US4675295A
Expired / Lapsed
US
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Process for producing subculturable lymphokine-producing human T cell hybridomas
Assignee: Denka Co Ltd
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Filing: 1984-01-26
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Granted: 1987-06-23
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Expires: 2004-01-26
Abstract
Abstract A process for preparing human T cell hybridomas which are subculturable and produce lymphokines comprising the steps of: (1) treating a human acute leukemia cell with a protein and/or RNA synthesis inhibitor; (2) independently activating a human T cell with a mitogen or antigen; (3) fusing the thus-treated human acute leukemia cell with the thus-activated human T cell in the presence of a fusion accelerator; and (4) isolating the thus-formed hybridoma. An in vivo process for producing lymphokines using the hybridomas is also described.
Inventors
- 👤 Toshiaki Osawa
- 👤 Yoshiro Kobayashi
- 👤 Makoto Asada
- 👤 Masahiro Higuchi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 05:03
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/525; C12; C12N; C12N5/00; C12N5/10; C12N5/12; C12N5/16; C12N5/163; C12N2510/00; C12N2510/02
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
159 words
A process for preparing a subculturable lymphokine-producing human T cell hybridoma comprising the steps of: (1) treating a human acute leukemia cell with an irreversilbe protein synthesis inhibitor or a combination of an irreversible protein synthesis inhibitor and an irreversible RNA synthesis inhibitor so that the growth of the human acute leukemia cell is completely inhibited; (2) independently activating a human T cell with a mitogen or antigen so that blast transformation of the human T cell occurs and the desired lymphokine is produced; (3) fusing the treated human acute leukemia cell of step (1) with the activated human T cell of step (2) in the presence of a fusion accelerator; and (4) isolating the product hybridoma by incubating in a nutrient medium on which human acute leukemia cella can grow, wherein said irreversible protein synthesis inhibitor is selected from the group consisting of emetine and emetine hydrochloric acid salt and said irreversible RNA synthesis inhibitor is actinomycin D.
Claim 2
Dependent Claim
159 words
A process for preparing a subculturable lymphokine-producing human T cell hybridoma comprising the steps of: (1) treating a human acute leukemia cell with an irreversilbe protein synthesis inhibitor or a combination of an irreversible protein synthesis inhibitor and an irreversible RNA synthesis inhibitor so that the growth of the human acute leukemia cell is completely inhibited; (2) independently activating a human T cell with a mitogen or antigen so that blast transformation of the human T cell occurs and the desired lymphokine is produced; (3) fusing the treated human acute leukemia cell of step (1) with the activated human T cell of step (2) in the presence of a fusion accelerator; and (4) isolating the product hybridoma by incubating in a nutrient medium on which human acute leukemia cella can grow, wherein said irreversible protein synthesis inhibitor is selected from the group consisting of emetine and emetine hydrochloric acid salt and said irreversible RNA synthesis inhibitor is actinomycin D.
Claim 3
Dependent Claim
19 words
The process as claimed in claim 1, wherein said human acute leukemia cell line is T line tumor cell.
Claim 4
Dependent Claim
26 words
The process as claimed in Claim 2, wherein said human acute leukemia cell is selected from the group consisting of CEM, TALL and MOLT-4 cell line.
Claim 5
Dependent Claim
39 words
The process as claimed in claim 1, 2 or 3, wherein 10 6 to 10 7 cells/ml of said human acute leukemia cell line is treated with 10 -4 to 10 -5 M of irreversible said protein synthesis inhibitor.
Claim 6
Dependent Claim
21 words
The process as claimed in claim 1, wherein said mitogen is selected from the group consisting of phytohemagglutinin-P and concanavalin A.
Claim 7
Dependent Claim
43 words
The process as claimed in claim 1, wherein 10 6 to 10 7 cells/ml of said irreversible acute leukemia cell line is treated with 10 -4 to 10 -5 M of said protein synthesis inhibitor and 0.05 to 0.2 μg/ml of actinomycin D.
