EP0293785A2
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EP
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Cloning and expression of simian transforming growth factor-SS1
Assignee: Oncogen LP
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Filing: 1988-05-27
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Granted: —
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Expires: 2008-05-27
Abstract
Abstract Recombinant transforming growth factor-β1 (TGF-β1) is expressed to high levels in Chinese hamster ovary (CHO) cells using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-β1 and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the SV40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines expressing amplified TGF-β1 nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate selected transfectants. The major proteins secreted by these cells consisted of latent TGF-β1 and TGF-β1 precursor polypeptides as judged by immunoblots using site-specific anti-peptide antibodies derived from various regions of the TGF-β1 precursor. Levels of recombinant TGF-β1 protein secreted by these cells approached 30 ug/24 hour/10 7 cells.
Inventors
- 👤 Antony F. Purchio
- 👤 Larry Gentry
- 👤 Daniel Twardzik
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 15:57
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/495; A; A61; A61P; A61P35/00; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; A61K; A61K38/00
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
61 words
A method for producing transforming growth factor-β1, comprising: (a) culturing a eucaryotic cell containing a nucleotide sequence encoding simian transforming growth factor-p1 under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein having transforming growth factor-81 activity is produced by the eucaryotic cell; and (b) recovering the transforming growth factor-β1 from the culture.
Claim 2
Independent Claim
61 words
A method for producing transforming growth factor-β1, comprising: (a) culturing a eucaryotic cell containing a nucleotide sequence encoding simian transforming growth factor-p1 under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein having transforming growth factor-81 activity is produced by the eucaryotic cell; and (b) recovering the transforming growth factor-β1 from the culture.
Claim 3
Dependent Claim
33 words
The method according to claim 1 in which the nucleotide sequence encoding the simian transforming growth factor-,81 comprises the nucleotide sequence substantially as depicted in FIG. 1 from nucleotide number 1 to 1173.
Claim 4
Dependent Claim
17 words
The method according to claim 1 in which the eucaryotic cell comprises a Chinese Hamster Ovary cell.
Claim 5
Dependent Claim
20 words
The method according to claim 1 in which the second nucleotide sequence which controls gene expression comprises an SV40 promoter.
Claim 6
Dependent Claim
46 words
The method accoding to claim 1 in which the second nucleotide sequence comprises a promoter and a sequence encoding a selectable marker for which the eucaryotic cell is deficient, so that the eucaryotic cell containing the simian transforming growth factor-,81 coding sequence can be identified. -
Claim 7
Dependent Claim
14 words
The method according to claim 5 in which the selectable marker comprises dihydrofolate reductase.
Claim 8
Dependent Claim
36 words
The method according to claim 6 further comprising exposing the eucaryotic cell to methotrexate, so that resistant colonies are selected which contain amplified levels of the coding sequences for dihydrofolate reductase and simian transforming growth factor-β1.
Claim 9
Dependent Claim
20 words
The method according to claim 7 in which the eucaryotic cell comprises a dihydrofolate reductase- deficient Chinese Hamster Ovary cell.
Claim 10
Independent Claim
33 words
A method for producing transforming growth factor-f1, comprising (a) culturing transfectant CHO-TGF-β1-3/2000 as deposited with the ATCC and assigned accession number CRL 9434; and (b) recovering the transforming growth factor-β1 from the culture.
Claim 11
Independent Claim
33 words
A method for producing transforming growth factor-f1, comprising (a) culturing transfectant CHO-TGF-β1-3/2000 as deposited with the ATCC and assigned accession number CRL 9434; and (b) recovering the transforming growth factor-β1 from the culture.
Claim 12
Dependent Claim
17 words
The method according to claim 9 in which the transfectant is cultured in the presence of methotrexate.
Claim 13
Dependent Claim
34 words
A eucaryotic cell containing a nucleotide sequence encoding simian transforming growth factor-81 under the control of a second nucleotide sequence that regulates gene expression so that the eucaryotic cell produces active transforming growth factor-β1.
Claim 14
Dependent Claim
34 words
A eucaryotic cell containing a nucleotide sequence encoding simian transforming growth factor-81 under the control of a second nucleotide sequence that regulates gene expression so that the eucaryotic cell produces active transforming growth factor-β1.
Claim 15
Dependent Claim
18 words
The eucaryotic cell according to claim 11 as used and defined in anyone of claims 2 to 8.
Claim 16
Dependent Claim
16 words
A cell line comprising CHO-TGF-β1-3/2000 as deposited with the ATCC and assigned accession number CRL 9434.
Claim 17
Dependent Claim
16 words
A cell line comprising CHO-TGF-β1-3/2000 as deposited with the ATCC and assigned accession number CRL 9434.
Claim 18
Dependent Claim
39 words
A substantially pure polypeptide comprising the amino acid sequence substantially as depicted in FIG. 1, preferably comprising amino acid residue numbers 1 to 390; amino acid residue numbers 30 to 390; or amino acid residue numbers 30 to 278.
Claim 19
Dependent Claim
39 words
A substantially pure polypeptide comprising the amino acid sequence substantially as depicted in FIG. 1, preferably comprising amino acid residue numbers 1 to 390; amino acid residue numbers 30 to 390; or amino acid residue numbers 30 to 278.
Claim 20
Dependent Claim
18 words
The substantially pure polypeptide of claim 14 which is glycosylated, or phosphorylated, preferably containing at least one mannose-6-phosphate.
Claim 21
Dependent Claim
23 words
The substantially pure polypeptide of claim 15 in which the mannose-6-phosphate is linked to an asparagine linked sugar chain of the rTGF-β1 precursor.
Claim 22
Dependent Claim
21 words
The substantially pure polypeptide of claim 16 in which the asparagine site is amino acid residue number 82, 136 or 176.
Claim 23
Dependent Claim
16 words
The use of transforming growth factor-β1 for preparing a pharmaceutical composition for the treatment of tumors.
Claim 24
Dependent Claim
16 words
The use of transforming growth factor-β1 for preparing a pharmaceutical composition for the treatment of tumors.
References Cited (Backward Citations)
EP0200341A1
Cited
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