JPH0679553B2 - Novel hybrid cell - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel hybrid cell (hybridoma) and a method for producing the same. More specifically, a novel fusion of tumor cells other than bone marrow cell-derived tumor cells, in particular, a new cell line for producing hybrid cells derived from human tumor cells and an animal cell containing human (active lymphocyte) as a parent cell The present invention relates to a hybrid cell (hybridoma) and a method for producing the hybrid cell.

The hybrid cells of the present invention include tumor cells other than bone marrow cell-derived tumor cells, particularly human tumor cells, for example, human melanoma, which is a novel cell line derived by artificial or natural mutation from a parent cell, while human Cells, eg, B cells of an allogeneic or xenogeneic animal sensitized with a particular immunogen,
Can be prepared by fusing both as another parent cell. The hybrid cell of the present invention can be used for production of monoclonal antibodies and other lymphokines having useful physiological activities.

In recent years, hybrid cells (hybridomas) capable of proliferating in vitro and capable of producing antibodies against a specific immunogen (antigen)
Has been extensively studied to produce uniform and highly specific antibody (monoclonal antibody) secreted by the hybridoma as a diagnostic agent or therapeutic agent, and immunology, biology, medicine, pharmacy Has received much attention in such fields.
The method of producing such hybridomas and the concept of using hybridomas were generalized by the University of Cambridge.
Ceaser Milstein et al. (Milstein C.et.al Nature
256, 495, 1975). They first prepared a mutant strain (P3-X63-Ag8) from mouse myeloma (PBK strain) given by Leo sacks of the Salk Institute, and then fused it with mouse splenocytes immunized with sheep red blood cell antigen. Hybridomas were prepared. It was clarified that the hybridoma was able to grow in vitro and produced a monoclonal antibody against sheep erythrocytes (MoAb).

Since the research by Milstein et al. In 1975, many researchers have conducted research on hybridomas that produce MoAbs against specific antigens. In both cases, parent cells used for hybridoma production are myeloma (myeloma) cells and B cells. Tumor cells derived from so-called bone marrow cells derived from cells. The genetic characteristics of tumor cells such as myeloma and the principle of hybridomas, which are necessary for the production of hybridomas, which are generalized below, will be described in detail.

In vitro proliferative parent cells (tumor cells) used for the production of hybridomas are all hypoxanthine-guanine phospho-olivosyl transfruase (Hypoxanthine).
-Guanine phosphoribosyl transferase (hereinafter abbreviated as HGPRT) deficient strain or thymidine kinase
se) deficient strain. Since the genetic biochemical mechanisms of both defective strains are the same in principle, here we describe general HGPRT defective strains. HGPRT is one enzyme responsible for DNA synthesis by the salvage cycle in the DNA synthesis pathway of all cells. In other words, HGPRT is a DNA synthesis (d) that uses purine and pyrimidine as substrates.
When the e novo cycle) is inhibited by some kind of inhibitor (eg aminopterin), the salvage cycle acts as a rescue pathway to perform DNA synthesis and maintain cell life. To do. Therefore, the HGPRT-deficient strain cannot survive in hypoxanthine-aminobuterin (inhibitor of de novo cycle) -thymidine medium (hereinafter abbreviated as HAT medium) because aminopterin is present in the medium. On the other hand, the other parental cell used for hybridoma production, such as splenocytes (B cells)
Has DNA synthesis pathways for both de novo and salvage circuits. Therefore, the hybridoma obtained by cell fusion with HGPRT-deficient strain grows by hypoxanthine utilizing DNA synthesis by the salvage cycle derived from splenocytes even when the de novo cycle is inhibited by aminopterin in HAT medium. You can That is, a hybridoma has both in vitro proliferation ability from parent cells such as myeloma and DNA synthesizing ability from splenocytes by a salvage circuit, and moreover, from splenocytes (B cells) Since it inherits the information to produce immunoglobulin (antibody) against the antigen, it can grow in HAT medium and secrete immunoglobulin.

