Patent US4574116A successfully refreshed from Google Patents.
US4574116A
Expired / Lapsed
US
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Methods and cell lines for immortalization and monoclonal antibody production by antigen-stimulated B-lymphocytes
Assignee: Leland Stanford Junior University
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Filing: 1983-01-13
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Granted: 1986-03-04
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Expires: 2003-01-13
Abstract
Abstract Methods are provided for producing fusion partners which involve employing an immortalized human myeloma cell line sensitive to HAT and having an additional dominant selectable resistance marker and fusing the doubly marked human myeloma cells with a stable immortalized rodent myeloma cell line, desirably previously subjected to substantial chromosome damage, and isolating cells having a substantially complete chromosomal complement of the rodent cell and at least about one chromosome of the human cell having a gene expressing said resistance, thereby being resistant to a selective agent. The resulting heteromyeloma may be fused with high efficiency with human lymphocytes to produce monoclonal antibodies. The cell lines designated as A6 and 36 were deposited at the A.T.C.C. on Jan. 11, 1983 and given accession numbers CRL8192 and CRL8193, respectively.
Inventors
- 👤 Henry S. Kaplan
- 👤 Nelson N. H. Teng
- 👤 Kit S. Lam
- 👤 Francisco Calvo-Riera
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 21:33
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/10; C12N5/12; C12N5/16; C12N5/166; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
150 words
A method for producing a heteromyeloma comprising: fusing in a fusing medium (a) first human myeloma donor cells, which cells are sensitive to a first selective means and have a gene providing resistance to a second selective means originating from said gene being introduced into said donor cells in vitro with (b) rodent recipient myeloma cells capable of stable maintenance in vitro without significant secretion of their own immunoglobulin proteins and significantly less sensitive than said donor cells to said first selective means but sensitive to the second selective means to which said gene provides resistance, to provide a mixture of cells including recipient cells having at least one donor chromosome containing said resistance gene; growing said mixture of cells under selective conditions for sensitivity to said first and second selective means; and isolating heteromyelomas comprising recipient cells having at least one chromosome of said donor cells bearing said gene.
Claim 2
Independent Claim
150 words
A method for producing a heteromyeloma comprising: fusing in a fusing medium (a) first human myeloma donor cells, which cells are sensitive to a first selective means and have a gene providing resistance to a second selective means originating from said gene being introduced into said donor cells in vitro with (b) rodent recipient myeloma cells capable of stable maintenance in vitro without significant secretion of their own immunoglobulin proteins and significantly less sensitive than said donor cells to said first selective means but sensitive to the second selective means to which said gene provides resistance, to provide a mixture of cells including recipient cells having at least one donor chromosome containing said resistance gene; growing said mixture of cells under selective conditions for sensitivity to said first and second selective means; and isolating heteromyelomas comprising recipient cells having at least one chromosome of said donor cells bearing said gene.
Claim 3
Dependent Claim
54 words
A method according to claim 1, wherein said donor and recipient cells are HAT-sensitive and said recipient cells irradiated or otherwise treated prior to fusing so as to introduce significant damage to one or more essential chromosomes, wherein said recipient cells can retain a higher number of donor chromosomes as compared to undamaged cells.
Claim 4
Dependent Claim
18 words
A method according to claim 2, wherein at least one of said selective means is a cytocidal drug.
Claim 5
Dependent Claim
15 words
A method according to claim 2, wherein said first selective means is sensitivity to ouabain.
Claim 6
Independent Claim
19 words
A method according to any of claims 1, 2, 3 or 4, wherein said rodent cells are mouse cells.
Claim 7
Dependent Claim
23 words
A hybridoma prepared by fusing a heteromyeloma produced according to the method of claim 1 with an activated B-lymphocyte or EBV transformed B-lymphocyte.
