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Cytokine-like proteins

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20120264143 patent thumbnailZoom

Cytokine-like proteins


A full-length cDNA corresponding to an EST (AA418955), which does not show any homology to other proteins in the database but has a weak homology to G-CSF, has been successfully isolated by synthesizing primers based on the EST sequence, and effecting PCR-cloning from a human fetal spleen library. Sequencing of the thus-isolated cDNA and analysis of its structure revealed that the cDNA has typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. It is also found out that the culture supernatant of said sequence-transfected CHO cells shows a proliferation supporting activity towards bone marrow cells in the coexistence of kit ligand.
Related Terms: Spleen

Inventor: Yuichi Hirata
USPTO Applicaton #: #20120264143 - Class: 435 792 (USPTO) - 10/18/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Assay In Which An Enzyme Present Is A Label >Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20120264143, Cytokine-like proteins.

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/830,043, filed on Jul. 2, 2010, which is a divisional of U.S. application Ser. No. 11/716,808, filed on Mar. 12, 2007, which is a divisional of U.S. application Ser. No. 10/440,066, filed May 15, 2003, issued as U.S. Pat. No. 7,252,967, which is a application of U.S. application Ser. No. 09/687,637, filed Oct. 13, 2000, issued as U.S. Pat. No. 6,610,285, which is a continuation-in-part of International Patent Application No. PCT/JP99/01997, filed Apr. 14, 1999, and claims priority from Japanese Patent Application No. 10-121805, filed Apr. 14, 1998. The disclosures of the foregoing applications are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a novel cytokine-like protein and the encoding gene.

BACKGROUND OF THE INVENTION

Cytokines are multi-functional cell growth/differentiation inducing factors controlling immune and hematopoietic reactions. The series of factors composing cytokines, are mainly produced by activated T cells, macrophages, or stromal cells and connect the cells of the lymphoid system and the hematopoietic system in a network, regulating the proliferation, differentiation, and functions of these cells. So far, a number of factors have been isolated as cytokines and apart from the factors themselves, antibodies and receptor molecules of those factors, or antibodies against those receptors, have been developed as therapeutic drugs and are in actual use.

For example, G-CSF, which has a neutrophil-proliferating function, is already in use as a drug for many diseases and leukopenia resulting from the treatment of these diseases (K. Welte et al., the first 10 years Blood Sep. 15 1996; 88:1907-1929 and also refer GENDAIKAGAKU ZOUKAN no. 18, Cytokine, edited by TOSHIAKI OHSAWA, 1990, published by TOKYO KAGAKU DOUJIN). Furthermore, an antibody against the receptor of IL-6, which acts in immune functions and inflammation, is being developed as a potential therapeutic drug against rheumatism and leukemia.

SUMMARY

OF THE INVENTION

The present invention provides a novel cytokine-like protein and the encoding DNA. It also provides a vector into which the DNA has been inserted, a transformant carrying the DNA, and also a method for producing a recombinant protein using the transformant. Furthermore, screening methods for a compound, which binds to the protein and regulates the activity, and the uses of the protein and the compounds regulating its function as pharmaceutical drugs, are also provided by the present invention.

Most cytokines known so far, have a conserved characteristic such as a WS Motif (Idzerda, R L et al., J Exp Med 1990 Mar. 1; 171 (3) 861-873), and form a super-family of cytokine receptors. Although the cytokine itself, which is the ligand, does not have a conserved characteristic or homology compared to the receptor, some groups have an extremely weak homology, hinting of a close stereoscopic structure. EPO/TPO family and IL-6/G-CSF/MGF family can be taken as examples. The present inventors, thinking that unknown, yet unidentified genes may exist in these families, attempted to isolate an unknown cytokine belonging to these families.

Specifically, it was found that the EST (AA418955) sequence, which does not show any homology to other proteins in the Database, has a weak homology to G-SCF and constructed primers based on that sequence, and did PCR cloning from a human fetal spleen library. As a result, the present inventors succeeded in isolating a full-length cDNA corresponding to the EST (this clone was named SGRF). Also, the isolated SGRF cDNA was sequenced and the structure analyzed to find that the isolated cDNA has the typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. Furthermore, the inventors analyzed the activity of the SGRF protein to find that the culture supernatant of SGRF-transfected CHO cells has a proliferation supporting activity towards specific bone marrow cells in the presence of mouse kit ligand. The isolated SGRF protein itself may be applicable for the prevention and treatment of diseases of the lymphoid and hematopoietic systems and for diseases related to defective cell growth. Also it is possible to use this protein for the screening of other factors related to the lymphoid and hematopoietic systems and as a drug candidate compound for diseases of those systems.