The HGPRT-deficient strain, which is a parent strain necessary for hybridoma production, is selected as a strain that is resistant to 8-azaguanine (8-azaguanine), which is usually produced by an appropriate mutation treatment, and has a property that it cannot grow in a HAT medium. As the parent cell,
It has become established that bone marrow-derived tumor cells such as myeloma and leukemia B cells are used. However, these tumor cells that can be converted into cell lines for producing hybridomas are mainly mouse or rat, and there are very few cases of human myeloma. The reason for this is that in human myeloma, it is difficult to prepare a cell line that can be used for HAT medium selection (hereinafter referred to as HAT selection), and because the original intended growth capacity of parent cells is weak, hybridomas are prepared. Even if it does, the hybridoma has a very weak proliferative ability and is unstable, and the amount of the target immunoglobulin secreted is small.

Therefore, at present, the use of immunoglobulins from so-called animal-animal hybridomas such as mice and rats is unavoidable. However, since the immunoglobulin produced by an animal-animal hybridoma is a protein of a different animal for humans, its antigenicity becomes a serious problem when used for humans. Therefore, human-human (hu
Immunoglobulins from (human-human) hybridomas are expected. In addition, large-scale culture of hybridomas from human myeloma presents many problems in terms of instability of hybridomas, as described above.

Usually, myeloma cells secrete a large amount of immunoglobulin called myeloma protein in the culture medium (however, the secreted immunoglobulin is unknown to which antigen, and produces an antibody to a specific antigen). It is impossible to select myeloma cells.) Recently, it has been revealed that the presence of myeloma proteins complicates the screening of hybridomas and the purification of antibodies, so that myeloma cells secreted to non-secreted may be used. It has become. That is, as a property of a parent cell for producing a hybridoma, the growth ability in vitro has been focused.

From the above situation, when a large amount of immunoglobulin is stably produced in vitro in a human-derived cell line that is capable of HAT selection after cell fusion with strong proliferative ability as a parent cell for hybridoma production, The appearance of hybridomas is expected. Parental cells include myeloma and B cell-derived cancerous cells (Epstein Barr virus)
(Including transformed cells by EBV) have been established, but these parental cells have various drawbacks, as mentioned above.

Therefore, the present inventors have found that any cells can be used for hybridoma production as long as the parent cells have high proliferative ability in vitro, and as a result of earnest research, they completely compensate for the defects of conventional parent cells. While establishing a new type of human cell line, it was clarified that the cell line is practically applicable as a parent cell of a hybridoma that stably produces an antibody in vitro. The novel cell line in the present invention is a cancerous cell derived from a different source from bone marrow cells and B cells, skin cancer (melanoma), liver cancer, gastric cancer, intestinal cancer, lung cancer,
Tumor cells of breast cancer, etc. Particularly preferably, human melanoma-derived strains induced by natural or artificial mutation (specifically, genetically deficient strains lacking the production of one of the enzyme groups involved in the DNA synthesis pathway). Is. These strains have stronger in vitro proliferative ability than conventional cell lines derived from tumor cells derived from bone marrow, and further, for example, the hybridoma of the present invention obtained by fusion with antibody-producing cells (B cells) is It has the same proliferative capacity as parent cells and can stably secrete immunoglobulin.

As described above, the tumor cell line, which is a parent cell used for preparing the hybridoma of the present invention, particularly the human tumor cell line, is obtained by fusion with splenocytes (B cells) from a heterologous animal sensitized with a specific antigen. Not only is it very useful for the production of hybridomas that produce specific antibodies, but since this cell line is of human origin, it can also be used for the production of human-human hybridomas among the hybridomas included in the present invention. In this case, the cells sensitized as one of the parent cells include human peripheral blood, tonsil, lymph nodes,
B cells from the spleen or the like may be used. Further, since the immunoglobulin secreted from human-human hybridoma is a human protein, there is no problem of antigenicity when used in human. Furthermore, when a cell line for producing the hybridoma of the present invention is used, the human hybrid T-cell line capable of producing lymphokines such as gamma-type interferon is used as a parent cell for the hybridoma of the present invention (T-ce).
ll hybridoma) can also be produced. As described above, the cell line of the present invention can be widely used, and the activated lymphocytes in the present specification are B cells, which are cells derived from animals including humans, which have been sensitized by the immunogen. In the case of T cells, it refers to cells that can produce lymphokines.

