EP0207147A4 - Procede d'immortalisation electrique de cellules lymphoides. - Google Patents

Procede d'immortalisation electrique de cellules lymphoides.

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Publication number
EP0207147A4
EP0207147A4 EP19860900562 EP86900562A EP0207147A4 EP 0207147 A4 EP0207147 A4 EP 0207147A4 EP 19860900562 EP19860900562 EP 19860900562 EP 86900562 A EP86900562 A EP 86900562A EP 0207147 A4 EP0207147 A4 EP 0207147A4
Authority
EP
European Patent Office
Prior art keywords
cells
cell
oncogene
human
myc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19860900562
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German (de)
English (en)
Other versions
EP0207147A1 (fr
Inventor
Robert L Lundak
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TECHNICLONE RES PARTNERS
Original Assignee
TECHNICLONE RES PARTNERS
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Application filed by TECHNICLONE RES PARTNERS filed Critical TECHNICLONE RES PARTNERS
Publication of EP0207147A1 publication Critical patent/EP0207147A1/fr
Publication of EP0207147A4 publication Critical patent/EP0207147A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • This invention relates to a method for preparing immortalized lymphoid cell lines through the introduction of cloned cellular oncogenes by high voltage electric fields, and to immortalized lymphoid cells prepared by that method.
  • Immortalized cells have the ability to grow indefinitely in vitro. These immortalized cells often retain the ability to produce specialized proteins, such as im unoglobulins. A culture grown from a single immortalized antibody-producing cell produces a monoclonal antibody. Human lymphoid cells have proven to be remarkably recalcitrant to immortalization by many of the techniques that have been successful in rodent and other models.
  • Such fusions have generally been performed using polyethylene glycol or dextran sulfate as mediators.
  • the resulting hybrids (hybridomas) are separated and grown in individual cultures which individually produce monoclonal antibody.
  • Another fusion technique that has recently been employed is electrofusion.
  • electrofusion In electrofusion the two types of parental cells are placed in a non-conducting isoosmotic environment. An alternating current is used to electrically align the cells to be fused. The membranes are compressed, and fusion occurs when the cells are subjected to brief pulses of high voltage direct current pulses. See, e.g., U. Zimmerman & J. Greyson, "Electric Field-Induced Cell Fusion", Biotechniques, September/October 1983: 118-122.
  • fusion techniques have significant limitations.
  • immortality of the antibody-producing cells is the result of the introduction of a vast amount of genetic information, only a minute amount of which is required for growth and immortality.
  • a few genes are responsible for the immortality of the resulting hybridoma, virtually all the genetic material of the immortal parent is transferred to the hybrid.
  • Another disadvantage of the fusion technique is that fusion is not selective, and unproductive cell fusion occurs between either of the two parents and between more than two cells.
  • Epstein Barr Virus transformation and RNA tumor virus transformation.
  • EBV Epstein Barr Virus
  • RNA tumor virus transformation Another method for immortalizing human lymphocytes.
  • EBV Epstein Barr Virus
  • N. Brown & G. Miller "Immunoglobulin Expression by Human B-Lymphocytes Clonally Transformed by Epstein Barr Virus", J. Immunology 128: 24- 29 (1982).
  • the human lymphocyte is infected with a B-cell-specific virus.
  • the viral transformation leads to immortalization and growth of the ⁇ cells.
  • a disadvantage of this method is the possible synthesis of contaminating viral products as well as the virus itself and, as yet, little antibody synthesis.
  • current data demonstrate that long-term growth resulting from EBV infection is the result of only a small amount of the viral genome.
  • retrovirus vectors such as RNA tumor viruses which have been genetically manipulated so that they carry the gene of interest but are unable to reproduce themselves.
  • the viruses used can infect and stably integrate with the human B-lymphoblast genome.
  • the use of this method while a great improvement over those described above, has received criticism for several reasons.
  • the viruses are produced in the presence of a helper virus which could conceivably lead to continued propagation and reinfection of cells.
  • cells may contain latent, quiescent, nonreplicative virus of unknown origin and virulence.
  • the presence of RNA virus genome from the infecting virus could conceivably cause these latent or nonreplicative virus to become corrected or activated.
