume of 20 ⁇ l was allowed to react at 37 °C for one hour.
- the reaction solution underwent the phenol extraction followed by the ethanol precipitation, the obtained pellets were dissolved in a mixed solution of 20 mM Tris-hydrochloric acid buffer solution (pH 7.5), 100 mM KCl, 4 mM MgCl 2 , 10 mM (NH ⁇ ) 2 S0 ⁇ , and 50 ⁇ g/ l bovine serum albumin. Thereto were added 60 units of Escherichia coli DNA ligase and the resulting solution was allowed to react at 16°C for 16 hours.
- the cDNA-synthesis reaction solution was used to transform Escherichia coli DH12S (GIBCO-BRL) .
- the transformation was carried out by the electroporation method. A portion of the transformant was inoculated on a 2xYT agar culture medium containing 100 ⁇ g/ l ampicillin, which was incubated at 37 °C overnight. A colony grown on the culture medium was randomly picked up and inoculated on 2 ml of the 2xYT culture medium containing 100 ⁇ g/ml ampicillin, which was incubated at 37 °C overnight. The culture medium was centrifuged to separate the cells, from which a plasmid DNA was prepared by the alkaline lysis method.
- the product was subjected to 0.8% agarose gel electrophoresis to determine the size of the cDNA insert.
- the sequence reaction using Ml3 universal primer labeled with a fluorescent dye and Taq polymerase was carried out and the product was analyzed by a fluorescent DNA-sequencer (Applied Biosystems Inc.) to determine the base sequence of the cDNA 5 '-terminal of about 400 bp.
- the sequence data were filed as a homo-protein cDNA bank data base.
- the base sequence registered in the homo-protein cDNA bank was converted to three frames of amino acid sequences and the presence or absence of an open reading frame (ORF) beginning from the initiation codon. Then, the selection was made for the presence of a signal sequence that is characteristic to a secretory protein at the N-terminal of the portion encoded by ORF . These clones were sequenced from the both 5 ' and 3 ' directions by using the deletion method to determine the whole base sequence.
- the hydrophobicity/hydrophilicity profiles were obtained for proteins encoded by ORF by the Kyte-Doolittle method [Kyte, J. & Doolittle, R. F., J. Mol . Bio.
- Two oligo DNA linkers LI ( 5 ' -GATCCCGGGTCACGTGGGAT-3 ' ) and L2 (5'-ATCCCACGTGACCCGG-3' ) , were synthesized and phosphorylated by T4 polynucleotide kinase . After annealing of the both linkers, followed by ligation with the previously-prepared pSSDl fragment by T4 DNA ligase, Escherichia coli JM109 was transformed. A plasmid pSSD3 was prepared from the transformant and the objective recombinant was confirmed by the determination of the base sequence of the linker-inserted fragment.
- Figure 1 illustrates the structure of the thus-obtained plasmid.
- the present plasmid vector carries three types of blunt-end formation restriction enzyme sites, Smal, P aCI, and EcoRV. Since these cleavage sites are positioned in succession at an interval of 7 bp, selection of an appropriate site in combination of three types of frames for the inserting cDNA allows to construct a vector expressing a fusion protein.
- Hindlll Digestion with Hindlll was further carried out and a DNA fragment containing the SV40 promoter and a cDNA encoding the secretory sequence at the downstream from the promoter was separated by agarose gel electrophoresis . This fragment was inserted between the pSSD3 Hindlll site and a restriction enzyme site selected so as to match with the urokinase-coding frame, thereby constructing a vector expressing a fusion protein of the secretory signal portion of the target cDNA and the urokinase protease domain (refer to Figure 2).
- Escherichia coli (host: JM109) bearing the fusion- protein expression vector was incubated at 37 °C for 2 hours in 2 ml of the 2xYT culture medium containing 100 ⁇ g/ml ampicillin, the helper phage M13K07 (50 ⁇ l ) was added and the incubation was continued at 37°C overnight.