Hereinafter, the production of the hybridoma of the present invention and the isolation of the monoclonal antibody (MoAb) from this hybridoma culture will be described. For simplification of description, a method for producing a hybridoma using human tumor cells and human splenocytes is disclosed, but the present invention is not limited thereto.

The production of a parent cell line for producing hybridomas will be described below.

First, the first step required to obtain human parent cells that can be used for hybridoma production is the salvage cycle enzyme (HGP) in the DNA synthesis pathway that can be used by HAT selection.
RT) deficient strain production. One is 8-
Azaguanine (8-Azaguanine) was added to the cell culture medium, and the emergence of the 8-azaguanine resistant strain was observed.
This is the end of step. Another method of the first step is to use ultraviolet light or a mutagenic agent such as ethylmenthane sulfonate, (EMS) N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) depending on the type of cancer cells.
After irradiation with an appropriate amount (GL-15, directly under 30 cm for 15 seconds to 10 minutes) or addition (0.05 to 10 μg / ml), the cells are transferred to a medium containing 8-azaguanine. Many of the mutagens are unstable, but the mutagens are cultured for several hours to 3 days, and after treatment, they are thoroughly washed with a serum-free medium and the cells are transferred to a medium containing 8-azaguanine. Also in this case, the first step is terminated with the appearance of the 8-azaguanine resistant strain.

Next, we will examine whether HAT selection can be used. After culturing the cells, the medium is removed by centrifugation, washed twice with HAT medium and replaced with the same HAT medium (10 ⁵ / ml). Observation is carried out for 5 days to determine life or death. Judgment is made by Trypane blue staining. If most of the cells die by this method, replace the cells (10 ⁵ / m 2
l) and feeder layer cells (spleen cells, etc.) are prepared in a 96-well microwell (Costar No.359).
6) Spread 10 ⁵ feeder cells per well and spread the treated cells at a probability of about 0.1, that is, so that the number of treated cells does not exceed 1 per well, about 1
Incubate for a week. The number of clones generated is increased sequentially, 10 ⁵ / ml
When the cells have grown to a certain extent, they are replaced with HAT medium again, and it is confirmed that the cells are completely killed, and the second step is completed. However, it is desirable that this second step be performed at least twice. Again 8
-Azaguanine must always be added to the medium until the cell line is established. It is desirable to add them appropriately even after establishment.

The usual medium may be the medium used before mutating, and when it is used as a parent cell of a hybridoma, it is not necessary to prepare a medium having any special composition.

The cells can be preserved by a conventional method. That is, 10% FCS (fetal cal
f serum) and 10% DMSO (dimet-hyl sulfoxide) are added to the medium and stored in liquid nitrogen or in a freezer at about -80 ° C without any problem.

Next, the hybridoma production method of the present invention using this cell line as a parent cell will be described.

Hybridoma production using the above cell line was carried out by using Eagle's MEM, Dulbecco's improved MEM, RPMI16 as a medium.
10% CS (calf s
erum) or 5% FCS + 5% CS, or 10% FCS. Normal maintenance of parent cells may be any of the above,
10% FCS is preferred for hybridoma production.

First, parental human tumor cells and splenocytes are prepared at a ratio of 1: 5. As a fusion agent, HVJ (Hemagglutinating
Virus of Japan, also known as Sendai virus, and polyethylene glycol (PEG) are used. Especially 30% of PEG1000 ~
About 50% is good. The method of fusing agent HAT selection is
Since it is already generalized, it is omitted. The screening method for the resulting hybridomas mainly uses the culture supernatant and uses SPA.
-Bind-SRBC method (SPA: Staphylococcus aureus protein
A, SRBC: Sheep Red Blood Cell: Immune experiment procedure VIIP237
5) Using the ELISA method (Dynatech method), clones of hybridomas secreting immunoglobulin were selected. Clones are gradually expanded, and when they reach 10 ⁵ / ml, subcloning is performed. Subsequently, in order to examine the homogeneity of the hybridoma, normal splenocytes were approximately used as a feeder lazer in 96-well microwells.
10 ⁵ cells / well are spread on the cells so that the number of hybridomas per well does not exceed 1 (-hole average probability is 0.1), and the antibody-producing ability of the clones that grow after culturing for about 1 week is examined. By repeating this procedure, a single hybridoma is obtained.

