NL8401779A - Tumour cells promoting hybridoma division - useful in hybridisation, cloning and/or mono-clonal antibody prodn. - Google Patents

Abstract

Cells and/ord cell supernatants capable of promoting division of hybridoma cells are claimed. The cells are tumour cells which secrete one or more factors which promote hybridoma division. S ecifically the tumour-cell line is ES-1, isolated from Ewing sarcoma cells by culturing in a 2% agarose tissue-culture medium comprising 90% PRMI-1640 Hepes, 10% foetal calf serum, 2 mM glutamine, 1 mM pyruvate, 0.005 mM 2-mercaptoerhanol 2-mercaptoethanol and antibodies, followed by isolating individual colonies and growing them in the tissue-culture medium. The resulting conditioned medium is designated ESGF.

Description

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Cells; supernatant of cells; method for promoting in vitro cell hybridization, cloning and / or production of monoclonal antibodies.

The invention relates to cells and / or supernatants of cells that promote the division of hybridoma cells, and to a method for promoting in vitro cell hybridization, cloning and / or production of monoclonal antibodies.

5 This is an article by Astaldi et al., J. Immunol. 125, no. 4, 1411-1414 (1980), human primary endothelial cells and their supernatant (HECS) are known to greatly enhance the growth of hybridoma cells and as feeder cells in culturing hybridoma cells in cultures containing a small number of hybrid cells , can service 10.

The hybridoma technique described by Kohler and Milstein, Nature 256, p. 495 et seq. (1975) allows the production of monoclonal antibodies.

The technique of producing monoclonal antibodies mainly comprises 3 phases; 1) fusion of cells of a myeloma cell line and lymphocytes from an animal or human immunized with the desired antigen to obtain hybridomas which secrete the desired antibodies and which further exhibit permanent growth in vitro.

2) Selection of the desired hybridoma clone by culturing a single cell (ie, "limiting dilution").

3) Live and share while retaining function of the selected monoclonal cell line in vitro for a longer time.

It has been found that the above-mentioned phases present a number of problems: 1) the fusion does not always proceed in the same way and in fact appears to be not very efficient (i.e. the number of hybrids obtained is often low).

2) "limiting dilution" requires the presence of "feeder" -30 cells; these often have a highly variable activity. Mouse macrophages, mouse thymocytes and human primary endothelial cells have been proposed as feeder cells.

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9 ¢. -2- 3) The hybridomas, obtained by fusion of human cells with mouse myeloma cells, often show very limited in vitro survival.

These problems are significantly alleviated when human primary endothelial cells and / or their supernatant are used according to the directions in the literature cited above.

The invention now provides another product to overcome the identified drawbacks, which product is characterized in that the cells are tumor cells capable of secreting one or more factors which promote the division of hybridoma cells.

It has been found that some tumor cells are able to secrete one or more factors that promote the division of hybridoma cells. Such cells in particular have tumor cells of the tumor cell line ES-t «. The said ability to promote the division of hybrido-15 cells is also inherent in the supernatant of such tumor cells.

The invention further provides a method for promoting in vitro cell hybridization, cloning and / or production of monoclonal antibodies, characterized in that hybridomas are grown in the presence of tumor cell supernatants, which may secrete one or more factors. promote division of hybridoma cells, preferably tumor cells of the tumor cell line ES-1, and / or in the presence of such tumor cells themselves, after they have been deactivated. The use of supernatant is highly preferred over the use of deactivated cells.

In comparison with the cells previously proposed as "feeder" cells, which are all primary cells, the tumor cells used according to the invention have the great advantage that they can be multiplied as a cell line with unlimited division activity in tissue culture, so that an unlimited and constant production of the hybridadamination promoting factor (s) can be realized. The previously proposed primary cells, on the other hand, cannot divide or only divide a limited number of times and then die, so cells with the ability to promote hybridoma have to be isolated again and again.

35 This is a laborious and expensive procedure.

In addition, the tumor cells and / or supernatant thereof used according to the invention appear to further improve the fusion, cloning and monoclonal antibody production compared to the known "feeder" cells, including the human endothelial cells. phases. A very high efficiency cloning is achievable 5 (80-100%).

The time required for the isolation of a stable hybrid (as a rule: a fusion fed by two cloning procedures) can be somewhat reduced in all these phases, from the application of the invention, from ± 2 months to 1 è. 1 $ month, because the hybridomas grow faster.

The invention will be explained in more detail by means of an example.