- a supernatant separated by centrifugation underwent precipitation with polyethylene glycol to obtain single-stranded phage particles. These particles were suspended in 100 ⁇ l of 1 mM Tris-0.1 mM EDTA, pH 8 (TE) .
- the simian-kidney-origin culture cells, C0S7 were incubated at 37 °C in the presence of 5% C0 2 in the Dulbecco's modified Eagle's culture medium (DMEM) containing 10% fetal calf albumin.
- DMEM Dulbecco's modified Eagle's culture medium
- the cell surface was washed with a phosphate buffer solution and then washed again with DMEM containing 50 mM Tris- hydrochloric acid (pH 7.5) (TDMEM) .
- TMEM Tris- hydrochloric acid
- Table 2 shows the restriction enzyme site used for cutting off the cDNA fragment from each clone, the restriction enzyme site used for cleavage of pSSD3, and the presence or absence of a clear circle. Except for pSSD3 used as the control, each of the samples formed a clear circle to identify that urokinase was secreted in the culture medium. That is to say, it is indicated that each of the cDNA fragments codes for the amino acid sequence that functions as the secretory signal sequence . Table 2
- the plasmid vector carrying the cDNA of the present invention was utilized for the in vitro transcription/translation by the T N T rabbit reticulocyte lysate kit (Promega Biotec). In this case, [ 35S]methionine was added and the expression product was labeled with the radioisotope. All reactions were carried out by following the protocols attached to the kit.
- Two micrograms of the plasmid was allowed to react at 30°C for 90 minutes in total 25 ml of a reaction solution containing 12.5 ⁇ l of the T N T rabbit reticulocyte lysate, 0.5 ⁇ l of the buffer solution (attached to the kit), 2 ⁇ l of an amino acid mixture (methionine-free) , 2 ⁇ l (0.37 MBq/ ⁇ l) of [ 35S]methionine (Amersha Corporation),
- GenBank using the base sequence of the present cDNA revealed that any EST possessing the homology of 90% or more was not found.
- GenBank using the base sequence of the present cDNA revealed that there existed some ESTs possessing the homology of 90% or more and containing the initiation codon (for example, Accession No. F3872), but any of the sequences thereof did not allow to predict the present protein.
- Reticulocalbin is a protein localized on the membrane surface of the endoplasmic reticulum and has been considered to participate in the protein folding. Accordingly, the protein of the present invention is considered to be applicable to the folding process of recombinant proteins.
- Table 5 indicates the comparison of the amino acid sequences between the human protein of the present invention (HP) and the rattlesnake lectin (CL) (Swiss-PROT Accession No. P21963).
- HP human protein of the present invention
- CL rattlesnake lectin
- - represents a gap
- * represents an amino acid residue identical to that in the protein of the present invention
- . represents an amino acid residue analogous to that in the protein of the present invention.
- the both proteins possessed a homology of 35.3%.
- GenBank using the base sequence of the present cDNA revealed that any EST possessing the homology of 90% or more was not found .
- a plasmid pET876 was prepared from the transfor ant and the objective recombinant was confirmed from the restriction enzyme cleavage map .
- the present expression vector expresses a protein in which methionine-alanine was inserted before a protein starting from serine at position 29 in the protein encoded by the clone HP00876.
- a suspension of pET876/BL21 (DE3) in 5 ml of the LB culture medium containing 100 ⁇ g/ml ampicillin was incubated in a shaker at 37 °C and isopropylthiogalactoside was added to make 1 mM when A 600 reached to about 0.5. After the incubation was continued at 37 °C for 6 hours, cells were collected by centrifugation and suspended in 25 ml of a column buffer solution for the amylose column (10 mM Tris- hydrochloric acid, pH 7.4, 200 mM NaCl, and 1 mM EDTA). The resulting suspension was sonicated and then the insoluble fraction was subjected to SDS-polyacrylamide electrophoresis to identify a band originating from the expression of the present vector at a position of about 14 kDa.
