CH658070A5 - CELL LINE DERIVED FROM HUMAN HYBRIDOMAS, ITS PREPARATION METHOD AND ITS USE FOR THE PRODUCTION OF MONOCLONAL HUMAN ANTIBODIES. - Google Patents

Description

The present invention belongs to the field of immunology. It relates to a human cell line capable of fusing with human lymphocytes.

Normally, human myelomatic cells do not merge satisfactorily but, by means of the present invention, genetically stable cells capable of forming hybrids are provided.

The production of antibodies specific for the antigenic determinants of viruses, tumors and foreign agents is important both for immunotherapy and for medical research.

Monoclonal antibodies for antigenic determinants have been produced in vitro in cultures of spleen cells infected with a virus, as illustrated by the Epstein-Barr technique. Antibodies have also been produced using pooled hybrid cells, or hybridomas, between mouse spleen cells and mouse myeloma cells: see U.S. Patent Nos. 4,172,124 and 4,196,265. Hybrid cells pooled from BALB / c mouse spleen cells and BALB / c myelomatic cells have also been described in the literature by Kohler et al. in Nature, Vol. 256, 495-497 (1975), and in Eur. J. Immunol., Vol. 6, 511-519 (1976). The spleen cells used for the production of these hybrids were taken from mice immunized with red blood cells from sheep. Other articles revealing the production of hybrid cells from animal spleen cells and animal myelomatic cells include those of Milstein et al. in Nature, Vol. 266, 550-552 (1977); Koprowski et al. in Proc. Nat. Acad. Sci., Vol. 74, 2985-2988 (1977) and Welsh in Nature, Vol. 266, 495 (1977).

Hybridoma technology has proven capable of satisfactory yields for antibody production rates. However, the antibodies produced by hybridomas consisting of somatic cells hybrid between spleen cells producing antibodies and myelomatic cells of BALB / c mice are necessarily foreign agents when injected into the human body. As a result, the natural human immune response system makes antibodies to fight the antibodies produced by hybridomas after their first introduction into the human body. Repeated injections of these antibodies can result in shock.

Certain human chromosomes are normally lost when human lymphocytes are joined with mouse myelomatic cells for the formation of mouse-human hybrids. Such hybrids must retain the two human chromosomes that carry the rearranged genes for the human light and heavy immunoglubin chains in order to remain effective. Consequently, it is difficult to obtain hybridomas between species which secrete human antibodies.

It is profitable to provide a hybridoma cell formed by the union of a human myelomatic cell with a human lymphocyte. Such a hybridoma cell could produce human antibodies with a reduced risk of shock resulting from repeated injections into a human. However, in general, human myelomatic cells do not meet well with human lymphocytes.

The present invention provides a stable myelomatic cell line, which can be grown and subcultured ad infinitum, and which is capable of hybridization with human lymphocytes and with lymphocytes from other anipials to form hybrid cells. together stable.

In addition, the present invention provides a process for producing hybridomas from human myelomatic cells, which

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hybridomas are capable of expressing useful antibodies which would be accepted by the human immune response system.

The present invention also aims to provide a method of producing antibodies which would be used for the treatment of human diseases, with which the antibodies produced would be accepted by the human immune system.

In accordance with the present invention, a human myelomatic cell is provided which is capable of hybridization with human and animal cells producing antibodies, which cell line is a mutant of human cells GM 1500 B and a deficiency of phosphoribosyl transferase hypoxanthine (HPRT).

Means for producing hybrid cells using stable human myelomatic cell lines, deficient in HPRT, are provided in one of the embodiments of the present invention.

The means of production for antibodies using such hybrid cells are provided in another embodiment.

This invention provides, among other things, a production process for new cells which are genetically stable, which can be grown and subcultured endlessly and which produce large amounts of antibodies against viruses, tumors and foreign agents, as well as their antigenic determinants. These cell lines are hybrid cells joined together (a) of a stable line of human myelomatic cells deficient in HPRT, for example a line of malignant cells derived from a primary tumor of the bone marrow, and (b) of lymphocytes producing antibodies, such as those of the spleen or lymphatic nodules, or peripheral lymphocytes. A particularly preferred cell line is a hybrid of cells united between peripheral human lymphocytes and human myelomatic cells. These cell lines can be stored considerably for an indefinite period in a selective culture medium such as hypoxanthine-aminopterin-thymidine (HAT), and can continue to produce specific antibodies indefinitely for antigenic determinants.

The myelomatic cells of this invention were derived from the human cell line B type GM 1500 which can be obtained from the Human Genetic Mutant Cell Repository of the Medical Research Institute in Camden, N & w Jersey (Institute for Medical Research).