Namely, this invention relates to a novel cytokine-like protein SGRF and the encoding gene, their production as well as the use of the protein in the screening of drugs and drug candidate compounds. More specifically,

1. a protein comprising the amino acid sequence of SEQ ID NO:1, or said sequence in which one or more amino acids are replaced, deleted, added, and/or inserted,

2. a protein encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2, which is functionally equivalent to the protein having the amino acid sequence of SEQ ID NO:1,

3. a DNA encoding the protein of (1) or (2),

4. the DNA of (3), which contains the coding region of the nucleotide sequence of SEQ ID NO:2,

5. a vector in which the DNA of (3) or (4) is inserted,

6. a transformant carrying, in an expressible manner, the DNA of (3) or (4),

7. a method for producing the protein of (1) or (2), which comprises the culturing of the transformant of (6),

8. a method for screening a compound which can bind to the protein of (1) or (2), the method comprising the steps of:

(a) exposing a test sample to the protein of (1) or (2) or its partial peptide;

(b) detecting the binding activity between the test compound and said protein or its partial peptide; and

(c) selecting a compound having a binding activity to said protein,

9. a compound which can bind to the protein of (1) or (2),

10. the compound of (9) which is obtainable by the method of (8),

11. a method for screening a compound which can promote or inhibit activity of the protein of (1) or (2), the method comprising the steps of:

(a) exposing the protein of (1) or (2) and the kit ligand to mammalian bone marrow cells under the absence of a test compound;

(b) detecting the proliferation of said bone marrow cells; and

(c) selecting a compound which promotes or inhibits the proliferation of bone marrow cells in comparison with the assay under the presence of the test sample,

12. the method of (11), wherein the bone marrow cells are Lin negative, Sca-1 positive, c-kit positive, and CD34 positive,

13. a compound which promotes or inhibits the activity of the protein of (1) or (2),

14. the compound of (13) which is obtainable by the method of (11) or (12),

15. a pharmaceutical composition comprising the protein of (1) or (2) as an active component,

16. a promoter or inhibitor of the protein of (1) or (2) wherein the active component is the compound of (13) or (14),

17. an antibody which can bind to the protein of (1) or (2), and

18. a DNA comprising at least 15 nucleotides, which can specifically hybridize with the DNA comprising the nucleotide sequence of SEQ ID NO:2.

The present invention relates to a novel cytokine-like protein. The nucleotide sequence of the cDNA encoding the protein named “SGRF”, which is included in the protein of the present invention is shown in SEQ ID NO: 2; the amino-acid sequence of said protein in SEQ ID NO:1.

So far, in mammals, IL-6 and G-CSF have been reported as factors thought to belong to the IL-6/G-CSF/MGF family. The “SGRF” cDNA isolated by the present inventors, had in its 3′ non-coding region, four mRNA destabilizing sequences (Lagnando C A, Brown C Y, Goodall G J (1994) Mol. Cell. Biol. 14, 7984-7995) called ARE (AT Rich element), often seen in cytokine mRNAs. The consensus sequence preserved in the IL-6/G-CSF/MGF family was also roughly maintained (FIG. 3). From these facts, “SGRF” can be assumed to be a novel factor belonging to the IL-6/G-CSF/MGF family.

The “SGRF” expression in human normal tissue as detected by northern-blot analysis is extremely localized, and was seen in the testis, lymph nodes, and thymus, being not present in a detectable level in other tissues (FIG. 4). Even in tissues where expression was detected, the expression level was assumed to be very low. An EST (U38443), a partial fraction of “SGRF”, which is normally hardly expressed, is reportedly induced following activation in a T cell-line (Jurkat) (Yatindra Prashar, Sherman M. Weissman (1996) Proc. Natl. Acad. Sci. USA 93:659-663). From this fact and from the results of northern-blot analysis, it can be assumed that in vivo, “SGRF” is mainly expressed in activated T cells.

Furthermore, the culture supernatant of “SGRF” transfected CHO cells showed an activity, which supported the proliferation of bone marrow cells (FIG. 12), in the presence of the kit ligand.

The characteristics of “SGRF” such as those above, suggest that it is a kind of a typical interleukin. “SGRF”, as are most cytokines isolated so far, is thought to be involved in the lymphoid and hematopoietic systems. Therefore, it can be applied as a therapeutic or preventive drug in diseases of the lymphoid and hematopoietic systems, and also in diseases associated with defects in cell proliferation.