  • construction of retrovirus vectors is a highly sophisticated technology requiring a significant amount of expertise. Accordingly, one of the objects of the present invention is to introduce genes into cells which convey immortality without risk of viral replication.
  • Another object of the present invention is to provide a technique for immortalizing B-lymphoid cells using only the DNA necessary for immortalization.
  • Still another object of the present invention is to provide stable, monoclonal antibody-producing cell lines that have been immortalized with an oncogene.
  • the present invention provides a method for immortalizing lymphoid cells by electrically inserting immortalizing DNA into a target cell.
  • This method comprises the steps of placing the lymphoid cells between two electrodes, adding deoxyribonucleic acid segments containing an oncogene to the cells, and immortalizing at least some of the cells by applying an electric potential to the electrodes to create an electric field of from about 1 to about 40 kV/cm in a pulse of about 0.1 nanoseconds to about 10 milliseconds, to introduce the deoxyribonucleic acid into at least some of the cells.
  • immortalized cells produced according to the foregoing methods are provided.
  • This invention also includes human lymphoid cells having an exogenous cellular oncogene (preferably human c-myc) incorporated into the genome thereof to immortalize the cell.
  • Those immortalized cells are preferably in the form of individual cell lines capable of producing monoclonal antibody.
  • DNA or "DNA polymer” shall mean a polymer of deoxyribonucleic acids.
  • naked DNA shall mean DNA that is not contained in an organism or structure such as a cell, a virus, or a vesicle. Naked DNA is usually free floating DNA in a solution or suspension.
  • exogenous DNA when used in reference to a cell shall mean DNA from a source external of that cell or of the progenitors thereof.
  • cellular when used to modify "oncogene” shall mean an oncogene taken from a cellular (as opposed to a viral) source.
  • immortal or “immortalized”, when used to describe a cell, shall mean that the cell has acquired the ability to grow indefinitely in vitro without exogenous growth factors. Immortalization requires stable growth for a period of at least several months. Mere production of foci does not demonstrate immortalization. Such immortal cells are usually grown in suspension in vitro.
  • the oncogenes used in the present invention are isolated from mammalian cells, preferably human cells.
  • the ability of any particular oncogene to immortalize lymphoid cells when incorporated into the genome of those cells using the techniques of the present invention can be readily determined by cloning the gene using standard techniques, inserting the cloned gene into a lymphoid cell using the electrical technique described herein, culturing the lymphoid cells so treated, and testing the resulting cell lines for stable growth and protein production.
  • the preferred immortalizing oncogene for use in the present invention is the c-myc oncogene. Procedures have been described for the isolation of the human c-myc oncogene (Dalla-Favera, R. Et al., (Nature 299:61-63
  • the human c-myc gene has been mapped to chromosome #8 and restriction enzyme fragments compared to mouse (Dalla Favera, R. et al Proc Nat'l Acad. Sci. U.S.A. 79:7824-7827 (1982)) and the nucleotide sequence and restriction enzyme digestion and fragment analysis described and compared to having c-myc and v-myc (Watson, D.K. et al Proc Nat' 1 Acad. Sci. U.S.A. 80:3642-3645 (1983)).
  • the human c-myc gene is isolated by the knowledge that high molecular weight human genomic DNA prepared by cell lysis, proteinase K digestion, extraction with phenol, and ethanol precipitation (Levis, R. and S. Penman J. Molec. Biol. 121:219-239 (1978); Wahl, G.M. et al. Proc Nat'l Acad. Sci. U.S.A.
  • the 8.2 Kb EcoR1/Hind III fragment is isolated from the majority of the DNA by adaptation of standard molecular cloning procedures (Maniatis, T. et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) and cloned into pUC8 or pBR322 plasmids using Escherichia coli DH1.
  • the restriction enzyme digest is separated by low melting agarose gel electrophoresis.
  • the 8.2Kb area is cut out, heated to 65° to melt agarose, and the DNA fragments extracted with phenol and recovered by ethanol precipitation.
  • the EcoR1/Hind III fragments are identified by using conventional 0.8% agarose gel electrophoresis for separation and Southern blot transfer (Southern E.M. J. Mol Biol. 98:503-517 (1975)) using acid pretreatment (Wahl, G.M. et al. Proc Nat'l Acad. Sci. U.S.A. 76:3683- 3687 (1979)) and hybridization to nick-translated (Ridby, P. . J. Mol. Biol. 113:237-251 (1977)) myc-gene probe (Oncor, Gaithersburg, M.D.). Optimum stringency conditions (hybridization temperature and formamide concentrations) for reaction of this probe against the 8.2 Kb fragment area of the EcoR1/Hind III are developed according to manufacturers suggestions.
