Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Fusion of cells
Reexamination Certificate
1999-04-01
2001-01-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Fusion of cells
C435S325000, C435S344000, C435S346000, C435S366000, C435S372000, C435S372200, C435S377000, C435S451000, C435S455000, C435S456000
Reexamination Certificate
active
06174726
ABSTRACT:
PATENT SPECIFICATION
The invention relates to a method for the preparation of a conditionally immortalized helper cell (fuseme), the hybridoma cells prepared using said fusemes as well as a method for immortalization of mammalian cells using the fuseme cells. Furthermore, the invention relates to the generation of T cells directed against tumor cells using a fuseme cell.
BACKGROUND OF THE INVENTION
Many medical or biological problems involve the requirement or desire to generate a great number of cells in vitro out of a starting material of few cells in order to conduct scientific experiments or to work with these cells in the context of a therapeutical intervention. The expansion of cells frequently imposes a problem since cells generally have a limited growth capacity. For example, this is also true for many tumor cells which often show a growth disadvantage in vitro as compared to normal cells making an expansion of these cells difficult [Lange (1998), Visonneau et al. (1995)]. One possibility to expand cells in vitro is to stimulate the cells with appropriate stimuli (cytokines, suitable feeder cells, stimulation of appropriate receptors on the cell surface, etc.) to enable the growth or the survival, respectively, of said cells. However, also in this case it is generally impossible to expand the cells to any number, and the cells will cease to grow after some time. Moreover, the stimulation conditions for many cell types are unknown to date so that this method may not be universally employed. Although the “culture” of different tumor cells in living immunodeficient experimental animals may be performed for a prolonged time [Visonneau et al. (1995)], this method is elaborate and, moreover, must be rejected principally for ethical reasons.
An elegant way to overcome this problem is genetical immortalization of the desired cells [Hubbard & Ozer (1995), MacDonald (1994)]. Indeed, some tumor cell lines have the capability of dividing practically indefinitely also in vitro, i.e. are immortalized. Previously, this led to the development of the technique of cell fusion between the tumor cells and the cells for which immortalization was desired resulting in the pioneering development of monoclonal antibodies [Köhler & Milstein (1975), Peters et al. (1988)). However, particularly for the immortalization of human cells no optimal fusion partners are available to date [Gordon (1989)]. For this reason, the method of fusing human cells with suitable cells of rodents is frequently used but the resulting hetero-species hybridomas have the unfavorable property of being cytogenetically unstable and to lose individual chromosomes (often the important ones and in the case of human/rodent hybridomas preferably the human ones) by time.
A group of promising human fusion partners is represented by Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs). In particular, these cells may be easily established [Walls and Crawford (1987)]. However, for many applications these cells have undesirable properties. Thus, the expression of some viral proteins results in an inhibition of the synthesis of immunoglobulins, a property which e.g. hinders the generation of human monoclonal antibodies. Besides, the antigens expressed by EBV in LCLs induce a strong immune response which may exceed all other immune responses. This is disadvantageous if it is desired to perform a fusion between a LCL and a tumor cell to employ the hybridoma afterwards in the stimulation of an immune reaction against antigens. However, another property of these cells has proven to be particularly favorable in the induction of an immune reaction. Regarding their immunostimulatory capabilities LCLs belong to the most potent cells available. Therefore, it may be expected that hybridomas of a tumor cell and a LCL also have all co-stimulatory molecules in addition to the tumor-specific antigens to efficiently induce an immune response.
It is an object of the present invention to provide a method enabling the preparation of cells which may be used in transferring the information for immortalization to another cell.
SUMMARY OF THE INVENTION
According to the invention, this object has been solved by the method characterized in more detail in claim
1
which serves to provide conditionally immortalized cells, in following referred to a “fuseme”. This cell may be regarded as a helper cell since it “helps” to immortalize another cell. The method of preparation of a conditionally immortalized immortalization-helper cell consists of at least the following steps:
(a) Introducing immortalizing genes into mammalian cells in a way that at least the expression and/or function of at least one of said genes may be controlled in order to obtain conditionally immortalized mammalian cells;
(b) introducing a least two selectable markers for positive and negative selection to enable the selection between fuseme cells, the cells to be immortalized using the fuseme cells and the cells immortalized by the fuseme cells;
(c) selecting for such cells (fusemes) carrying the selectable marker introduced in step (b) and being conditionally immortalized.
As the mammalian cells there may be employed any cell capable of being immortalized. Preferably, those cells are human cells or rodent cells. Examples for such cells are lymphocyte cells or fibroblast cells. As the lymphocyte cells for example B cells or T cells may be conditionally immortalized.
Conditional immortalization of mammalian cells is known per se [Kempkes et al. (1995), Wyllie et al. (1993)]. For this purpose, the genes necessary for the immortalization of a cell must be introduced into a mammalian cell. Introduction of the genes may be performed e.g. by infection with a virus containing the genes or by transfection of the DNA.
Genes capable of immortalizing cells are known per se. These include for example the immortalizing genes of EBV, adenoviruses, HTLV-1 or oncogenes. These genes may be introduced into the mammalian cells to be conditionally immortalized by means of vectors which vectors include viruses, plasmids, cosmids etc. according to the invention. The genes responsible for immortalization are in each case engineered to enable a conditional expression or regulation of the function of the immortalizing genes. For example, these genes may be deleted from a viral genome on which they are naturally present, for example from EBV, the information required for immortalization is introduced to a plasmid and the expression of the immortalizing genes on the plasmid or the function of these genes may be conditionally regulated using suitable methods which are presented by way of example in this application. Examples for vectors for introduction of the immortalizing genes are EBV, adenoviruses, retroviruses, foamy viruses, pox viruses or SV40 as well as vectors derived from said viruses. An example for a vector derived from EBV are mini-EBV vectors. An example for a pox virus is the vaccinia virus.
The immortalizing genes of the aforementioned viruses or the oncogenes enabling an immortalization, respectively, are examples for oncogenes known per se useful in the immortalization of mammalian cells which are c-myc, c-abl, c-ras, and combinations of these oncogenes; immortalizing genes of EBV are for example EBNA2 and LMP1.
The method according to the present invention provides a fuseme cell at least characterized by the following properties:
(a) mammalian cell which is conditionally immortalized;
(b) having at least two selectable markers for positive and negative selection enabling the selection between fuseme cells, cells to be immortalized and immortalized cells.
The fuseme cell described above can be used for preparing monoclonal antibodies producing cell line and for preparing a T/B hybridoma cell line.
The fuseme cells obtained by the invention described above are employed as helper cells to immortalize mammalian cells. For this purpose, a fuseme cell and a mammalian cell to be immortalized using the fuseme cell are contacte
Bornkamm Georg
Kempkes Bettina
Staege Martin
GSF-Forschungszentrum fuer Umwelt und Gesundheit GmbH
Schwartzman Robert A.
Townsend and Townsed and Crew LLP
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