The protein of the present invention can be prepared by methods commonly known to one skilled in the art, as a recombinant protein made using genetic engineering techniques, and also as a natural protein. For example, a recombinant protein can be prepared by, inserting DNA encoding the protein of the present invention (for example, DNA comprising the nucleotide sequence in SEQ ID NO:1) into a suitable expression vector, introducing this into a host cell, and purifying the protein from the resulting transformant or the culture supernatant. The natural protein can be prepared by immobilizing in a column, antibodies taken from immunizing a small animal with the recombinant protein, and performing affinity chromatography for extracts of tissues or cells (for example, testis, lymph nodes, thymus, etc.) expressing the protein of the present invention.

Also, this invention features a protein, which is functionally equivalent to the “SGRF” protein (SEQ ID NO:1). The method of inserting a mutation into the amino acids of a protein is a well-known method for isolating such proteins. In other words, for a person skilled in the art, the preparation of a protein functionally equivalent to the “SGRF” protein, is something which can be generally done using various methods such as the PCR-mediated, site-specific-mutation-induction system (GIBCO-BRL, Gaithersburg, Md.), oligonucleotide-mediated, site-specific-mutation-induction method (Kramer, W. and Fritz, H J (1987) Methods in Enzymol., 154:350-367) suitably replacing amino acids in the “SGRF”protein shown in SEQ ID NO:1, which do not influence the function. Mutations of amino acids can occur spontaneously as well. Therefore, the protein of the invention includes those proteins that are functionally equivalent to the “SGRF” protein, having an amino acid sequence in which one or more amino acids in the amino acid sequence of the “SGRF” protein (SEQ ID NO:1) have been replaced, deleted, added, and/or inserted. The term “functionally equivalent” as used herein, refers to a protein having a cytokine activity equivalent to that of the “SGRF” protein. The cytokine activity of the “SGRF” protein includes, for example, a proliferation-supporting activity (Example 11) towards cells which are Lin negative, Sca-1 positive and c-kit positive.

The number of amino acids that are mutated is not particularly restricted, as long as a cytokine activity equivalent to that of the “SGRF” protein is maintained. Normally, it is within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids and even more preferably within 5 amino acids. The site of mutation may be any site, as long as a cytokine activity equivalent to that of the “SGRF” protein is maintained.

A “conservative amino acid substitution” is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In the present invention, the protein having numerous depletions in the amino acid sequence of the “SGRF” protein (SEQ ID NO:1) includes a partial peptide. The partial peptide includes, for example, a protein of which the signal peptide has been excluded from the “SGRF” protein of SEQ ID NO:1.

Also, a fusion protein can be given as a protein comprising the amino acid sequence of the “SGRF” protein and several amino acids added thereto. Fusion proteins are, for example, fusions of the above described proteins and other peptides or proteins, and are included in the present invention. Fusion proteins can be made by techniques commonly known to a person skilled in the art, such as linking the DNA encoding the protein of the invention and with DNA encoding other peptides or proteins, so as the frames match, inserting this into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the protein of the present invention.

Commonly known peptides, for example, FLAG (Hopp, T. P. et al., Biotechnology (1988) 6:1204-1210), 6×His constituting six H is (histidine) residues, 10×His, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment can be used as peptides that are fused to the protein of the present invention. Examples of proteins that are fused to protein of the invention are, GST (glutathione-S-transferase), HA (Influenza agglutinin), Immunoglobulin constant region, β-galactosidase and MBP (maltose-binding protein). Fusion proteins can be prepared by fusing commercially available DNA encoding these peptides or proteins with the DNA encoding the protein of the present invention and expressing the fused DNA prepared.

The hybridization technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to one skilled in the art as an alternative method for isolating a protein functionally equivalent to the “SGRF” protein (SEQ ID NO:1). In other words, for a person skilled in the art, it is a general procedure to prepare a protein functionally equivalent to the “SGRF” protein, by isolating DNA having a high homology with the whole or part of the DNA encoding the “SGRF” protein used as a base for the preparation of the protein. Therefore, the protein of the present invention also includes proteins, which are functionally equivalent to the “SGRF” protein and are encoded by DNA hybridizing with DNA encoding the “SGRF” protein. The term “functionally equivalent” as used herein, means as mentioned above, proteins that show a cytokine activity equivalent to that of the “SGRF” protein. Apart from humans, for example, mice, rats, cows, monkeys and pigs can be used as animals from which functionally equivalent proteins can be isolated. One skilled in the art can suitably select the stringency of hybridization for isolating DNA encoding a functionally equivalent protein, but normally, it is equilibrium hybridization at about 42° C., 2×SSC, 0.1% SDS (low stringency); about 50° C., 2×SSC, 0.1% SDS (medium stringency); or about 65° C., 2×SSC, 0.1% SDS (high stringency). If washings are required to reach equilibrium, then the washings are performed using the same buffer as the original hybridization solution, a listed above. In general, the higher the temperature, the higher is the homology of the DNA obtainable. “High homology” refers to, in comparison with the amino acid sequences of the “SGRF” protein, normally a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 95% or higher. The homology of a protein can be determined by following the algorithm in Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80:726-730.

