US4407945A
Expired / Lapsed
US
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Constitutive production of interleukin 2 by a T cell hybridoma
Assignee: Immunex Corp
•
Filing: 1981-04-29
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Granted: 1983-10-04
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Expires: 2001-04-29
Abstract
Abstract A process for constitutively producing murine IL-2 from hybridoma cells generated by fusing nitrogen stimulated malignant murine cells with drug sensitive murine thymoma driver cells. A fusing agent is used to fuse the two parent cells together. After fusion, the hybrid cells are cultured in vitro in a supplemented, serum containing tissue culture medium to thereby constitutively produce IL-2. The medium also includes a group of suppressing compounds which will prevent unfused driver cells from replicating, and feeder cells used to nurture the growth of competent hybrid cells.
Inventors
- 👤 Steven Gillis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 08:52
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 51
Claim 1
Independent Claim
75 words
A process for constitutively producing murine interleukin-2, comprising: stimulating murine mononuclear leukocytes in a first culture medium containing a T cell mitogen stimulant; marking murine neoplastic cells with a sensitizing drug to produce driver cells; fusing said murine mononuclear leukocyte cells with said driver cells to produce a hybridoma capable of constitutively producing interleukin-2; and culturing the hybridoma cells in a second culture medium, containing selective suppressing agents which allow only hybrid cells to proliferate.
Claim 2
Dependent Claim
75 words
A process for constitutively producing murine interleukin-2, comprising: stimulating murine mononuclear leukocytes in a first culture medium containing a T cell mitogen stimulant; marking murine neoplastic cells with a sensitizing drug to produce driver cells; fusing said murine mononuclear leukocyte cells with said driver cells to produce a hybridoma capable of constitutively producing interleukin-2; and culturing the hybridoma cells in a second culture medium, containing selective suppressing agents which allow only hybrid cells to proliferate.
Claim 3
Dependent Claim
14 words
The process of claim 1, wherein the murine mononuclear leukocytes include malignant murine cells.
Claim 4
Dependent Claim
17 words
The process of claim 2, wherein said malignant cells are T leukemic cells or T lymphoma cells.
Claim 5
Dependent Claim
13 words
The process of claim 3, wherein said T lymphoma cells are LBRM-33 cells.
Claim 6
Dependent Claim
16 words
The process of claim 4, wherein said T lymphoma cells are clones of said LBRM-33 cells.
Claim 7
Dependent Claim
16 words
The process of claim 3, wherein said malignant cells are clones of said T lymphoma cells.
Claim 8
Dependent Claim
20 words
The process of claim 6, wherein said clones of T lymphoma cells are clones of LBRM-33 lymphoma murine T cells.
Claim 9
Dependent Claim
30 words
The process of claim 1, wherein said T cell mitogen in said first culture medium is a compound selected from the group consisting of phytohemagglutinin, concanavalin A, and pokeweed mitogen.
Claim 10
Dependent Claim
17 words
The process of claim 8, wherein said first culture medium includes Roswell Park Memorial Institute medium 1640.
Claim 11
Dependent Claim
18 words
The process of claim 8, wherein said first culture medium includes Click's medium or Dulbecco Modified Eagle's medium.
Claim 12
Dependent Claim
32 words
The process of claim 1, wherein said first culture medium is a compound selected from the group consisting of Roswell Park Memorial Institute medium 1640, Click's medium and Dulbecco Modified Eagle's medium.
Claim 13
Dependent Claim
30 words
The process of claim 2, wherein said T cell mitogen in said first culture medium is a compound selected from the group consisting of phytohemagglutinin, concanavalin A and pokeweed mitogen.
Claim 14
Dependent Claim
17 words
The process of claim 12, wherein said first culture medium includes Roswell Park Memorial Institute medium 1640.
Claim 15
Dependent Claim
18 words
The process of claim 12, wherein said first culture medium includes Click's medium or Dulbecco Modified Eagle's medium.
Claim 16
Dependent Claim
30 words
The process of claim 3, wherein said T cell mitogen in said first culture medium is a compound selected from the group consisting of phytohemagglutinin, concanavalin A and pokeweed mitogen.
Claim 17
Dependent Claim
17 words
The process of claim 15, wherein said first culture medium includes Roswell Park Memorial Institute medium 1640.
Claim 18
Dependent Claim
18 words
The process of claim 15, wherein said first culture medium includes Click's medium or Dulbecco Modified Eagle's medium.
Claim 19
Dependent Claim
20 words
The process of claim 1, wherein said murine driver cells include drug marked, ouabain or 8-azaguanine resistant, malignant T cells.
Claim 20
Dependent Claim
15 words
The process of claim 18, wherein said driver cells include drug marked BW 5147 thymocytes.
Claim 21
Dependent Claim
22 words
The process of claim 18, wherein said sensitizing drug is a compound selected from the group consisting of ouabain, glutathione and 8-azaguanine.
Claim 22
Dependent Claim
51 words
The process of claim 1, wherein the step of fusing the murine mononuclear leukocyte cells with drug sensitive driver cells includes mixing the murine mononuclear leukocyte cells with the drug sensitive driver cells, and adding a fusion compound selected from the group consisting of polyethylene glycol and deoxyribonucleic acid transforming viruses.
Claim 23
Dependent Claim
20 words
The process of claim 21, wherein said deoxyribonucleic acid transforming viruses include Sendai virus or the fusion protein obtained therefrom.