- lectins Since lectins recognize and then bind to sugar chains, lectins are useful as sugar-chain detection reagents and as affinity carriers for purification of glycoproteins . In addition, extracellular secretory lectins play important roles also in intercellular signal transduction and thereby are useful as medicines.
- Determination of the whole base sequence for the cDNA insert of clone HP01134 obtained from the human liver cDNA libraries revealed the structure consisting of a 5 ' -non- translation region of 116 bp, an ORF of 1131 bp, and a 3'- non-translation region of 502 bp.
- the ORF codes for a protein consisting of 376 amino acid residues with a hydrophobic region of a putative secretory signal sequence at the N- terminal.
- Figure 6 depicts the hydrophobicity/hydrophilicity profile of the present protein obtained by the Kyte-Doolittle method.
- Table 6 indicates the comparison of the amino acid sequences between the human protein of the present invention (HP) and the tangerine cysteine proteinase (CP) (GenBank Accession No. Z47793).
- HP human protein of the present invention
- CP tangerine cysteine proteinase
- - represents a gap
- * represents an amino acid residue identical to that in the protein of the present invention
- . represents an amino acid residue analogous to that in the protein of the present invention.
- the both proteins possessed a homology of 49% among the N-terminal region of 286 amino acid residues.
- the search of the protein data base using the amino acid sequence of the present protein revealed that the protein was not homologous with any of known proteins.
- GenBank using the base sequence revealed that there existed some ESTs possessing the homology of 90% or more (for example, Accession No. H87021), but they were shorter than the present cDNA and any molecule containing the initiation codon was not identified.
- the search of the protein data base using the amino acid sequence of the present protein revealed that the protein was not homologous with any of known proteins.
- GenBank using the base sequence revealed that there existed some ESTs possessing the homology of 90% or more and containing the initiation codon (for example, Accession No. N56270), but a frame shift had occurred and the same ORF as that in the present cDNA was not identified.
- laminin As an extracellular matrix, laminin deeply participates in the proliferation and differentiation of cells. Accordingly, laminin has been employed as an additive for the cell culture and so on.
- Determination of the whole base sequence for the cDNA insert of clone HP10298 obtained from the human stomach cancer cDNA libraries revealed the structure consisting of a 5 '-non-translation region of 137 bp, an ORF of 369 bp, and a 3 ' -non-translation region of 580 bp.
- the ORF codes for a protein consisting of 122 amino acid residues with a hydrophobic region of a putative secretory signal sequence at the N-terminal.
- Figure 10 depicts the hydrophobicity/hydrophilicity profile of the present protein obtained by the Kyte-Doolittle method.
- Determination of the whole base sequence for the cDNA insert of clone HP10368 obtained from the human stomach cancer cDNA libraries revealed the structure consisting of a 5 '-non-translation region of 72 bp, an ORF of 528 bp, and a 3 '-non-translation region of 266 bp.
- the ORF codes for a protein consisting of 175 amino acid residues with a hydrophobic region of a putative secretory signal sequence at the N-terminal.
- Figure 11 depicts the hydrophobicity/hydrophilicity profile of the present protein obtained by the Kyte-Doolittle method.
- the present invention provides human proteins having secretory signal sequences and cDNAs encoding said proteins. All of the proteins of the present invention are putative proteins controlling the proliferation and differentiation of the cells, because said proteins are secreted outside the cells and exist in the extracellular liquid or on the cell membrane surface. Therefore, the proteins of the present invention can be used as pharmaceuticals or as antigens for preparing antibodies against said proteins. Furthermore, said DNAs can be used for the expression of large amounts of said proteins .
- polynucleotides and proteins of the present invention may exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
- Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA) .
- the polynucleotides provided by the present invention can be used by the research community for various purposes .
- the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodiesusing DNA immunization techniques
- the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
- the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al . , Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
- the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
- the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
- the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
- Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements . Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
- the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
- the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
- Cytokine and Cell Proliferation/DifferentiationActivity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
- the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9 , B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5 , DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. 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; Bertagnolli et al . , J. Immunol.
- Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Po lyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
- Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 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 .
- Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. 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. Sci.
- a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
- a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
- These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial orfungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses , mycobacteria, Leishmania spp . , malaria spp . and various fungal infections such as candidiasis .
- a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
- Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
- a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
- Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention .
- T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
- Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
- Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
- Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7 ) ) , e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
- B lymphocyte antigen functions such as , for example, B7
- GVHD graft-versus-host disease
- blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
- rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant .
- a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody
- B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
- Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant .
- the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
- Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
- the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
- appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
- murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
- Blocking antigen function may also be therapeutically useful for treating autoimmune diseases .
- Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases .
- Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
- Administration of reagents which block costimulation of T cells by disrupting receptor : ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.
- blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
- the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
- Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the commoncold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
- anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
- Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
- the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
- up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
- Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
- a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
- tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/or B7-3-like activity.
- the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
- gene therapy techniques can be used to target a tumor cell for transfection in vivo.
- tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class
- Il chain protein and an MHC class Il ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
- Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.
- a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
- the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. 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 et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
- T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
- MLR Mixed lymphocyte reaction
- Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al . , Journal of Virology 67:4062-4069, 1993; Huang et al .
- lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al . , Cancer Research 53:1945-1951, 1993; Itoh et al .
- Assays for proteins that influence early steps of T-cell commitment and development include,without limitation, those described in: Antica et al . , Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al . , Blood 85:2770-2778, 1995; Toki et al . , Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
- a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
- erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and onocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for proliferation and differentiation of various hematopoietic lines are cited above.
- Assays for embryonic stem cell differentiation include, without limitation, those 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 . , Blood 81:2903-2915, 1993.
- Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al . eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hiraya a et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K.
- a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
- a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals .
- Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
- a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
- a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc. ) mediated by inflammatory processes .
- tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
- a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
- Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
- compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
- the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
- the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
- the compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
- the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
- Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
- a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium) , muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.
- organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
- muscle smooth, skeletal or cardiac
- vascular including vascular endothelium
- a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
- a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
- Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps . 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
- a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
- FSH follicle stimulating hormone
- the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example. United States Patent 4,798,885.
- a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs .
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for activin/inhibin activity include, without limitation, those described in: Vale et al . , Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
- a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
- Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
- Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
- a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
- the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell che otaxis .
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
- Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al . J. Clin. Invest.
- a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
- a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al . , J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al . , Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
- a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
- receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
- Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
- a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
- the activity of a protein of the invention may, among other means, be measured by the following methods:
- Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al . , Proc. Natl. Acad. 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; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994? Stitt et al . , Cell 80:661-670, 1995.
- Proteins of the present invention may also exhibit anti-inflammatory activity.
- the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
- Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of ytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
- a protein of the invention may exhibit other anti-tumor activities.