Claim 8
Dependent Claim
24 words
The process as claimed in claim 1, wherein said cell fusion accelerator is a polyethylene glycol having a molecular weight of 1,000 to 4,000.
Claim 9
Dependent Claim
38 words
The process as claimed in claim 1 or 7, wherein upon cell fusion, said antigen- or mitogen-treated human T cell is mixed with said human acute leukemia cell line in a cell number proportion of 1:1 to 20:1.
Claim 10
Dependent Claim
38 words
The process as claimed in claim 1 or 7, wherein upon cell fusion, said antigen- or mitogen-treated human T cell is mixed with said human acute leukemia cell line in a cell number proportion of 2:1 to 15:1.
Claim 11
Dependent Claim
47 words
The process as claimed in claim 1 or 7, wherein after cell fusion the resulting hybridoma cells are cultivated in a medium which does not permit non-protein and/or RNA synthesis inhibitor-treated human acute leukemia cell line to proliferate, thereby allowing for the isolation of the hybridoma cell.
Claim 12
Dependent Claim
21 words
The process as claimed in claim 1, wherein said cell fusion accelerator is Sendai virus (HVJ) or inactivated Sendai virus (HVT).
Claim 13
Dependent Claim
20 words
A process as claimed in claim 1, wherein the lymphokine produced by the product hybridoma is migration inhibitory factor (MIF).
Claim 14
Dependent Claim
18 words
The process as claimed in claim 1, wherein the lymphokine produced by the product hybridoma is lymphotoxin (LT).
Claim 15
Dependent Claim
19 words
The process as claimed in claim 1, wherein the product hybridoma is then subcultured in vitro or in vivo.
Claim 16
Dependent Claim
41 words
The process claimed in claim 1, further comprising the steps of: (5) transplanting said product hybridoma to a warm-blooded animal other than a human; (6) multiplying said hybridoma in said animal, and (7) cultivating the multiplied hybridomas in a nutrient medium.
Claim 17
Dependent Claim
33 words
The process as claimed in claim 15, wherein said warm-blooded animal other than a human is a hamster to which an immunosuppressive agent is administered prior to or after transplantation of the hybridoma.
Claim 18
Dependent Claim
37 words
The process as claimed in claim 16, wherein said immunosuppressive agent is anti-lymphocyte serum obtained by immunizing a rabbit with at least one member selected from the group consisting of cortison acetate, cyclophosphamide and a hamster thymocyte.
Claim 19
Dependent Claim
34 words
The process as claimed in claim 15, wherein said nutrient medium contains at least one member selected from the group consisting of sugars, amino acids, vitamins, hormones, proteins, antibiotics, growth factors and inorganic salts.
Claim 20
Dependent Claim
21 words
The process as claimed in claim 18, wherein animal serum, an antigen and/or a mitogen is incorporated into said nutrient medium.
Claim 21
Dependent Claim
31 words
The process as claimed in claim 19, wherein said animal serum is selected from the group consisting of fetal calf serum, neonatal calf serum, bovine serum, horse serum and human serum.
Claim 22
Dependent Claim
24 words
The process as claimed in claim 19, wherein said antigen is selected from the group consisting of purified tuberculin, diphtheria toxoid and herpes virus.
Claim 23
Dependent Claim
26 words
The process as claimed in claim 19, wherein said mitogen is selected from the group consisting of phytohemagglutinin, concanavalin A, staphylococcal enterotoxin A and galactose oxidase.
Claim 24
Dependent Claim
16 words
The process as claimed in claim 15, wherein said lymphokine is a macrophage migration inhibitory factor.
Claim 25
Dependent Claim
15 words
The process as claimed in claim 15, wherein said lymphokine is a macrophage activation factor.
Claim 26
Dependent Claim
15 words
The process as claimed in claim 15, wherein said lymphokine is a colony stimulating factor.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-06-22 | 1990-12-22 | 1991-06-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-22 | 1994-12-22 | 1995-06-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-22 | 1998-12-22 | 1999-06-23 | $7700.00 | Window Closed / Lapsed |