The GM 1500 myelomatic cells were treated with methyl ethyl sul-fonate to increase their mutation rate, and they were selected for their deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in a medium containing thioguanine-6 with a concentration of approximately 30 micrograms / ml [the basic technique for selection is described in Nature, Vol. 256, 495-497 (1975)]. This process kills most myelomatic cells; human B cells normally die in a medium containing thioguanine-6. Furthermore, even among the surviving cells, most are not capable of hybridization with lymphocytes.

Two publications have revealed the hybridization of human GM 1500 myelomatic B cells with non-human myelomatic cells. The GM 1500 cells were not subjected to mutagenic treatment or any other selection process before the meeting in the processes revealed by these articles. These articles are: Croce et al. in Proc. Nat. Acad. Sci. U.S.A., Vol. 76, 3416-3419 (1974) and Croce et al. in Eur. J. Immunol., Vol. 10, 486-488 (1980).

Although methyl sulfonate may be favorably used as a mutagen in the process described above, it is understood that any other mutagen can be used to cause mutation of human cells B GM 1500 as long as the agent does not adversely affect cells.

The treatment with the mutagen and the selection process gave two "survivors", making it possible to propagate modified, stable, HPRT-deficient myelomatic cell lines: the two survivors were designated by the name GM 1500 6TG-Al- 1 and GM 1500 6TG Al-2. The two survivors, who appear to function interchangeably, form continuous cell lines which are interposed in the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852-US, under numbers CRL-8032 and CRL-8038, respectively. These myelomatic cell lines have the distinct characteristic of being able to be hybridized with human lymphocytes as well as with lymphocytes from other animals. These cells secrete IgG (immunoglobulin gamma-2, K) like the parent cells GM 1500, and they are positive for the nuclear antigen of the Epstein-Barr virus. The cells were stored in Eagle minimum essential medium (MEM), RPMI (Rosewell Park Memorial Institute) 1640 medium could be used as an alternative, with 10% fetal bovine serum. The growth of myelomatic cells is inhibited by the selective medium of hypoxanthine-aminopterin-thymidine. This medium is described in Science, Vol. 154, 709-710 (1964). The rest of this talk refers to GM 1500 6TG-A1-2 cells; the GM 1500 6TG-A1-1 cell line can be used as an alternative.

The lymphocytes used can be any lymphocyte from a human, considered to have the required antibodies. It is also possible to stimulate human lymphocytes in vitro to obtain the desired antibodies. For example, lymphocytes can be taken from a patient infected with an active virus such as rabies, influenza, measles, rubella, polio, etc. However, it goes without saying that these are only examples; it is also possible to use lymphocytes creating antibodies attacking other viruses or other non-viral antigens, such as pollen. It is preferable that the lymphocytes are peripheral lymphocytes separated from the patient's blood, or cells of the spleen.

In one implementation of the invention, the source of lymphocytes used was heparinized blood plasma from a patient suffering from subacute sclerosing panencephalitis (SSPE), an infection of the brain measles virus. In order to check for the presence of anti-SSPE lymphocytes in this blood plasma, a serum sample was diluted 1:10 000 000 with a phosphate buffer solution and was found to bind using titration. dioimmune to target cells infected with measles. These data and the evidence of histological brain lesions characteristic of the disease confirmed the clinical diagnosis of PTSD. 45 A 10 ml sample of heparinized blood plasma was taken from this patient and a suspension of lymphocytes purified with Ficoll was prepared as taught by Gerhard et al., Eur. J.

Immunol., 5, 720-725 (1975). The red blood cells were lysed by incubating the blood sample for 15 minutes at 4 ° C in so NH4.CI (0.83%). The resulting cell suspension was washed by centrifugation (800Xg) using heat-inactivated bovine serum and centrifugation in protein-free medium (RPMI 1640, buffered with 7.5 mM HEPES, pH 7.2) . The production of the hybrids was accomplished by mixing approximately ten million selected myelomatic cells with HPRT deficiency of this invention with one to ten million lymphocytes. The cell mixture was centrifuged at 800Xg and the cells were suspended again, for reunification in a 50% solution (weight / volume) of polyethylene-1000 glycol (PEG) diluted in essential medium 60 minimum (MEM) without serum . Following the meeting procedures taught by Davidson et al., Soamt., Cel Genet. 2.175-176, the polyethlyene glycol was first diluted in MEM without serum, and then in MEM with serum before the cells were seeded in the wells of Linbro plates in a selective medium 65 dehypoxanthine-aminopterin-thymidine. The cultures were incubated at 37 ° C. in an atmosphere of 95% air / 5% CO 2 and every 7 to 10 days the culture medium was partially replaced by a fresh medium (HAT) (from 1/2 to 1/3) .

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The cell growth was observed in 20 of the 24 wells 15 to 21 days after incubation of the cultures produced by the union of lymphocytes with the myelomatic cells.

