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Novel proteins, gene encoding the same and method of utilization thereof

a technology of gene encoding and protein, applied in the direction of peptide/protein ingredients, dna/rna fragmentation, depsipeptides, etc., can solve the problems of ostalgia as a side effect, large pain and burden on patients and physicians, and the risk of unknown side effects

Inactive Publication Date: 2008-02-28
SHA SHIKEN +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach enables the production of G-CSF without the need for frequent drug administration, reducing pain and burden, and potentially minimizing side effects by using antibodies to stimulate neutrophil production, providing a more effective and safer treatment for neutropenia.

Problems solved by technology

Because of its low stability in the blood, however, it requires frequent administration, and because its administration is limited to the intravenous route, this has resulted in a great deal of pain and burden to the patient and physician.
Furthermore, administration of G-CSF as a drug has been reported to cause ostalgia as a side-effect.
The alternative option of direct administration of macrophages or stroma cells that produce G-CSF will produce the risk of unknown side-effects since the cells contain numerous proteins and other substances, and therefore such treatment has not been practiced.
Because administration of G-CSF itself for differentiation and proliferation of neutrophils provoke ostalgia as a side-effect, and it also requires frequent administration and increases the pain and burden to the patient and physician, it has been strongly desired to develop an alternative treatment method; however, no such method has yet been established.
However, the antigens recognized by the G-CSF inducing antibodies have not yet been discovered.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Antigen Gene Recognized by the Monoclonal Antibody from Macrophage Cell Line

[0132] (1) Preparation of poly(A)+ RNA from Macrophage Cells (RAW264.7)

[0133] Guanidium thiocyanate-phenol-chloroform single-step extraction (Laboratory Manuals of Genetic Engineering, 3rd Edition, pp. 83-84, 1996) was used to prepare approximately 0.3 mg of total mRNA from 2×108 mouse macrophage cells (RAW264.7). This was purified using an oligo(dT) cellulose column to obtain 5 μg of poly(A)+ RNA.

[0134] (2) Synthesis of Double-Stranded cDNA from poly(A)+ RNA

[0135] A reaction solution (50 μl) containing the poly(A)+ RNA as obtained above (1) (5 μg), a reverse transcriptase (MMLV-RTase; product of STRATAGENE Corp.; 70 units) and dNTPs (0.6 mM) was incubated at 37° C. for 60 minutes to synthesize of a first strand cDNA. Next, a reaction solution containing the aforementioned reaction solution (45 μl), DNA polymerase (product of STRATAGENE Corp.; 100 units) and dNTPs (0.3 mM) was incubated at 16°...

example 2

Expression of the Protein (MMR-CAM) of the Invention

[0148] The clone (MMR19) obtained in Example 1(4) was inserted into an expression vector (λZAPII) following a common procedure and transformed E. coli (XL1-Blue), then a transformant cell line was constructed. The transformed E. coli cells were cultured, and the culture supernatant was dot blotted and allowed to with the same monoclonal antibodies used as shown in (3), which was produced by hybridoma deposited as FERM BP-6103 as a probe. Following this process, it was confirmed that the culture supernatant contained the protein that bound to the monoclonal antibodies.

example 3

Comparison of the Mouse-Derived Protein with Other Homologous Proteins Using Database Search

[0149] A data-search was conducted for human genes homologous to the nucleotide sequence and amino acid sequence listed as SEQ ID NO:1 and determined in Example 1 on both the amino acid level and the DNA level databases using (DNA DATA BANK of JAPAN (DDBJ): Dept. of Education, National Institute of Genetics, Center for Information Biology). The results are shown in Tables 1 and 2. These results suggest that the gene of the invention is also conserved in humans with high homology.

TABLE 1Homology on amino acid levelPosition within amino acidMatching insequence of SEQ ID NO: 1human homologue 1 to 9183 / 91 (91%) 50 to 14683 / 97 (85%) 1 to 7870 / 78 (89%)200 to 24140 / 42 (95%)172 to 24167 / 70 (95%)103 to 15046 / 48 (95%)169 to 24158 / 73 (79%)

[0150]

TABLE 2Homology on DNA levelPosition within nucleotideMatching insequence of SEQ ID NO: 1human homologue519 to 736189 / 218 (86%)666 to 689 23 / 24 (95%)381 to 40...

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Abstract

The present invention provides, as a gene encoding an antigen recognized by G-CSF-inducing antibodies, a gene encoding: (a) a protein having the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing; (b) a protein having the amino acid sequence listed as SEQ ID NO:2 of the Sequence Listing with one or more amino acid deletions, substitutions, additions or insertions and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor; or (c) a protein having at least 50% homology with the amino acid sequence listed as SEQ ID NO:2 and also binding to an antibody or its fragment that is active to induce granulocyte colony-stimulating factor.

Description

[0001] This application is a Divisional of co-pending application Ser. No. 09 / 937,905, filed on Oct. 1, 2001, and for which priority is claimed under 35 U.S.C. § 120. application Ser. No. 09 / 937,905 is the national phase of PCT International Application No. PCT / JP00 / 02080 under 35 U.S.C. § 371, which has an International filing date of Mar. 31, 2000, and which claims priority from Japanese Application No. 95092 / 1999 filed on Apr. 1, 1999. The entire contents of each of the above-identified applications are hereby incorporated by reference.TECHNICAL FIELD [0002] The present invention relates to a protein which is reactive with antibodies that are active to induce granulocyte colony-stimulating factor, to a gene encoding it and to a method for their use. BACKGROUND ART [0003] Granulocyte colony-stimulating factor (G-CSF) has a molecular weight of approximately 18,000 to 22,000 and consists of 174 (in rare cases 178) amino acids in the case of humans and 178 amino acids in the case of ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7088A61K38/16A61K39/395A61P7/00C07H21/04C07K14/00C07K16/00G01N33/00A61P31/00C07K14/47C07K14/535C07K14/705C07K16/28C12N1/21C12N15/12C12P21/08
CPCC07K14/47C07K14/535Y10T436/143333C07K16/28A61K2039/505C07K14/705A61P31/00A61P7/00C12N15/11
Inventor SHA, SHIKENAOKI, YOSHIKONISHI, YOSHISUKE
Owner SHA SHIKEN