Claim 8
Independent Claim
115 words
A method for producing a heteromyeloma of human and mouse cells comprising fusing in a polyethylene glycol containing fusing medium (a) human myeloma cells which are sensitive to HAT and have a gene providing resistance to G-418 as a result of introduction of said gene in vitro with (b) mouse HAT-sensitive myeloma cells capable of stable maintenance in vitro without secretion of mouse immunoglobulins and sensitive to G-418 to provide heteromyeloma cells containing a major complement of mouse chromosomes and at least one human chromosome bearing said gene; growing said mixture of cells in the presence of ouabain and G-418, whereby only heteromyeloma cells, which are HAT-sensitive, survive; and isolating and cloning said human-mouse heteromyelomas.
Claim 9
Independent Claim
115 words
A method for producing a heteromyeloma of human and mouse cells comprising fusing in a polyethylene glycol containing fusing medium (a) human myeloma cells which are sensitive to HAT and have a gene providing resistance to G-418 as a result of introduction of said gene in vitro with (b) mouse HAT-sensitive myeloma cells capable of stable maintenance in vitro without secretion of mouse immunoglobulins and sensitive to G-418 to provide heteromyeloma cells containing a major complement of mouse chromosomes and at least one human chromosome bearing said gene; growing said mixture of cells in the presence of ouabain and G-418, whereby only heteromyeloma cells, which are HAT-sensitive, survive; and isolating and cloning said human-mouse heteromyelomas.
Claim 10
Dependent Claim
58 words
A heteromyeloma cell derived from a recipient rodent myeloma cell and a donor human myeloma cell, comprising a cell having at least a substantially complete rodent genome, and at least one chromosome from a human cell carrying a gene originating from in vitro introduction imparting resistance to a selective means to which said rodent myeloma cell is sensitive.
Claim 11
Dependent Claim
58 words
A heteromyeloma cell derived from a recipient rodent myeloma cell and a donor human myeloma cell, comprising a cell having at least a substantially complete rodent genome, and at least one chromosome from a human cell carrying a gene originating from in vitro introduction imparting resistance to a selective means to which said rodent myeloma cell is sensitive.
Claim 12
Dependent Claim
16 words
A heteromyeloma cell according to claim 8, wherein said rodent myeloma cell is a mouse cell.
Claim 13
Dependent Claim
45 words
A heteromyeloma according to claim 8, wherein the genome of said rodent myeloma cell has sustained significant chromosome damage originating from exposure to ionizing radiation or radiomimetic chemical agents, wherein said cell can retain a higher number of donor chromosomes as compared to undamaged cells.
Claim 14
Dependent Claim
37 words
A heteromyeloma according to any of claims 8, 9 or 10, wherein said rodent cell is the mouse myeloma line P3X63-Ag 8.653-NP and said human chromosome is a human chromosome from mutant human myeloma cell line U-266.
Claim 15
Dependent Claim
18 words
A heteromyeloma according to any of claims 8, 9 or 10, wherein said gene imparts resistance to G-418.
Claim 16
Dependent Claim
19 words
A hybridoma prepared by fusing a heteromyeloma according to claim 8 with an activated B-lymphocyte or EBV transformed B-lymphocyte.
Claim 17
Independent Claim
42 words
A method for producing human monoclonal antibodies which comprises: fusing a heteromyeloma having at least one chromosome from a human myeloma cell in a fusing medium with a lymphocyte from a human; and selecting for hybridomas secreting the desired human monoclonal antibodies.
Claim 18
Independent Claim
42 words
A method for producing human monoclonal antibodies which comprises: fusing a heteromyeloma having at least one chromosome from a human myeloma cell in a fusing medium with a lymphocyte from a human; and selecting for hybridomas secreting the desired human monoclonal antibodies.
Claim 19
Dependent Claim
16 words
A method according to claim 14, wherein said heteromyeloma is derived from mouse myeloma recipient cells.
References Cited (Backward Citations)
GB2079313A
Cited
EP0057107A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-03-03 | 1989-09-02 | 1990-03-04 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-03-03 | 1993-09-02 | 1994-03-04 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-03-03 | 1997-09-02 | 1998-03-04 | $7700.00 | Window Closed / Lapsed |