- a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
- a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis ) , by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth
- a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolis , processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component ( s ) ; effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing
- Sequence No. 1 Sequence length: 154 Sequence type: Amino acid Topology: Linear Sequence kind: Protein Hypothetical : No Original source:
- Organism species Homo sapiens Cell kind: Fibrosarcoma Cell line: HT-1080 Clone name: HP00658 Sequence description Met Lys Val Ser Ala Ala Ala Leu Ala Val He Leu He Ala Thr Ala
- Sequence No. 2 Sequence length: 315 Sequence type: Amino acid Topology: Linear Sequence kind: Protein Hypothetical : No Original source:
- Organism species Homo sapiens Cell kind: Epidermoid carcinoma Cell line: KB Clone name: HP00714 Sequence description Met Asp Leu Arg Gin Phe Leu Met Cys Leu Ser Leu Cys Thr Ala Phe
- Organism species Homo sapiens Cell kind: Stomach cancer Clone name: HP00876 Sequence description Met Ala Ser Arg Ser Met Arg Leu Leu Leu Leu Leu Leu Ser Cys Leu Ala
- Sequence No. 4 Sequence length: 376 Sequence type: Amino acid Topology: Linear Sequence kind: Protein Hypothetical : No Original source:
- Organism species Homo sapiens Cell kind: Liver Clone name: HP01134 Sequence description Met Val Trp Lys Val Ala Val Phe Leu Ser Val Ala Leu Gly He Gly
- Sequence No . 5 Sequence length: 173 Sequence type: Amino acid Topology : Linear Sequence kind: Protein Hypothetical : No Original source:
- Organism species Homo sapiens
- Sequence No. 6 Sequence length: 73 Sequence type : Amino acid Topology : Linear Sequence kind: Protein Hypothetical: No Original source :
- Organism species Homo sapiens
- Organism species Homo sapiens
- Sequence No. 9 Sequence length: 175 Sequence type: Amino acid Topology : Linear Sequence kind: Protein Hypothetical : No Original source:
- Organism species Homo sapiens Cell kind: Stomach cancer Clone name: HP10368 Sequence description Met Glu Lys He Pro Val Ser Ala Phe Leu Leu Leu Val Ala Leu Ser
- Organism species Homo sapiens
- Organism species Homo sapiens
- Sequence No. 12 Sequence length: 474 Sequence type: Nucleic acid Strandedness : Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens
- GAGTACATAA GTGGCTATCA GAGAAGCCAG CCGATATGGA TTGGCCTGCA CGACCCACAG 300
- Organism species Homo sapiens
- Organism species Homo sapiens Cell kind: Epidermoid carcinoma Cell line: KB Clone name: HP10029 Sequence description
- Sequence No. 15 Sequence length: 219 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens
- Organism species Homo sapiens
- Organism species Homo sapiens Cell kind: Stomach cancer Clone name: HP10298
- Sequence No. 18 Sequence length: 525 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens
- Organism species Homo sapiens
- Sequence No. 20 Sequence length: 3311 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens Cell kind: Epidermoid carcinoma Cell line: KB Clone name: HP00714 Sequence characteristics: Code representing characteristics: CDS Existence site: 57.. 1004 Characterization method: E Sequence description
- AAA TTT GCA CAA AAG CGC TGG ATT TAC GAG GAT GTA GAG CGA CAG TGG 395 Lys Phe Ala Gin Lys Arg Trp He Tyr Glu Asp Val Glu Arg Gin Trp
- AAA AAT GCC ACC TAC GGC TAC GTT TTA GAT GAT CCA GAT CCT GAT GAT 491 Lys Asn Ala Thr Tyr Gly Tyr Val Leu Asp Asp Pro Asp Pro Asp Asp 130 135 140 145