  • the 8.2Kb EcoR1/Hind III restriction fragments are ligated into plasmids pUC8 or pBR322 by cutting the parent plasmid vector with EcoR1 and Hind III restriction enzymes and carrying out the ligation reaction with an excess of the 8.2 Kb fragment (i.e., 10 ⁇ g/2 ⁇ of plasmid) and transforming Escherichia coli DH1 (Hanahan, D. J. Mol. Biol. 166:557-580 (1983)) and selecting for ampicillin resistance.
  • the DH1 host is plated at 100,000 cells/20 cm plate and viable colonies are replicated to filter paper (Gergen, J.P. Nucleic Acid Research 7:2115-2135 (1979)) and hybridized against the myc gene probe.
  • the positive areas are scraped into broth cultures and recultured on a picillin containing plates to give spacing.
  • the clones are arrayed and rescreened against the myc gene probe and single colonies are isolated.
  • the 8.2 Kb fragments from the EcoR1/Hind III areas of soft agarose electrophoresis gels can be cloned before plasmid construction by using coliphage lambda charon vector 28 (Rimm, D.L. et al. Gene 12:301-309 (1980)).
  • the appropriate colonies carrying the plasmids containing the 8.2 Kb c-myc gene insert are identified by Southern blot analysis against the myc gene probe of gel electrophoresed samples of EcoR1/Hind III digested plasmid minipreps.
  • the identity of the c-myc gene is established by diagnostic restriction fragment analysis (Watson, D.K. Proc Nat'l Acad. Sci. U.S.A. 80:3642-3645 (1983)); Dalla-Favera, R. Proc Nat'l Acad. Sci. U.S.A. 79:6497-6501 (1982)).
  • Plasmid DNA containing the gene or gene fragments to be used in the electroporation method are prepared by mass culture of the appropriate DH1 stains in ampicillin containing broth, and chloramphenicol amplification of the culture, lysozy e-Triton X-100 lysis of cells (Katz, L.D. et al. J. Bacteriol. 114:577-591 (1973)) and cesium chloride-ethidium bromide equilibrium centrifugation (Radloff, R.W. et al. Proc Nat'l Acad. Sci U.S.A. 57:1514- 1521 (1967)).
  • the isolated naked DNA containing the desired oncogene may be inserted into the lymphoid cell in the plasmid vector. However, transformation and immortalization is more effective when the oncogene is inserted as a linear segment of naked DNA. Appropriate restriction enzymes may be used to linearize and isolate the cloned gene from the plasmid.
  • the c-myc gene may be isolated from pBR322 as an 8.2 Kb segment using Hind III and EcoR1.
  • Cells that can be immortalized using the techniques of the present invention include lymphoid cells, preferably of human origin.
  • Preferred cells are human B-lymphocytes that produce a desired protein or polypeptide.
  • proteins and polypeptides include antibodies to particular antigens, T-cell growth factor, and B-cell growth factor.
  • a cell suspension containing the desired lymphoid cells is prepared.
  • a relatively nonconducting suspension such as about 0.3M mannitol, sorbitol, glucose, sucrose, or histidine is preferred.
  • the naked DNA segments containing the oncogene are added to the suspension, and the suspension is placed between two electrodes. Good results are obtained when the electrodes are spaced 0.3-1.0 cm apart.
  • a standard electrofusion chamber containing such electrodes, such as the electrofusion chamber manufactured by Maxwell Manufacturing Co. of San Diego, can be used.
  • Insertion of the DNA into the lymphoid cells is accomplished by delivering at least 1 and preferably 2 to 7 electric pulses to the cell suspension containing the naked oncogene.
  • the use of from 3 to 5 pulses is
  • the pulse length may be from about 0.1 ns to about 10 ms. Usually the pulse length is about five to about fifty ⁇ s.
  • the pulse may be generated by any conventional technique, such as by use of a pulse transformer and/or capacitor discharge. Several commercially available pulse generators in standard
  • electrofusion equipment such as that developed by Maxwell Manufacturing Co. of San Diego, can provide suitable electric pulses.
  • Another suitable pulse generator is Hewlett Packard Model 214B.
  • the cell suspension is allowed to sit for about 10 to 15 minutes at a low temperature, e.g., 4°C or at room temperature. It is believed that the rest period is required for a return of the electroporated cell membrane to a normal condition, during which time the cell membrane remains permeable to the naked exogenous DNA.
