Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-09
2003-02-04
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S235100, C435S239000, C435S325000, C435S349000, C435S455000, C435S069100
Reexamination Certificate
active
06514728
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for the preparation of a cytokine by genetic recombination technology. More particularly, the present invention relates to a process for preparing a cytokine useful as a medicine inexpensively in large amounts by expressing the cytokine in eggs using Sendai virus vector.
BACKGROUND OF THE INVENTION
Cytokines such as interferons (IFN), interleukins, colony-stimulating factors (CSF) and tumor necrosis factors (TNF) are proteinous physiologically active biogenic substances which control cellular responses such as cell replication, differentiation, maintenance of existence, cell death, or expressions of functions, regulate immune system and inflammation reaction, and maintain homeostasis in cells or tissues. A cytokine network is formed because a single cytokine has many activities (pleiotropy), some cytokines have redundant activities (redundancy), and many cytokines have interactions in a single cell.
The most commonly known interferons are interferon &agr;, &bgr; and &ggr;, which have antivirus activities, inhibitory activities on cell proliferation, and regulatory activities on immunity, etc.
Interleukins are a group of biologically active substances which are produced by immunocompetent cells such as lymphocytes, monocytes, or macrophages. At present interleukins 1-18 have been elucidated. Colony-stimulating factors include a granulocyte colony-stimulating factor (G-CSF) having an activity for forming neutrophil colonies, a-macrophage colony-stimulating factor (M-CSF) having an activity for forming a macrophage colony, and a granulocyte macrophage colony-stimulating factor (GM-CSF) having an activity for forming a granulocyte/macrophage mixed colony. Other cytokines include a tumor necrosis factor (TNF), a transforming growth factor (TGF-&bgr;) super family including TGF-&bgr; as a representative, growth factors such as an epidermal growth factor (EGF) or a platelet-derived growth factor (PDGF), and hematopoietic factors such as erythropoietin or thrombopoietin.
Some of the cytokines described above have been commercially manufactured by genetic recombination technology or are in the course of development since they are useful as agents for treatment of various infectious diseases, tumors, thrombocytopenia, neuropenia, aplastic anemia, etc.
Generally, when medications comprising proteins or peptides are prepared from organs or blood, there are problems of shortage and high cost of materials, risk of infection, etc. In order to solve these problems, methods for preparing proteins or peptides as medications by utilizing genetic engineering have been developed. As mass expression systems of proteins for medical use, there have been expression systems using a procaryote such as
E. coli
as a host, those using yeast which is eucaryotic and unicellular cell as a host, those using a baculovirus expression system and employing insect cells as a host, those using mammalian cells such as Chinese hamster ovary cells (CHO cells) as a host, etc. Also, recently it has become possible to produce transgenic animals which are animal individuals integrated by foreign genes, and methods for preparing useful proteins by using these animals have been developed. For example, a method in which a characteristic is expressed specifically in mammary gland cells such as those of a cow, a sheep, or a goat, a useful protein is secreted into milk, and the protein of interest is recovered is anticipated as a mass expression system for preparing useful proteins. At present, such a method is being developed as an inexpensive method for preparing blood preparations such as human serum albumin, and it has been attempted to secrete the thrombolytic drug, t-PA, into goat milk.
As to production of cytokines using genetic engineering, interferon-&agr;, interferon-&bgr;, interferon-&ggr;, interleukin-2, etc. have been produced or proposed to be produced in
E. coli
systems, G-CSF and interleukin-11 have been produced or proposed to be produced in CHO cell systems, and GM-CSF has been produced or proposed to be produced in yeast systems.
However, the above-described prior art expression systems for useful proteins have various problems, and especially when the protein of interest has sugar chains, there is a problem of whether sugar chains can be bound to the expressed protein in a host of an expression system. In
E. coli
systems, mass culturing can be carried out and the growth rate is high, but sugar chains are not bound to expressed proteins. Also, in
E. coli
, the proteins which are produced are usually accumulated within the cells, and therefore, isolation and purification including isolation of cells from the culture medium are expensive. In baculovirus expression systems using insect cells, sugar chains are bound, but are bound in a different way from in mammals.
Expression systems using mammalian cells as hosts are not practical since the yield is extremely low, the cost is high especially when CHO cells which require expensive fetal bovine serum are used, and there is a risk of contamination of retrovirus in the cells.
Methods using transgenic animals require much labor and money when producing transgenic animals for the first time, and the amount of expression is still inadequate.
In order to put a method for preparation of a useful protein by recombinant DNA technology to practical use, it is important whether the produced protein is naturally occurring, what are the effects and side effects of the protein as a medication, and whether the protein can be expressed in large amounts and effectively. The ease and cost of isolation and purification, the maintenance of protein activity during a purification process, and the ease of formulation of drugs are also important.
As mentioned above, it is difficult to produce cytokines having sugar chains in a way similar to mammals in the form of naturally occurring proteins in large amounts and inexpensively by prior art expression systems for proteins.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a process for producing a naturally occurring cytokine in large amounts and inexpensively.
As a result of investigation to solve the problems of the prior art expression systems for proteins described above, the present inventors found that proteins having sugar chains close to those of mammals can be produced in large amounts and inexpensively by using Sendai virus vector and expressing the proteins in hen's eggs, and also found that in the present system the proteins are released into the chorioallantoic fluid of hen's eggs in large amounts, so isolation and purification can be easily conducted, thereby stablishing a mass production system for cytokines.
In one aspect, the present invention provides a process for preparing a cytokine, comprising infecting an egg with recombinant Sendai virus containing a gene coding the cytokine and recovering the expressed cytokine.
The recombinant Sendai virus can be produced in a cell by insertion of a gene coding the cytokine into a Sendai virus genome to produce a recombinant Sendai virus genome containing said cytokine gene and introduction of the resulting construct into the cell. The cell to be used for production of the recombinant Sendai virus may be a cell that expresses nucleocapsid protein (NP), phosphoprotein (P/C) and large protein (L) of the Sendai virus.
The expressed cytokine may be recovered from the chorioallantoic fluid of the infected egg.
The cytokine obtained in accordance with the present process has sugar chains, or the expressed cytokine is glycosylated.
The present process can be preferably applied to the process for preparation of interleukins.
REFERENCES:
patent: 3560611 (1971-02-01), Chany et al.
patent: 4276282 (1981-06-01), Sugimoto et al.
patent: 4285929 (1981-08-01), Sugimoto et al.
patent: 5162215 (1992-11-01), Bosselman et al.
patent: 5518899 (1996-05-01), Kurimoto et al.
patent: 5641656 (1997-06-01), Sekellick et al.
patent: 0863202 (1998-09-01), None
patent: 0864645 (1998-09-01), None
patent:
Kai Chieko
Kato Atsushi
Bunner Bridget E.
Burns Doane , Swecker, Mathis LLP
Kunz Gary L.
Nippon Biocaptal Limited
LandOfFree
Process for preparation of cytokines using Sendai virus... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for preparation of cytokines using Sendai virus..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for preparation of cytokines using Sendai virus... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3129271