- GAG CCA GAA TGG GTA AAG ACA GAG CGA GAG CAG TTT GTT GAG TTT CGG 779 Glu Pro Glu Trp Val Lys Thr Glu Arg Glu Gin Phe Val Glu Phe Arg 230 235 240 GAT AAG AAC CGT GAT GGG AAG ATG GAC AAG GAA GAG ACC AAA GAC TGG 827 Asp Lys Asn Arg Asp Gly Lys Met Asp Lys Glu Glu Thr Lys Asp Trp
- ATATGTATAT ATAACCTTTA TTATTGCTAT
- ATCTTTGTGG ATCTTTGTGG ATAATACATT CAGGTGGTGC 2700
- TTTCCTGCCC TCTGGGTTCC CCATTTTTAC TATTAAGAAG ACCAGTGATA ATTTAATAAT 2940
- Sequence No. 21 Sequence length: 1152 Sequence type : Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens Cell kind: Stomach cancer Clone name: HP00876 Sequence characteristics: Code representing characteristics: CDS Existence site: 147.. 623 Characterization method: E Sequence description
- Sequence No. 22 Sequence length: 1749 Sequence type: Nucleic acid Strandedness : Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens Cell kind: Liver Clone name: HP01134 Sequence characteristics: Code representing characteristics: CDS Existence site: 117.. 1247 Characterization method: E Sequence description
- GGC AAA GTC CTG AAG AGT GGC CCC CAG GAT CAC GTG TTC ATT TAC TTC 551 Gly Lys Val Leu Lys Ser Gly Pro Gin Asp His Val Phe He Tyr Phe 130 135 140 145
- Organism species Homo sapiens
- TGT ATG TTC ACT TAC GCC TCT CAA GGA GGG ACC AAT GAG CAA TGG CAG 242 Cys Met Phe Thr Tyr Ala Ser Gin Gly Gly Thr Asn Glu Gin Trp Gin
- GTG ACC AAA ACA GCA GTG GCT CAC AGG CCC GGG GCA TTC AAA GCT GAG 482 Val Thr Lys Thr Ala Val Ala His Arg Pro Gly Ala Phe Lys Ala Glu 145 150 155 CTG TCC AAG CTG GTG ATT GTG GCC AAG GCA TCG CGC ACT GAG CTG 527 Leu Ser Lys Leu Val He Val Ala Lys Ala Ser Arg Thr Glu Leu
- Sequence No. 24 Sequence length: 390 Sequence type : Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source :
- Organism species Homo sapiens
- Sequence No. 25 Sequence length: 4667 Sequence type: Nucleic acid Strandedness: Double Topology: Linear Sequence kind: cDNA to mRNA Original source:
- Organism species Homo sapiens
- CATTTAGTTA CTCTGCTCAT TTCTCTTAAG CTTTCCTTGG ATGAGTTGAG CTTTGAATCC 60 TTCCTGATGA ACCTTGCCTT TTAAGGATCC TCCAAATGCC CCAAGAAGCT GGGATTTTTC 120 ATTTTTTTTT TCACTGGGGA GGGGAATGGT GCTTTCCAGG GTCCTGGATG TTTGAGTCTT 180 CTCACCTTCC AGCCCGGTGA TATGTCTGGA GCTTTAACTC TCTATATAAG CCCTAATCTT 240 TGTGTTCTCT GCCTGATCTT CTGTCTGGGG TGGTCCAGGT CACAAGAAGA AGCTGACCCC 300 TGCTGGCTTT GGGAAAATGC TGAGTTCATT GCCTGGCACA AATGCAAGGG CCCTTCCCCA 360 CCCTGTGAAT TCTGGTCTCT GATGATCACT TACATGTGCC TTGTGCTTTC TGTTTGAGGG 420 GCCCCTTGCA GCCCACAG
- GGT AGA CTC CGC AAT GCC ACC GCC AGC CTG TGG TCA GGG CCT GGG CTG 2598 Gly Arg Leu Arg Asn Ala Thr Ala Ser Leu Trp Ser Gly Pro Gly Leu 600 605 610 615
- GAC AGC CGG AGA GAG GCA GAG AGG CTG GTG CGG CAG GCG GGA GGA GGA 3030 Asp Ser Arg Arg Glu Ala Glu Arg Leu Val Arg Gin Ala Gly Gly Gly
Abstract
Description
Claims
Priority Applications (4)
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EP97940374A EP0932676A2 (en) | 1996-09-13 | 1997-09-12 | HUMAN PROTEINS HAVING SECRETORY SIGNAL SEQUENCES AND DNAs ENCODING THESE PROTEINS |
CA002265923A CA2265923A1 (en) | 1996-09-13 | 1997-09-12 | Human proteins having secretory signal sequences and dnas encoding these proteins |
AU42207/97A AU4220797A (en) | 1996-09-13 | 1997-09-12 | Human proteins having secretory signal sequences and DNAs encoding these prot eins |
JP51350998A JP2001506484A (en) | 1996-09-13 | 1997-09-12 | Human protein having secretory signal sequence and DNA encoding the same |