The method of classifying the immunoglobulin secreted by the hybridoma and the subclass can be easily identified by the octterlony method performed using agarose. That is,
The expected anti-globulin antibody is appropriately prepared, and the determination is made by using the blocking line after 24 hours (see Example 6).

As described above, the present invention overturns the theorem from Milstein, which was previously considered to be usable only when the most important parent cell was a myeloma- or B-cell-derived tumor cell in producing a hybridoma. That is, it was demonstrated that tumor cells that can proliferate in vitro are used as parent cells for hybridoma production. Further, the human-human hybridoma of the present invention is produced from the established human tumor cell line as a parent cell which is simpler in culture and more stable and proliferative than the conventional myeloma in the present invention.

The method of producing the hybridoma of the present invention will be described by the following examples, and the identification of the antibody produced by the hybridoma thus produced will be described by the examples.

Example 1 Method for preparing parental cells for producing hybridoma from human tumor cell Colo38 (No. 1) All cultures in the following experiments were CO ₂ 5%, air 95%, and humidity about 10
It was performed at 37 ° C. under 0% conditions.

Human malignant melanoma Colo38 was cultivated in advance, and cells in the proliferative phase on the 2nd to 3rd days were prepared. The final concentration is 0.05-10
Add μg / ml mutagen MNNG (N-methyl-N'-nitro-N-nitrosoguanidine: Sigma) and add
The time and concentration at which 20% of dead cells appeared were set (5
μg / ml: 36 hours). Next, after washing with RPMI1640 (Nissui Pharmaceutical Co., Ltd.) containing no serum three times to remove MNNG, 2 × 10 ⁵ cells were added to the medium containing 8-azaguanine at a final concentration of 1 to 50 μg / ml. / ml (10 ml total volume) MNNG treated cells were added. Set a concentration (20 μg / ml) at which almost all cells (99% or more) die within 1 week, and observe this for at least 2 weeks or more every day in this state, and check for the presence or absence of cell proliferation. investigated. After about 2 weeks, the cells began to grow rapidly, so the cells were collected by centrifugation and added in the presence of 8-azaguanine at a final concentration of 50 μg / ml (2 × 10 ⁵ cells as in the first treatment). / ml).

In this state, culturing for another 2 weeks
The strain was an azaguanine resistant strain.

To examine whether HAT selection can be used, 8-
The cells in the presence of azaguanine were washed by repeatedly centrifuging three times in a serum-free medium, and HAT medium (RPMI1640 medium, 1
Hypoxanthine 10 ^-4 M, aminopterin 4 x 10 ^-7 , thymidine 1.6 in 0% FCS (CSL, made in Australia) at final concentration.
× 10 ⁻⁵ M was added). The appropriate number of cells was 10 ⁵ cells / ml (total volume 10 μm).

Observation was carried out for 5 days, and life or death was judged. Judgment is made by trypan blue staining. Even when the cells are completely dead by this method, 10 ⁵ cells / ml of the same 8-azaguanine-treated cells and the feeder cells (spleen cells) before replacement with HAT are prepared, and 1-well of 96-well microplate is prepared. After supplying 10 ⁵ cells per well, the cells were seeded so that the number of treated cells was about 0.1, and the cells were cultured for about 1 week. The number of generated clones (6 in 96 wells) was sequentially increased, and after further culturing for 1 to 2 weeks, HAT selection was performed again on all the clones when they grew to about 10 ⁵ cells / ml. In this method, the cells used in the above 6 clones all died within 5 days. However, among the 6 clones, 1 clone was further seeded on a 96-well microplate by the above method, and further subcloned to obtain 8 clones. In the same manner, the cell numbers of these clones were gradually increased, and it was confirmed that each cell was completely killed in HAT medium.

The thus obtained human tumor cells (human malignant melanoma) that are resistant to 8-azaguanine and HAT selection are named ME1 series, and are usually called ME1.