  • the cell suspension is then diluted and seeded into an appropriate culture medium. After incubation at 37°C for a number of days, growth of the immortalized cells will be observed. Single colony isolates may then be obtained and the particular cells producing the desired protein may be identified and cultured using standard techniques.
  • This technique is particularly important in obtaining monoclonal antibody-producing cell lines of human origin. Because of ethical constraints, conventional techniques for obtaining cell samples from animals in which a comparatively large numbers of lymphoid cells produce the desired antibody cannot be used for obtaining human cells. Therefore, a technique that exhibits high transformation efficiency is particularly valuable, because the concentration of the desired cell in the human cell suspension is extremely low.
  • the cells produced according to the present invention have an exogenous cellular oncogene incorporated into the genome thereof, making the cell immortal.
  • Such immortalized human lymphoid cells are considered a part of the present invention.
  • the antibody-producing cells resulting from this invention differ from those resulting from previous methods.
  • No known transforming virus or viral genome has been introduced.
  • Oncogenes are thought to provide natural signals which are used during differentiation and maturation of cells. Normally, their expression is controlled by the location or proximity to normal control regions or signals in the chromosome. In the transformed cells of the present invention, it is believed that the oncogenes have simply integrated into a location where expression is not prevented by normal cellular control mechanisms. There have been no alterations in the chromosomal load; i.e., no additional chromosomes have been introduced. EXAMPLE
  • a fresh tonsil was dispersed into cell suspension by passing through a garlic press.
  • the resulting tonsilar cell suspension in Hank's buffered salt solution was stored at 4°C until use.
  • a recombinant pBR322 plasmid containing the human c-myc gene isolated from the promyelocytic leukemia cell line HL-60 was obtained as described in D. Favera, et al., "One Gene Amplification in Promyelocytic Leukemia Cell in HL-60 and Primary Leukaemic Cells of the Same Patient" , Nature 299: 61-63 (1982) and D.
  • Tonsil cells prepared as above were centrifuged at 500 xG for five inutes.
  • Hanks buffer was removed by aspiration and the cell pellet was resuspended in cold, sterile 0.3M mannitol, pH 7.2, to a final cell density of 2-5 x 10° cells/ml.
  • the c-myc gene or plasmid containing the gene [approximately 1 mg/ml in 10mM Tris-HCl, 0.5mM sodium EDTA, pH 7.4] was added to the cell suspension. Generally 10 ⁇ l was added, which represents 10 ⁇ -** to 10 ⁇ genes per ml.
  • the suspension was gently mixed and transferred to a multiple pre-cooled stainless steel electrofusion chamber made by Maxwell Corporation of San Diego.
  • the lymphocytes were rendered permeable to the c-myc genes or plasmids by four electric pulses creating a field strength between the electrodes of of 4 kV/cm to 7 kV/cm. The pulse lengths were 5 to 50 ⁇ sec. The suspension was gently mixed for 10 to 20 seconds between pulses.
  • the pulsed cell suspension was allowed to sit on ice at about 4°C for approximately 15 minutes.
  • the suspension was then diluted 1:10 to 1:15 with RPMI-1640 medium containing 10 percent fetal calf serum (FCS) .
  • FCS fetal calf serum
  • the resulting diluted suspension W s carefully centrifuged and resuspended in the RPMI-10% FCS to 5 x 10 5 to 10 6 cells/ml.
  • One ml aliquots then were seeded into each of the wells of a 24-well culture dish.
  • the cultures were incubated at 37°C under a five percent CO2 atmosphere until growth was apparent; generally 7 to 10 days.
  • the cultures were expanded, were media tested for presence of immunoglobulin, and permanent collections were established by freezing at -70°C.
  • Single colony isolates were obtained by standard soft agar or limiting- dilution cloning techniques. The clones were expanded and characterized with regard to growth properties, antibody production
  • the immortalized monoclonal-antibody producing cell lines have exhibited stable growth and antibody production for over four months and continue to exhibit such growth and antibody production.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
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  • Wood Science & Technology (AREA)
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  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
EP19860900562 1984-12-21 1985-12-19 Procede d'immortalisation electrique de cellules lymphoides. Withdrawn EP0207147A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US68467184A 1984-12-21 1984-12-21
US684671 1996-07-22