The “percent identity” of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the)(BLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

This invention also relates to a DNA encoding the above protein. There is no particular restriction as to the DNA of the present invention as long as it encodes the protein of the present invention and includes cDNA, genomic DNA and chemically synthesized DNA. cDNA can be prepared by, making a primer using the nucleotide sequence of the “SGRF” cDNA, disclosed in SEQ ID NO:2, and performing RT-PCR using the mRNA prepared from cells expressing the “SGRF” protein as the template. In the case of genomic DNA, preparation can be done by the plaque hybridization method using a genomic DNA inserted λ phage library and the cDNA probe obtained. The nucleotide sequence of the DNA acquired can be decided by ordinary methods in the art using the commercially available “dye terminator sequencing kit” (Applied Biosystems). The DNA of the present invention, as stated later, can be utilized for the production of a recombinant protein and gene therapy.

An “isolated nucleic acid” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.

The term “substantially pure” as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The present invention also features a vector into which the DNA of the present invention has been inserted. There is no restriction as to the vector to which DNA is inserted, and various vectors such as those for expressing the protein of the present invention in vivo and those for preparing the recombinant protein can be used according to the objective. To express the protein of the present invention in vivo (especially for gene therapy), various viral vectors and non-viral vectors can be used. Examples of viral vectors are, adenovirus vectors (pAdexLcw and such) and retrovirus vectors (pZlPneo and such). Expression vectors are especially useful when using vectors for the objective of producing the protein of the invention. For example, when using Escherichia coli the “pQE vector” (Qiagen, Hilden, Germany), when using yeast “SP-Q01” (Stratagene, La Jolla, Calif.), when using insect cells “Bac-to-Bac baculovirus expression system” (GIBCO-BRL, Gaithersburg, Md.) are highly appropriate, but there is no restriction. Also, when using mammalian cells such as CHO cells, COS cells, NIH3T3 cells, for example, the “LacSwitch II expression system (Stratagene, La Jolla, Calif.) is highly suitable, but there is no restriction. Insertion of the DNA of the present invention into a vector can be done using ordinary methods in the art.

The present invention also refers to a transformant, carrying, in an expressible manner, the DNA of the present invention. The transformant of the present invention includes, those carrying the above-mentioned vector into which DNA of the present invention has been inserted, and those host genomes into which the DNA of the present invention has been integrated. As long as the DNA of the present invention is maintained in an expressible manner, no distinction is made as to the form of existence of the transformants. There is no particular restriction as to the cells into which the vector is inserted. When using the cells to express the protein of the present invention for the purpose of gene therapy by the ex vivo method, various cells (for example, various cells of the immune system) can be used as target cells according to the type of disease. Also, when the purpose is to produce the protein of the present invention, for example, E. coli, yeast, animal cells and insect cells can be used in combination with the vectors that are utilized. Introduction of a vector into a cell can be done using commonly known methods such as electroporation and calcium phosphate method.

The separation and purification of the recombinant protein from the transformant made to produce the protein can be done using ordinary methods. The recombinant protein can be purified and prepared by, for example, ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography where an antibody against the protein of the present invention has been immobilized in the column, or by combining two or more of these columns. Also when expressing the protein of the present invention inside host cells (for example, animal cells and E. coli) as a fusion protein with glutathione-S-transferase protein or as a recombinant protein supplemented with multiple histidines, the expressed recombinant protein can be purified using a glutathione column or nickel column. After purifying the fusion protein, it is also possible to exclude regions other than the objective protein by cutting with thrombin or factor-Xa as required.



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stats Patent Info
Application #
US 20120264143 A1
Publish Date
10/18/2012
Document #
13336560
File Date
12/23/2011
USPTO Class
435/792
Other USPTO Classes
530350, 435 29, 536 235, 4353201, 43525233, 4352542, 435325, 435348, 435 691, 5303879
International Class
/
Drawings
12


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