Patent EP0162699A2 successfully refreshed from Google Patents.
EP0162699A2
Expired / Lapsed
EP
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Interleukin-2 receptor and DNA encoded therefor and their preparation
Assignee: Immunex Corp
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Filing: 1985-05-21
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Granted: —
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Expires: 2005-05-21
Abstract
Abstract Interleukin-2 receptor derived from normal and malignant cells has been purified by use of various techniques including affinity chromatography in conjunction with a monoclonal antibody directed to the receptor. The purification process also includes reversed phased high performance liquid chromatography. By these techniques, interleukin-2 receptor has been purified to homogeneity. The high purification of the interleukin-2 receptor has made possible the sequencing of the amino acid residues at the N-terminal of this protein molecule. Double-stranded cDNA is prepared from polyadenylated RNA extracted from cell lines or other sources known to produce IL-2 receptor. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the purified IL-2 receptor. The cDNA clone isolated with the probe is characterized by restriction enzyme mapping and sequenced by chain-termination method. The particular DNA clone that actually contains the gene coding for the functional IL-2 receptor is identified by expressing the clones in COS-7 monkey kidney cells and assaying for the expressed IL-2 receptor by its ability to bind IL-2 or a monoclonal antibody directed against the IL-2 receptor.
Inventors
- 👤 Douglas P. Cerretti
- 👤 David J. Cosman
- 👤 Steven K. Dower
- 👤 Carl J. March
- 👤 David L. Urdal
- 👤 Alf D. Larsen
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 20:45
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/705; C07K14/715; C07K14/7155; C07K16/00; C07K16/18; C07K16/28; C07K16/2866
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
30 words
A homogenous receptor for interleukin 2 comprising a protein characterized as having a molecular weight of about 55,000 to 60,000 daltons and an N-terminal amino acid sequence as follows: Glu-Leu-Cys-Asp-Asp-Asp-Pro-Glu-Ile.
Claim 2
Dependent Claim
30 words
A homogenous receptor for interleukin 2 comprising a protein characterized as having a molecular weight of about 55,000 to 60,000 daltons and an N-terminal amino acid sequence as follows: Glu-Leu-Cys-Asp-Asp-Asp-Pro-Glu-Ile.
Claim 3
Dependent Claim
24 words
A homogenous receptor for interleukin 2 comprising an amino acid sequence extending from residue No. 1 to residue No. 251 inclusive in FIGURE 2.
Claim 4
Dependent Claim
24 words
A homogenous receptor for interleukin 2 comprising an amino acid sequence extending from residue No. 1 to residue No. 251 inclusive in FIGURE 2.
Claim 5
Dependent Claim
20 words
A homogenous receptor for interleukin 2 as claimed in claim 2 and further comprising the following N-terminal amino acid sequence:Met-Try-Gly-Leu-Leu-Thr-Phe-Ile-Met-Val-Pro-Gly-Cys-Glu-Ala.
Claim 6
Dependent Claim
36 words
Substantially pure DNA extracted from mammalian cells capable of producing IL-2 receptor molecules and comprising the nucleic acid sequence extending from nucleic acid base No. 64 to nucleic acid base No. 816 inclusive in FIGURE 2.
Claim 7
Dependent Claim
36 words
Substantially pure DNA extracted from mammalian cells capable of producing IL-2 receptor molecules and comprising the nucleic acid sequence extending from nucleic acid base No. 64 to nucleic acid base No. 816 inclusive in FIGURE 2.
Claim 8
Dependent Claim
30 words
DNA as claimed in claim 4 and further comprising the N-terminal nucleic acid sequence extending from nucleic acid base No. 1 to nucleic acid base No..63 inclusive in FIGURE 2.
Claim 9
Dependent Claim
18 words
Substantially pure DNA encoding for interleukin 2-receptor and comprising the nucleotide sequence underlined by dots in FIGURE 2.
Claim 10
Dependent Claim
18 words
Substantially pure DNA encoding for interleukin 2-receptor and comprising the nucleotide sequence underlined by dots in FIGURE 2.
Claim 11
Dependent Claim
21 words
An amino acid chain encoded by the nucleotide sequence of DNA as claimed in any one of claims 4 to 6.