Cells are 10% FCS, 10% DMSO (dimethyl sulfoxide)
And 80% normal medium composition were stored under liquid nitrogen or in a freezer at about -80 ° C.

Example 2 Method for Preparing Hybridoma-Producing Cells from Human Tumor Cell Colo38 (Part 2) In this Example, a method of utilizing mutation by ultraviolet rays instead of a mutagenizing agent will be described. 2 × 10 ⁵ cells / ml of human malignant melanoma (colo38) in the proliferative phase on a 10 cm diameter shearle
(10ml total volume), UV irradiation (underneath GL-35)
(30 cm) for about 5 minutes, and then 8-azaguanine was added to the final concentration of 20 μg / ml. 99.5% or more of the cells die in about 1 week, but the culture is continued in this state for about 2 more weeks and observed daily. About 3 weeks after the first attachment of 8-azaguanine, the number of cells rapidly increased, and the cells became uniform floating cells. Collect cells by centrifugation then 50 μg / final concentration
In the presence of ml 8-azaguanine, the cells (2 x 10 ⁵ cel
ls / ml) was added. In this state, the cells were further cultured for 2 weeks, and the proliferating cells were designated as 8-azaguanine resistant strains.

The HAT selection method is the same as in Example 1 below.
Three ME1 series cells were obtained by this method. Compared with Example 1, the probability that 8-azaguanine resistant strains appeared was reduced to about 1/5. However, when an 8-azaguanine resistant strain was obtained, the probability that the subsequent operation, HAT selection, was applied was the same.

Example 3 Method for preparing parental cells for hybridoma production from human tumor cell colo38 (3) In this example, a method for directly adding 8-azaguanine to a medium to produce a resistant strain will be described.

Proliferative human malignant melanoma (colo38) 2 × 10 ⁵ cells / ml
After culturing (total volume of 10 ml) at a final concentration of 5 μg / ml for 5 days,
Furthermore, 8-azaguanine was added so that the final concentration was 20 μg / ml. Almost all cells died in the period of 10 days from the beginning, but the culture was continued in this state for at least another 20 days. Probability of proliferation was low. In this case, cells out of 18 flasks could be seen to grow rapidly in one flask. Then, a part of the cells was further cultured for 2 weeks in the presence of 8-azaguanine at a final concentration of 50 μg / ml, and the cells with normal proliferative potential appeared 8-
The strain was an azaguanine resistant strain.

The HAT selection method was the same as in Example 1. By this method, 6 cells of ME1 series were obtained.

As described above, 17 types of ME1 series cell lines (SUN N-21-1) were selected from human malignant melanoma (melanoma colo38) by three methods.
~ SUN N-21-17) was obtained.

Example 4 Method for Preparing Hybridoma-Producing Parent Cell from Human Tumor Cell (M21) This example illustrates that a hybridoma-producing parent cell can be obtained from other types of human tumor cells as well as colo38.

The method is almost the same as in Example 1. That is, melanoma M21 was pre-cultured and cells in the growth phase after 60 hours were collected, and MNNG as a mutagenizing agent at a final concentration of 5 μg / ml was added and cultured for 36 hours. (Dead cells: about 35% occur) Next, serum-free RP
The MNNG was removed by washing with MI1640 three times. Viable cells were prepared in 2 × 10 ⁵ cells / ml (total volume: 10 ml) containing a final concentration of 20 μg / ml of 8-azaguanine, and then cultured. 99.5% or more of the cells died within 1 week, but the culture was continued in this state for 2 more weeks.

The growth of cells was observed under a microscope once a day. From around day 20, cells gradually proliferating appeared, and within 5 days thereafter, the cells proliferated rapidly. Next, the cells were collected by centrifugation, and further suspended in 2 × 10 ⁵ cells / ml (total amount 10 ml) in a medium containing 8-azaguanine at a final concentration of 50 μg / ml, and cultured for about 2 weeks to grow the cells. An 8-azaguanine resistant strain was used.

The HAT selection and repeated subcloning methods were the same as in Example 1.