Publications (2)

Publication Number Publication Date
EP0207147A1 EP0207147A1 (fr) 1987-01-07
EP0207147A4 true EP0207147A4 (fr) 1988-07-14

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DE3537261A1 (de) * 1985-10-19 1987-04-30 Gca Corp Verfahren und medium zum feldinduzierten einschleusen von makromolekuelen in lebende zellen
NZ219392A (en) * 1986-02-28 1989-05-29 Smithkline Beckman Corp Production of an immortalised primary cell line
DE3627326A1 (de) * 1986-08-12 1988-02-18 Boehringer Mannheim Gmbh Immortalisierung durch dns-uebertragung
US4956288A (en) * 1988-04-22 1990-09-11 Biogen, Inc. Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA
GB8928884D0 (en) * 1989-12-21 1990-02-28 Feldmann Marc Transformed cell lines
CA2030933A1 (fr) * 1990-05-04 1991-11-05 Rex G. Risser Plasmocytomes a antigene specifique et anticorps derive desdits plasmocytomes
US5244656A (en) * 1990-05-04 1993-09-14 Wisconsin Alumni Research Foundation Antigen specific plasmacytomas and antibodies derived therefrom
US5888791A (en) * 1996-01-31 1999-03-30 Ipr Institute For Pharmaceutical Research Ag Method of producing bacteriorhodopsin and carotenoids by electrostatic treatment of Halobacterium halobium
WO2001049839A2 (fr) * 2000-01-07 2001-07-12 Umeda Jimusho Ltd. Procede d'expression genique et son utilisation
US20030170898A1 (en) * 2001-12-04 2003-09-11 Gundersen Martin A. Method for intracellular modifications within living cells using pulsed electric fields
IN2014DN06624A (fr) 2005-10-18 2015-07-10 Univ Colorado
US8986702B2 (en) 2008-05-16 2015-03-24 Taiga Biotechnologies, Inc. Antibodies and processes for preparing the same
DK2966084T3 (en) 2008-08-28 2018-08-06 Taiga Biotechnologies Inc MODULATORS OF MYC, PROCEDURES FOR USING SAME AND PROCEDURES FOR IDENTIFYING SUBSTANCES MODULATING MYC
AU2013292330B2 (en) 2012-07-20 2018-07-12 Htyr Acquisition Llc Enhanced reconstitution and autoreconstitution of the hematopoietic compartment
US10272115B2 (en) 2013-03-11 2019-04-30 Taiga Biotechnologies, Inc. Production and use of red blood cells
CN113786476A (zh) 2016-12-02 2021-12-14 泰加生物工艺学公司 纳米颗粒调配物
US10149898B2 (en) 2017-08-03 2018-12-11 Taiga Biotechnologies, Inc. Methods and compositions for the treatment of melanoma
CN113874033A (zh) 2019-04-08 2021-12-31 泰加生物工艺学公司 用于冷冻保存免疫细胞的组合物和方法
JP2022532608A (ja) 2019-05-14 2022-07-15 タイガ バイオテクノロジーズ，インク． Ｔ細胞疲弊を処置するための組成物および方法

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DE3023787A1 (de) * 1980-06-25 1982-01-21 Studiengesellschaft Kohle mbH, 4330 Mülheim Verfahren zur erhoehung der inkorporation und der expression von genetischem material in die kerne von intakten zellen mit hilfe von liposomen
US4465769A (en) * 1981-12-11 1984-08-14 The United States Of America As Represented By The Department Of Health And Human Services Non-transformed thymidine kinaseless cell line and its use for testing tumorigenic potential of genes

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HYBRIDOMA, vol. 3, no. 2, 1984, pages 107-118, Mary Ann Liebert Inc. Publishers; Z.L. JONAK et al.: "Production of continuous mouse plasma cell lines by transfection with human leukemia DNA" *
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See also references of WO8603780A1 *

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JPS62501537A (ja) 1987-06-25
WO1986003780A1 (fr) 1986-07-03
EP0207147A1 (fr) 1987-01-07

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