Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2000-01-21
2002-05-21
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S070100, C930S090000
Reexamination Certificate
active
06391633
ABSTRACT:
The invention relates to human cells which are capable, on the basis of an activation of the endogenous human EPO gene, of producing EPO in sufficient amount and purity to permit economical preparation of human EPO as a pharmaceutical preparation. The invention furthermore relates to a method of preparing such human EPO-producing cells, DNA constructs for activating the endogenous EPO gene in human cells, and methods for the large-scale production of EPO in human cells.
Erythropoietin (EPO) is a human glycoprotein which stimulates the production of red blood cells. EPO occurs in the blood plasma of healthy persons only in very low concentrations, so that preparation in large amounts is not possible in this manner. EP-0148 605 and EP-B-0205 564 describe the preparation of recombinant human EPO in CHO cells. The EPO described in EP-B-0148 605 has a higher molecular weight than urinary EPO and no O-glycosylation. Meantime, the EPO described in EP-B-0 205 564 from CHO cells, is available in large amounts and in pure form, but it originates from nonhuman cells. Moreover, the ability of CHO cells to produce is often relatively limited.
Furthermore, the harvesting of human EPO from the urine of patients with aplastic anemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558-5564). Therein a seven-step process is disclosed which includes ion exchanger chromatography, ethanol precipitation, gel filtration and adsorption chromatography. An EPO preparation with a specific activity of about 70,000 U/mg of proteins obtained in a 21% yield. Disadvantages of this process and other methods of obtaining urinary EPO consist in the procurement of starting material in sufficient amounts and in repeatable quality. Furthermore, the purification from urine is difficult and even a purified product is not free of urinary contaminants.
GB-A-2085 887 describes a method for the preparation of human lymphoblastoid cells which are capable of producing EPO in small amounts. The economical production of a pharmaceutical with these cells is not possible.
WO 91/06667 describes a method for the recombinant preparation of EPO. In a first process step in primary human embryo kidney cells the endogenous EPO gene is brought by homologous recombination into operable linkage with a viral promoter and the DNA is isolated from these cells. In a second step the DNA thus isolated is transformed into nonhuman CHO cells and the expression of EPO in these cells is analyzed. No mention is found that production of EPO in human cells is possible.
WO 93/09222 describes the production of EPO in human cells, wherein a relatively high EPO production of up to 960,620 mU/10
6
cells/24 h is found in human fibroblasts which had been transfected with a vector containing the complete EPO gene. These cells contain an exogenous EPO gene which is not at the correct EPO gene locus, so that problems are to be expected with regard to the stability of the cell line. No information on a constitutive EPO production is found in WO 93/092222. Furthermore, neither is there any information as to whether the EPO produced can be obtained in a quality sufficient for pharmaceutical purposes.
Furthermore, in WO 93/09222 an activation of the endogenous EPO gene in human HT1080 cells is described. In it an EPO production is found of only 2,500 mU/10
6
cells in 24 h (corresponding approximately to 16 ng/10
6
cells/24 h). Such low production is entirely unsuited for the economical production of a pharmaceutical preparation.
WO 94/12650 and WO 95/31560 describe how a human cell with an endogenous EPO gene activated by a viral promoter is able, after amplification of the endogenous EPO gene, to produce up to approximately 100,000 mU/10
6
cells/24 h (corresponding to about 0.6 &mgr;g/10
6
cells/24 h) of EPO. This amount too is still not sufficient for the economical production of a pharmaceutical.
The problem on which the present invention is based thus consisted in eliminating at least partially the above-described disadvantage of the state of the art and to offer a technologically better method for the preparation of EPO in human cells. Especially, this is to make it possible to obtain a product in sufficient quantity and purity to permit economical production for pharmaceutical purposes.
This problem is solved by activation of the endogenous EPO gene in human cells and if desired by subsequent amplification of the activated human EPO gene. In this manner it has surprisingly been possible, by the selection of suitable starting cells, DNA constructs and selection strategies, to provide human cells which are capable of producing EPO in sufficient quantity, quality and purity to permit the economical production of pharmaceutical preparations. Especially after amplification of the activated endogenous EPO gene, cells can be obtained which have a definitely higher production output than the CHO production cells used previously for the preparation of recombinant EPO.
One subject matter of the invention is a human cell which contains a copy of an endogenous EPO gene in operable linkage with a heterologous expression control sequence active in the human cell and is capable without prior gene amplification of producing at least 200 ng of EPO/10
6
cells per 24 hours. Preferably the human cell according to the invention is capable of the production of 200 to 3000 ng EPO/10
6
cells/24 h, and most preferably for the production of 1000 to 3000 ng EPO/10
6
cells/24 h.
Another subject matter of the present invention is a human cell which is obtainable by gene amplification from the cell previously described and contains several copies of an endogenous EPO gene, each in operable linkage with a heterologous expression control sequence active in the human cell, and is capable of the production of at least 1,000 ng EPO/10
6
cells/24 h. With special preference the human cell line obtainable by gene amplification is capable of producing 1,000 to 25,000 ng EPO/10
6
cells/24 h, and most preferably for the production of 5,000 to 25,000 ng EPO/10
6
cells/24 h.
The human cell is any cell, provided it can be cultured in vitro. Especially preferred are human cells which can be cultured in a serum-free medium, and especially in suspension.
In this manner the production of EPO can be performed in a large fermenter with a culture capacity of, for example, 1,000 liters.
Especially preferred is a human cell which is an immortalized cell, for example an HT 1080 cell (Rasheed et al., Cancer 33 (1974), 1027-1033), a HeLa S3 cell (Puck et al., J. Exp. Med. 103 (1956), 273-284), a Namalwa cell (Nadkarni et al., Cancer 23 (1969), 64-79) or a cell derived therefrom. An example of a cell according to the invention is the clone “Aladin” which was deposited on Jul. 15, 1997 according to the prescriptions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig, under number DSM ACC 2320.
In the cell according to the invention, the endogenous EPO gene is linked with a heterologous expression control sequence which is active in the human cell. The expression control sequence comprises a promoter and preferably additional expression-improving sequences, e.g., an enhancer. The promoter can be a inducible or a constitutive promoter. Preferably the promoter is a strong viral promoter, e.g., an SV40 or a CMV promoter. The CMV promoter/enhancer is especially preferred.
Furthermore, to optimize the EPO expression it may be preferred for the endogenous EPO gene in the human cell, which is in operable association with the heterologous promoter, to have a signal peptide-coding sequence which is different from the natural signal peptide-coding sequence and codes preferably for a signal peptide with a modified amino acid sequence. Especially preferred is a signal peptide-coding sequence which codes for a signal peptide sequence modified in the region of the first four amino acids which is selected from
Met-X
1
-X
2
-X
3
,
wherein X
1
is Gly or Ser, X
2
is Ala, Val, Leu, Ile, Ser or Pro, and X
3
is Pro, Arg, Cys or His, on
Auer Johannes
Brandt Michael
Honold Konrad
Koll Hans
Stern Anne
Fulbright & Jaworski LLP
Roche Diagnostics GmbH
Yucel Remy
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