Any growth in HAT medium is an indication of successful hybridization between lymphocytes and myelomatic cells. These cells were reproduced continuously in HAT medium and were cloned into microplates (Linbro) by limiting the dilution. Each independent clone (derived from an independent well) was then tested for the production of human immunoglobulin chains and for the immunoprecipitation capacity of the measles virus proteins. The hybridomas were found to secrete IgM specific for the measles virus nucleocapsides, as described below.

The specificity of the antibodies for the measles virus was determined by immunoprecipitation and electrophoresis with 10% poly-acrylamide-SDS gel, which techniques are well known in the art (SDS is an abbreviation for sodium dodecyl sulfate). Generally, cell cultures were labeled with 100 microcuries of leucine-3H (70 curies per millimole) per milliliter of solution by being exposed to it for 12 hours of incubation. The immunoglobulin chains were then immunoprecipitated with anti-human immunoglobulin antisera according to established procedures. The labeled chains of immunoprecipitated human immunoglobulins were then separated by conventional electrophoresis on 10% polyacrylamide-SDS gel.

A comparison was made between (1) the immunoprecipitations of the immunoglobulin produced by GM 1500 6TG-A1-2 cells following a reaction with an antihuman gamma antiserum; (2) immunoprecipitations of immunoglobulin chains secreted by human-human hybridomas (obtained by the joining method described above) following a reaction with an antihuman mu antiserum; and (3) immunoprecipitations of immunoglobulin chains secreted with a human-human hybridoma following a reaction with antisera of specific mu and anti-human gamma chains, using the electrophoretic separation techniques explained above. The purpose of this comparison was to demonstrate that the human-human hybridomas produced according to the present invention expressed the two chains of human immunoglobulins mu and gamma.

Six samples of cloned hybrid cells were tested by immunoprecipitation of the immunoglobulins secreted by the hybrids with an anti-mu rabbit antiserum (IgG1). All hybrids expressed the chain of human mu immunoglobulin. Some of these hybridomas also expressed a light chain of immunoglobulin migrating differently from the light chain expressed by the myelomatic clone GM 1500 6TG-A1-2. One of these hybridoma clones was unable to produce the immunoglobulin light chain expressed by GM 1500 6TG-A1-2 cells. The culture fluid of the hybridomas which had been immunoprecipitated with the rabbit antisera of the mu chain and antihuman gamma expressed two heavy chains of immunoglobulin, the gamma chain of the parent cell GM 1500 6TG-A1-2 and the mu chain of the parent cell B of human SSPE (subacute sclerosing panencephalitis).

Another comparison was made by polyacrylamide gel electrophoresis to determine the specificity of the antibodies expressed by the hybridomas of the invention for the measles virus antigen. Two samples of hybridoma culture media were compared with two controls. The controls were: (a) serum taken from a patient recovering from an atypical measles infection and (2) culture fluid from the GM 1500 6TG-A1-2 human cell line. Serum from a patient with atypical measles was chosen to test the anti-measles virus antibodies. The results of the comparison were obtained using conventional analysis techniques. The procedures used in immunoprecipitation were as follows: (1) lyses of CV1 cells infected with the measles virus, labeled with methionine-S, were used as antigens; samples (25 microliters) of the antigen were mixed with

samples (100 microliters) of concentrated culture liquid and incubated at approximately 37 "C for 90 minutes and then at 4 for 4 hours; (3) samples (25 microliters) of total rabbit antihuman antibody were then added; (4 ) the incubations were repeated; (5) the precipitated polypeptides were collected by centrifugation in an Eppendorf centrifuge for 20 minutes at 10,000 rpm; (6) the visible pellet recovered was again suspended in phosphate buffer and washed three times; (7) the pellet was suspended in a lysis buffer and boiled for three minutes; (8) the resulting solutions were electrophoresed on a 10% polyacrylami-de-SDS gel according to the conditions described by Hall et al., Proc Nat Acad. Sci, USA; Vol 76, 2047-2051 (1979) After the fluorography, the dried gel was exposed on a Cronex radiographic film.

The fluids of the hybridoma cultures precipitated the NP virus polypeptide and changed the amounts of its dividing fragments. Antibodies in culture fluids are therefore specific for a single polypeptide, NP, which is the essential structural polypeptide of the virus nucleocapsid, and the primary antigen of the measles virus.

Another comparison was made, using electrophoretic analysis on polyacrylamide-SDS gel between (1) the measles virus polypeptides precipitated by the culture liquid (not concentrated) of a hybridoma subclone, obtained in limiting dilution, and (2) virus polypeptides precipitated by the same atypical measles serum as that used above. The results confirm the specificity of the antibodies in the culture fluid of the hybridoma clone, while the subclone thereof precipitates the NP polypeptide with an activity comparable to that of the parent hybridoma clone.

