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Cytokine-like proteins




Title: Cytokine-like proteins.
Abstract: 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. ...


USPTO Applicaton #: #20120264143
Inventors: Yuichi Hirata


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

CROSS-REFERENCE TO RELATED APPLICATIONS

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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

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

BACKGROUND

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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

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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.




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stats Patent Info
Application #
US 20120264143 A1
Publish Date
10/18/2012
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
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Drawings
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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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20121018|20120264143|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 |
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