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EP (1) | EP0932676A2 (en) |
AU (1) | AU4220797A (en) |
CA (1) | CA2265923A1 (en) |
WO (1) | WO1998011217A2 (en) |
Cited By (14)
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WO1998028423A2 (en) * | 1996-12-20 | 1998-07-02 | Board Of Regents, The University Of Texas System | Compositions and methods of use for osteoclast inhibitory factors |
WO1998041627A1 (en) * | 1997-03-19 | 1998-09-24 | Zymogenetics, Inc. | Secreted polypeptides with homology to xenopus cement gland proteins |
EP0929575A1 (en) * | 1996-08-23 | 1999-07-21 | Human Genome Sciences, Inc. | Novel human growth factors |
WO2000066731A2 (en) * | 1999-04-30 | 2000-11-09 | Biostatum, Inc. | Recombinant laminin 5 |
US6171816B1 (en) | 1996-08-23 | 2001-01-09 | Human Genome Sciences, Inc. | Human XAG-1 polynucleotides and polypeptides |
US6235477B1 (en) | 1997-08-08 | 2001-05-22 | Incyte Pharmaceuticals, Inc. | Human reticulocalbin isoforms |
EP1117833A1 (en) * | 1998-10-02 | 2001-07-25 | Diadexus LLC | A novel method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers |
WO2002014368A2 (en) * | 2000-08-16 | 2002-02-21 | Curagen Corporation | Proteins and nucleic acids encoding the same |
WO2002085937A2 (en) * | 2001-04-23 | 2002-10-31 | B.R.A.H.M.S Aktiengesselschaft | Inflammation-specific peptides and the uses thereof |
US6703363B1 (en) | 1999-04-30 | 2004-03-09 | Biostratum, Inc. | Recombinant laminin 5 |
US6962779B1 (en) | 1998-10-02 | 2005-11-08 | Diadexus, Inc. | Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers |
WO2007139972A2 (en) * | 2006-05-25 | 2007-12-06 | Wyeth | Expression of the cysteine protease legumain in vascular and inflammatory diseases |
WO2014111458A3 (en) * | 2013-01-17 | 2014-09-12 | Medizinische Hochschule Hannover | Factor 1 protein, factor 2 protein and inhibitors thereof for use in treating or preventing diseases |
WO2021148411A1 (en) * | 2020-01-21 | 2021-07-29 | Boehringer Ingelheim International Gmbh | Myeloid-derived growth factor for use in treating or preventing fibrosis, hypertrophy or heart failure |
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WO1995004158A1 (en) * | 1993-07-29 | 1995-02-09 | The Upjohn Company | Use of heparanase to identify and isolate anti-heparanase compound |
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- 1997-09-12 AU AU42207/97A patent/AU4220797A/en not_active Abandoned
- 1997-09-12 CA CA002265923A patent/CA2265923A1/en not_active Abandoned
- 1997-09-12 EP EP97940374A patent/EP0932676A2/en not_active Withdrawn
- 1997-09-12 WO PCT/JP1997/003239 patent/WO1998011217A2/en not_active Application Discontinuation
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WO1995004158A1 (en) * | 1993-07-29 | 1995-02-09 | The Upjohn Company | Use of heparanase to identify and isolate anti-heparanase compound |
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Cited By (27)
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EP0929575A4 (en) * | 1996-08-23 | 2004-09-08 | Human Genome Sciences Inc | Novel human growth factors |
EP0929575A1 (en) * | 1996-08-23 | 1999-07-21 | Human Genome Sciences, Inc. | Novel human growth factors |
US7611846B2 (en) | 1996-08-23 | 2009-11-03 | Human Genome Sciences, Inc. | Diagnostic methods involving human growth factor huXAG-1 |
US6171816B1 (en) | 1996-08-23 | 2001-01-09 | Human Genome Sciences, Inc. | Human XAG-1 polynucleotides and polypeptides |