Claim 12
Dependent Claim
80 words
A process for producing a homogeneous receptor for interleukin 2, comprising: (a) culturing cells capable of expressing interleukin 2 receptor molecules in culture medium; (b) harvesting said cells from the culture medium; (c) extracting the interleukin 2 receptor molecules from said cells; and (d) purifying said interleukin 2 receptor molecules with a reversed phase, high-performance liquid chromatography column containing methyl, octyl or octyldecyl groups covalently bonded to silica gel, whereby the interleukin 2 receptor molecules are retained by the column.
Claim 13
Dependent Claim
80 words
A process for producing a homogeneous receptor for interleukin 2, comprising: (a) culturing cells capable of expressing interleukin 2 receptor molecules in culture medium; (b) harvesting said cells from the culture medium; (c) extracting the interleukin 2 receptor molecules from said cells; and (d) purifying said interleukin 2 receptor molecules with a reversed phase, high-performance liquid chromatography column containing methyl, octyl or octyldecyl groups covalently bonded to silica gel, whereby the interleukin 2 receptor molecules are retained by the column.
Claim 14
Dependent Claim
25 words
A process as claimed in claim 8, wherein culturing said interleukin 2 receptor expressing molecules includes culturing T-cells in the presence of an activating agent.
Claim 15
Dependent Claim
25 words
A process as claimed in claim 8, wherein said interleukin 2 receptor expressing cells comprise malignant cells, e.g. T-lymphoma or T-leukemia cells, or normal T-cells.
Claim 16
Dependent Claim
92 words
A process as claimed in any one of claims 8 to 10, wherein said receptor molecules are initially partially purified by passing the interleukin 2 containing extract through an affinity chromatography column prior to the reversed phase high-performance liquid chromatography procedure, said column containing a gel substrate to which is bound an antibody that specifically binds to an antigenic epitope on the interleukin 2 receptor whereby the interleukin 2 receptor is retained by the chromatography column, eluting the retained interleukin 2 receptor from the column and pooling fractions exhibiting interleukin 2 activity.
Claim 17
Dependent Claim
72 words
A process as claimed in claim 11, wherein said antigenic epitope is included within the amino acid sequence extending from residue No. 1 to residue No. 10 inclusive in FIGURE 2, or is included within the amino acid sequence extending from residue No. 103 to residue No. 174 inclusive in FIGURE 2, or is included within the amino acid sequence-extending from residue No. 1 to residue No. 251 inclusive in FIGURE 2.
Claim 18
Dependent Claim
109 words
A process for producing DNA encoding for interleukin 2 receptor comprising employing a hybridization probe having a base sequence corresponding to the amino acid sequence at the N-terminal end of interleukin 2 receptor to isolate a DNA fraction from a cDNA library prepared by the action of reverse transcriptase from polyadenylated mRNA extracted from a cell line or other source known to produce interleukin 2 receptor, rendering double-stranded, using DNA polymerase, said DNA fraction , incorporating the resulting DNA into a cloning vector, using the resulting vector to transform a host, and isolating from the resulting transformants, using a synthetic oligonucleotide probe, cloned vector DNA.encoded for interleukin 2 receptor.
Claim 19
Dependent Claim
109 words
A process for producing DNA encoding for interleukin 2 receptor comprising employing a hybridization probe having a base sequence corresponding to the amino acid sequence at the N-terminal end of interleukin 2 receptor to isolate a DNA fraction from a cDNA library prepared by the action of reverse transcriptase from polyadenylated mRNA extracted from a cell line or other source known to produce interleukin 2 receptor, rendering double-stranded, using DNA polymerase, said DNA fraction , incorporating the resulting DNA into a cloning vector, using the resulting vector to transform a host, and isolating from the resulting transformants, using a synthetic oligonucleotide probe, cloned vector DNA.encoded for interleukin 2 receptor.
Claim 20
Dependent Claim
46 words
A process for producing interleukin 2 receptor which comprises cloning DNA which has been produced by a process as claimed in claim 13 or DNA as defined in any one of claims 4 to 6 in a mammalian cell system so as to express said DNA.
References Cited (Backward Citations)
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