1. A patient's lesion in the deep soft tissue of the back and associated with a vertebra was surgically removed. After histological editing of part of this lesion, the pathologist 15 was able to diagnose it as an Ewing Sarcoma based on the morphology. In . In accordance with this diagnosis, the cells of the lesion Factor VIII related antigen were detected using an immunoperoxidase staining. The lesion was processed into a substantially single-cell suspension using collagenase (0.125%), after which these cells were seeded in tissue culture medium (90% RFMI-1640 Hepes, 10% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 5.10 ^ B-mercaptoethanol and antibiotics) containing tissue culture vessel.

After adhering to the bottom of the tissue culture vessel, the cells so seeded divide, after which the bottom matured with cells. Then the cells were detached from the bottom using a phosphate buffer, in which dissolved trypsin (2.5 gr / lr) and EDTA (1 mM). After adding 10 ml of tissue culture medium, centrifugation was performed (5 min, 200 g) and the resulting cell pellet was resuspended in tissue culture medium which had been semi-solidified (agarose medium) by adding 2% (w / v) agarose.

The purpose of this operation is to effect that preferentially the tumor cells, and not the equally low fibrclasts, divide in the semisolid culture medium and then give rise to the formation of tumor cell colonies floating in the agarose medium. Any tumor cell. . colony will come from a single cell. One tumor cell colony 35 was then isolated using a capillary and then grown in a tissue culture medium containing tissue culture vessel. The on these 8 4 0 1 7 7 9 ............................

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In principle, tumor cell culture obtained in an unlimited manner can be cultured indefinitely, but can also be stored in ampoules in liquid nitrogen.

The cell line thus obtained was designated the ES-1 cell line, and this cell line has been in continuous tissue culture for more than two years. The cells in this tissue culture are derived from the tumor cells in the original Ewing Sarcoma lesion, as they are morphologically similar, while also containing factor VIII related antigen (demonstrated by immunoperoxidase staining). The cells have been characterized chromosomally as of human origin. They contain a pseudo-diploid 10 number of chromosomes with a characteristic translocation (45, XX, -13, T (1; 4), T (10; 13) ·). The factor or factors produced by ES-1 cells which promote the division of hybridoma cells are secreted into the tissue culture medium during growth of the ES-1 cells and thus can be isolated as conditioned tissue culture medium.

This tissue culture medium conditioned by ES-1 cells is called ESGF.

2. Application of ESGF

A biological effect of ESGF is illustrated below. 350 hybridoma cells are divided into 96 microcultures in tissue culture medium semi-solidified using 2% agarose (aga-rose medium). The semisolid culture medium is used in this case to quantify the number of hybridoma cells (clones) growing into colonies. Extra is added to the agarose medium, respectively, nothing, 10% (volume / volume) ESGF, 5% ESGF, 10% culture supplement of a random (irrelevant) tumor line (GLC-1, a small cell lung carcinoma derived cell line) and 10% HECS (this is a culture supernatant of primary human endothelial cells. This hybridoma division is known to secrete promoting factors). The results (Table 1) show that ESGF induces at least a similar number of clones to growth as HECS, while no addition or addition of irrelevant tumor cell culture supernatant yields only the growth of a very limited number of clones. In addition to this, in terms of numbers acting at least in a similar manner from ESGF to HECS, the use of ESGF 35 as an added benefit has been shown to promote faster clone outgrowth (see Table 1),

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nothing* . . 10% ESGF 5% ESGP 10% HECS

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Number of colonies grown, after sowing 10 14 310 346 271 of 350 (3%) (4%) (86%) (99%) (77%)

Number of days required for colonies to grow to ± 10 ± 10 ± 15 of a similar diameter (0 0.1 - 1 mm)

The absolute number of colonies grown has been indicated. The percentages indicated in brackets indicate the cloning percentage.

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Claims (4)

1. Cells and / or supernatant of tumor cells, which promote the division of hybridoma cells, characterized in that the cells are tumor cells, which can secrete one or more factors, which promote the division of hybridoma cells. 2. Tumor cells of the tumor cell line ES-1, and / or supernatant thereof. 3. A method of promoting in vitro cell hybridization, cloning and / or production of monoclonal antibodies, characterized in that hybridomas are grown in the presence of tumor cell supernatant according to claim 1 or 2, and / or in the presence of deactivated tumor cells according to claim 1 or 2. Method according to claim 3, characterized in that tumor cell supernatant according to claim 1 or 2 is used. 8401779