US7060801B2 (en) | 1996-08-23 | 2006-06-13 | Human Genome Sciences, Inc. | Antibodies to human growth factor huXAG-3 and methods of use |
US6818412B2 (en) | 1996-08-23 | 2004-11-16 | Human Genome Sciences, Inc. | Human growth factors |
WO1998028423A3 (en) * | 1996-12-20 | 1998-09-03 | Univ Texas | Compositions and methods of use for osteoclast inhibitory factors |
WO1998028423A2 (en) * | 1996-12-20 | 1998-07-02 | Board Of Regents, The University Of Texas System | Compositions and methods of use for osteoclast inhibitory factors |
WO1998041627A1 (en) * | 1997-03-19 | 1998-09-24 | Zymogenetics, Inc. | Secreted polypeptides with homology to xenopus cement gland proteins |
US6235477B1 (en) | 1997-08-08 | 2001-05-22 | Incyte Pharmaceuticals, Inc. | Human reticulocalbin isoforms |
EP1117833A1 (en) * | 1998-10-02 | 2001-07-25 | Diadexus LLC | A novel method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers |
EP1117833A4 (en) * | 1998-10-02 | 2002-08-14 | Diadexus Inc | A novel method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers |
US6962779B1 (en) | 1998-10-02 | 2005-11-08 | Diadexus, Inc. | Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancers |
WO2000066731A3 (en) * | 1999-04-30 | 2001-06-28 | Biostatum Inc | Recombinant laminin 5 |
WO2000066731A2 (en) * | 1999-04-30 | 2000-11-09 | Biostatum, Inc. | Recombinant laminin 5 |
US6703363B1 (en) | 1999-04-30 | 2004-03-09 | Biostratum, Inc. | Recombinant laminin 5 |
WO2002014368A2 (en) * | 2000-08-16 | 2002-02-21 | Curagen Corporation | Proteins and nucleic acids encoding the same |
WO2002014368A3 (en) * | 2000-08-16 | 2003-09-25 | Curagen Corp | Proteins and nucleic acids encoding the same |
WO2002085937A3 (en) * | 2001-04-23 | 2002-12-19 | B R A H M S Aktiengesselschaft | Inflammation-specific peptides and the uses thereof |
WO2002085937A2 (en) * | 2001-04-23 | 2002-10-31 | B.R.A.H.M.S Aktiengesselschaft | Inflammation-specific peptides and the uses thereof |
US7153662B2 (en) | 2001-04-23 | 2006-12-26 | B.R.A.H.M.S. Aktiengesellschaft | Inflammation-specific peptides and the uses thereof |
WO2007139972A2 (en) * | 2006-05-25 | 2007-12-06 | Wyeth | Expression of the cysteine protease legumain in vascular and inflammatory diseases |
WO2007139972A3 (en) * | 2006-05-25 | 2008-01-24 | Wyeth Corp | Expression of the cysteine protease legumain in vascular and inflammatory diseases |
WO2014111458A3 (en) * | 2013-01-17 | 2014-09-12 | Medizinische Hochschule Hannover | Factor 1 protein, factor 2 protein and inhibitors thereof for use in treating or preventing diseases |
US10369198B2 (en) | 2013-01-17 | 2019-08-06 | Medizinische Hochschule Hannover | Factor 1 protein, factor 2 protein and inhibitors thereof for use in treating or preventing diseases |
EP3747457A3 (en) * | 2013-01-17 | 2021-03-03 | Medizinische Hochschule Hannover | Factor 1 protein, factor 2 protein and inhibitors thereof for use in treating or preventing diseases |
WO2021148411A1 (en) * | 2020-01-21 | 2021-07-29 | Boehringer Ingelheim International Gmbh | Myeloid-derived growth factor for use in treating or preventing fibrosis, hypertrophy or heart failure |
Also Published As
Publication number | Publication date |
---|---|
CA2265923A1 (en) | 1998-03-19 |
EP0932676A2 (en) | 1999-08-04 |
AU4220797A (en) | 1998-04-02 |
WO1998011217A3 (en) | 1998-07-16 |
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