Claim 24
Dependent Claim
16 words
The process of claim 21, wherein said fusion compound further includes a third tissue culture medium.
Claim 25
Dependent Claim
25 words
The process of claim 23, wherein said third tissue culture medium includes Roswell Park Memorial Institute medium 1640, Click's medium or Dulbecco Modified Eagle's medium.
Claim 26
Dependent Claim
32 words
The process of claim 1, wherein said second culture medium includes a compound selected from a group consisting of Roswell Park Memorial Institute medium 1640, Click's medium or Dulbecco Modified Eagle's medium.
Claim 27
Dependent Claim
35 words
The process of claim 25, wherein said second culture medium contains one or more compounds selected from the group consisting of fetal calf serum, sodium pyruvate, Hepe's buffer, NaHCO 3 , penicillin, streptomycin and L-glutamine.
Claim 28
Dependent Claim
21 words
The process of claim 26, wherein said second culture medium includes suppressing agents to preclude the growth of unfused driver cells.
Claim 29
Dependent Claim
24 words
The process of claim 27, wherein said suppressing agents include one or more compounds selected from the group consisting of hypoxanthine, aminopterin and thymidine.
Claim 30
Dependent Claim
14 words
The process of claim 25, wherein said second culture medium further includes feeder cells.
Claim 31
Dependent Claim
30 words
The process of claim 29, wherein said feeder cells includes cells selected from the group consisting of murine thymocytes, murine spleen cells, irradiated murine peritoneal exudate cells and murine macrophages.
Claim 32
Dependent Claim
14 words
The process of claim 25, wherein said second culture medium further includes feeder cells.
Claim 33
Dependent Claim
14 words
The process of claim 1, further including the step of cloning said hybridoma cells.
Claim 34
Dependent Claim
16 words
The process of claim 32, wherein said hybridoma cells are cloned in a fourth culture medium.
Claim 35
Dependent Claim
32 words
The process of claim 33, wherein said fourth culture medium includes a compound selected from the group consisting of Roswell Park Memorial Institute medium 1640, Click's medium and Dulbecco Modified Eagle's medium.
Claim 36
Dependent Claim
35 words
The process of claim 34, wherein said fourth culture medium contains one or more compounds selected from the group consisting of fetal calf serum, sodium pyruvate, Hepe's buffer, NaHCO 3 , penicillin, streptomycin and L-glutamine.
Claim 37
Dependent Claim
14 words
The process of claim 34, wherein said fourth culture medium further includes feeder cells.
Claim 38
Dependent Claim
30 words
The process of claim 36, wherein said feeder cells include cells selected from the group consisting of murine thymocytes, murine spleen cells, irradiated murine peritoneal exudate cells and murine microphages.
Claim 39
Dependent Claim
46 words
A process for producing IL-2 from a hybrid murine T cell line, comprising culturing in a tissue culture medium, hybrid T cells capable of constitutively producing IL-2, said hybrid cells produced by fusing mitogen stimulated, malignant murine IL-2 producer cells with drug marked murine driver cells.
Claim 40
Dependent Claim
46 words
A process for producing IL-2 from a hybrid murine T cell line, comprising culturing in a tissue culture medium, hybrid T cells capable of constitutively producing IL-2, said hybrid cells produced by fusing mitogen stimulated, malignant murine IL-2 producer cells with drug marked murine driver cells.
Claim 41
Dependent Claim
18 words
The process of claim 38, whereinsaid malignant murine IL-2 producer cells are T leukemic or T lymphoma cells.
Claim 42
Dependent Claim
13 words
The process of claim 39, wherein said T lymphoma cells are LBRM-33 cells.
Claim 43
Dependent Claim
36 words
The process of claim 40, wherein said T lymphoma cells are clones of said LBRM-33 cells produced by separating said LBRM-33 cell line into individual cells and then culturing the individual cells in a culture medium.
Claim 44
Dependent Claim
18 words
The process of claim 38, wherein said murine driver cells are T leukemic cells or T lymphoma cells.
Claim 45
Dependent Claim
14 words
The process of claim 42, wherein said murine lymphoma cells are BW 5147 cells.
Claim 46
Dependent Claim
33 words
The process of claim 38, wherein said hybrid T cells are clones derived from parent hybrid T cells produced by fusion of murine, mitogen stimulated, IL-2 producer cells with drug-sensitive murine driver cells.
Claim 47
Dependent Claim
31 words
The process of claim 38, wherein said culture medium includes a compound selected from the group consisting of Roswell Park Memorial Institute medium 1640, Click's medium, and Dulbecco Modified Eagle's medium.
Claim 48
Dependent Claim
22 words
The process of claim 45, wherein said culture medium further includes driver cell suppressing agents to prevent proliferation of uncoupled driver cells.
Claim 49
Dependent Claim
14 words
The process of claim 46, wherein said suppressing agents include hypoxanthine, aminopterin and thymidine.
Claim 50
Dependent Claim
13 words
The process of claim 45, wherein said culture medium further includes feeder cells.
Claim 51
Dependent Claim
30 words
The process of claim 48, wherein said feeder cells include cells selected from the group consisting of murine thymocytes, murine spleen cells, irradiated murine peritoneal exudate cells and murine macrophages.
References Cited (Backward Citations)
US4196265A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-10-03 | 1987-04-04 | 1987-10-04 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-10-03 | 1991-04-04 | 1991-10-04 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-10-03 | 1995-04-04 | 1995-10-04 | $7700.00 | Window Closed / Lapsed |