According to this method, 6 strains (SUN N-22-1 to SUNN-22-) that can be used as parent cells for producing hybridomas that are resistant to 8-azaguanine of melanoma M21 and die in HAT medium are used.
6) Got it. This series will be referred to as the ME2 series, and is generally referred to as ME2.

The following examples explain that the parental cells established in the above examples form hybridomas by fusion with B cells such as splenocytes, and actually secrete immunoglobulin into the culture medium.

Example 5 Preparation of hybridoma of ME1 prepared in Example 1 and mouse splenocytes immunized with human lung cancer (A549) and confirmation of monoclonal antibody secretion SUN N-21-1 of ME1 series of parent cells Prepared by culturing 10 ⁷ cells, BALB / C mice (female 4-week-old) were inoculated once a week with human lung cancer (A549) for 10 ⁷ cells for 4 weeks, and spleen cells (5 × 10 ⁷ cells) were fused with polyethylene glycol (PEG1000 Wako Pure Chemical Industries, Ltd.) (40%). That is, each cell was mixed in a centrifuge tube, centrifuged, and the supernatant was discarded. About 0.5 ml of PEG1000 made up to 40% with RPMI1640 was added to the packed cells, and the mixture was left still for 3 minutes and then at 3 rpm at 500 rpm. Centrifugation was carried out for a minute, and then, about 5 ml of the medium was slowly added, the mixture was centrifuged again, and the supernatant was discarded.
Then, all the cells are gently transferred to T-75 (Falcon No. 3024), and the medium is added to about 40 ml, followed by culturing overnight. On the next day, all of this culture solution including cells was transferred to a centrifuge tube, centrifuged, and the supernatant was discarded. Next, after adding 40 ml of HAT medium and stirring well, 96
About 100 μl / well was added to the wells of the microplate.
In this way 4 plates were completely filled. When cultured for 1 week in this state, colonies were observed in about 10% of the holes. However, the colony in this case had a feature that it spreads in a planar shape as compared with the conventional colony. From 1 week onward, 1 drop (about 25 to 30 μl) of HT medium (medium obtained by removing aminopterin from HAT) was appropriately added to each well. After the hybridomas grew to some extent (about 10 to 14 days), it was examined whether or not the monoclonal antibody against the target human lung cancer (A549) was secreted by the Protein A-bind-SRBC method using the culture supernatant. . As a result, it was found that 4 clones out of 45 clones secreted a monoclonal antibody against A549. Therefore, one of the four clones was subcloned. The method was the same as the subcloning of Example 1. As a result, 4 clones grew and all secreted the monoclonal antibody.

The storage method for these cells is as usual: 10% DMSO10%
FCS-80% normal medium in the presence of liquid nitrogen or in a freezer at about -80 ° C.

Experimental Example 1 Examination of immunoglobulin class and subclass secreted by the hybridoma obtained in Example 5 by the Ouchterlony method A 15% agarose solution (containing 0.01% NaN ₃ ) was added to a Petri dish (inner diameter 52 mm) in an amount of 5 ml, and Hold for 30 minutes until solidified. Next, as shown in FIG. 1, holes were made in the coagulated agarose, 5 μl of each antibody shown in Table 1 below was added to each hole, and the monoclonal antibody secreted by the hybridoma obtained in Experimental Example 1 was added to the center hole ( 15 μl of a culture solution (10-fold concentrated) containing LAC1, 2, 3, and 4) was added. This state was left for 24 hours, and the monoclonal class was determined with the resulting inhibition line.

Table 1: Antibodies used No. antibody and origin 1 anti-human IgM rabbit (manufactured by Hoechst) 2 anti-human IgM rabbit (〃) 3 anti-mouse IgG rabbit (manufactured by Capel) 4 anti-mouse IgM Yagi ( Hoechst) 5 anti-human Ig (G + A + M) goat (〃) 6 anti-human IgA rabbit (〃) As a result, 4 of the monoclonal antibodies (LAC1 to 4) obtained
It was found that the type was mouse IgG because the inhibition line occurred only between 3 and 7.

Next, in order to examine the IgG subclass, the same experiment as described above was performed. That is, a hole is made on agarose as shown in FIG. 2 and 5 to 4 antibodies of each subclass (Table 2) are prepared from 1 to 4.
15 μl of LAC1-4 (one type each) monoclonal antibody was added to 5 μl in the center, and a total of 4 petri dishes were allowed to stand at room temperature for 24 hours.