The cell line produced by combining the stable human myelomatic cell line deficient in HPRT of this invention with lymphocytes producing human antibodies, as demonstrated by the anti-SSPE lymphocytes used in the implementation of the preceding invention, is a hybrid culture showing the characteristics of both normal lymphocytes and parent myelomatic cells, and appears to be derived from a single case of reunion. The cells have a hybrid nature since:

(a) the hybrid cell line has been maintained for several months in a HAT selective medium which inhibits the growth of parental myelomatic cells but not that of normal lymphocytes which, in turn, would probably not survive in vitro for more than 4 to 5 weeks ;

(b) the number of chromosomes in hybrid cells is close to the sum of that of normal lymphocytes and parent myelomatic cells;

(c) the hybridoma of new immunoglobulin chains such as the light and mu chains, and

(d) the hybrid produces specific antibodies, whereas the parental myelomatic cells do not produce this kind of antibody. Furthermore, the rate of production of such antibodies is higher than that observed in cultures of non-hybridized lymphocytes.

Similar methods can be performed by a person skilled in the art, using other lymphocytes, including human lymphocytes which express other antibodies, and lymphocytes from other animals. As an alternative to the culture of hybridomas in vitro, hybridoma cells can be injected into an immunosuppressed animal such as a Nude mouse for culture in vivo.

The antibodies produced by the hybrids can be recovered by the use of current techniques and can be used in analytical medical research for the identification of antigens in samples of blood and culture media. The antibodies are considerably homogeneous and are highly specific for the target antigen. A source of different homogeneous antibodies, "each highly specific to a particular antigen, allows researchers to quickly determine the specificity of an antigen and / or characterize the antigens. In addition, the antibodies can be administered to sick humans to assist fighting disease.

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Claims (21)

658,070 2 1. Cell line derived from a hybrid fused cell of (a) a cell originating from a human myelomatic cell line GM-1500 sensitive to a medium of hypoxanthine-aminopterin-thymidine and from (b) a lymphocyte producing d 'antibody. 2. Cell line according to claim 1 wherein said myelomatic cell line corresponds to the cell line deposited at ATCC under the number CRL-8032. 3. Cell line according to claim 1 wherein said myelomatic cell line corresponds to the cell line deposited at ATCC under the number CRL-8038. 4. The cell line according to claim 1, wherein the antibody-producing lymphocyte is a human lymphocyte. 5. Method for producing a hybrid cell line according to claim 1, by combining an antibody producing cell and a myelomatic cell, characterized in that the myelomatic cell is a mutant of a human B cell GM 1500 deficient in hypoxanthine phosphoribosyl transferase. 6. Method according to claim 5, in which the myelomatic cell is taken from a continuous line of cells produced by the treatment of human cells B GM 1500 with methyl ethyl sulfonate to increase the rate of mutation of the cells, and cells are then selected with thioguanine-6 for hypoxanthine phosphoribosyl transferase deficiency. 7. The method of claim 5 wherein the myelomatic cell is obtained from "the continuous cell line deposited at ATCC under the access number CRL-8032. 8. The method of claim 5 wherein the myelomatic cell is obtained from the continuous cell line deposited at the ATCC under the access number CRL-8038. 9. The method of claim 5 wherein the combined hybrid cell is cultured and the antibodies produced by the hybrid cell are collected. 10. The method of claim 9 wherein said hybrid is cultured in vitro. 11. The method of claim 9 wherein said hybrid is cultured in vivo. 12. The method of claim 9 wherein said hybrid is grown in a medium containing rhypoxanthin-aminopterin-thymide. 13. The method of claim 5 wherein said antibody producing cell is a lymphocyte selected from the group consisting of spleen cells, lymphatic nodule cells and peripheral lymphocytes. 14. The method of claim 13 wherein the antibody producing cell is a peripheral lymphocyte. 15. The method of claim 5 wherein the antibody producing cell is obtained from a human infected with a virus. 16. The method of claim 15 wherein the virus is selected from the group consisting of rabies, measles, influenza, rubella and poliomyelitis. 17. The method of claim 16 wherein the virus is that of measles. 18. Human myelomatic cell, capable of hybridization with human and animal cells producing antibodies for the implementation of the method according to claim 5, characterized in that it is a mutant of human cells B GM 1500 deficient in phosphoribosyl- hypoxanthine transferase. 19. The cell according to claim 18, wherein the cell line corresponds to the cell line deposited at the ATCC under the number CRL-8032. 20. The cell according to claim 18, wherein the cell line corresponds to the cell line deposited at the ATCC under the number CRL-8038. 21. Use of a cell line according to claim 1 for the production of human monoclonal antibodies, characterized in that the cell line according to claim 1 is cultivated and that the culture liquid is collected after growth of said line cellular.