Table 2: Antibody No. used and origin 1 anti-mouse IgG1 rabbit (Miles) 2 anti-mouse IgG2a rabbit (〃) 3 anti-mouse IgG2b rabbit (〃) 4 anti-mouse IgG3 rabbit (〃) these From the results, it was found that all the monoclonal antibodies of LAC1 to 4 were IgG2a because the blocking line occurred between 2 and 5.

The amount of secretion was also about the same as that of the monoclonal antibody secreted by the hybridoma prepared using mouse myeloma as the parent cell (about 30 μg / ml). Furthermore, the unity of the antibody secreted by the hybridoma was confirmed by gel electrophoresis.

From the above results, the human parental cells of the present invention (for example, ME1: SUN
N-21-1), if fused with B cells as an information source,
It has been proved that it secretes the immunoglobulin as informed.

Next, the human myeloma prepared for conventional hybridomas is said to be difficult to culture and have poor or unstable cell growth. On the other hand, the cell line of the present invention has an original cancer. It proves that it has a stable growth ability which is a property of cells.

Experimental Example 2 Growth curve of cancer cells and parent cells of the present invention (ME1) Human melanoma (Colo38) and parent cells for hybridoma production derived from Colo38 (ME1: SUN N-21-
Each of 1) was prepared at 2 × 10 ⁵ cells / ml (total amount: 10 ml) and put in a usual RPMI1640 + 10% FCS and cultured.

For ME1, the same number of cells were also cultured at a final concentration in RPMI1640 + 10% FCS containing 20 μg / ml 8-azaguanine. Economically sampled and viable cells counted.

The results are shown in Fig. 3. As shown in this figure, ME1 showed a growth curve similar to that of human melanoma (Colo38). In addition, there was almost no difference in growth even in the presence of 8-azaguanine.

Although not shown, the monoclonal antibody-producing hybridoma 4 clone obtained in Example 5 was also used as a parent cell (ME1:
SUN N-21-1) showed the same growth curve.

[Brief description of drawings]

FIG. 1 and FIG. 2 are plan views showing the octalonies used in Experimental Example 1. FIG. 3 is a graph showing growth curves of cancer cells and parental cells (ME1) for preparing the hybridoma of the present invention.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12P 21/08 8214-4B (C12P 21/00 C12R 1:91) (C12P 21/08 C12R 1:91) ( 72) Inventor Teruhisa Noguchi 2-8-11 Kugenuma Kaigan, Fujisawa City, Kanagawa Prefecture (56) Reference JP-A-57-39777 (JP, A) JP-A-57-208987 (JP, A) Nature, 256, 751 -753 (1975); 256, 495-497 (1975) J. Am. Immulol. , 128, 2714-2718 (1982) Proc. Natl. Acad. Sc i. USA, 78, 7717-7721 (1981)

Claims (8)

[Claims] 1. A hybrid derived from human melanoma cells, which is induced by the fusion of a cell line capable of selecting a HAT medium induced by natural or artificial mutation with activated lymphocytes derived from animals including humans. cell. 2. The human melanoma cell is Colo38 or
The hybrid cell according to claim 1, which is M21. 3. The hybrid cell according to claim 1 or 2, wherein the activated lymphocytes derived from animals including humans are B cells sensitized with an immunogen. 4. The hybrid cell according to claim 1 or 2, wherein the activated lymphocytes derived from animals including humans are T cells capable of producing lymphokines. 5. A cell line derived from human melanoma cells and capable of HAT medium selection induced by natural or artificial mutation, and induced by fusing activated lymphocytes derived from animals including humans. Method for producing hybrid cell. 6. The human melanoma cell is Colo38 or
The manufacturing method according to claim 5, wherein the manufacturing method is M21. 7. The method according to claim 5 or 6, wherein the activated lymphocytes derived from animals including humans are B cells sensitized with an immunogen. 8. The method according to claim 5 or 6, wherein the activated lymphocytes derived from animals including humans are T cells capable of producing lymphokines.