GB2145113A - Production of human monoclonal antibodies - Google Patents

Abstract

A process for the production of specific human monoclonal anti-bodies to a predefined antigen or hapten comprises: a. immunising B lymphocyte cells against the antigen or hapten, b. infecting the immunised B lymphocyte cells with Epstein-Barr (EB) virus, derived from a culture, preferably a mycoplasma free culture, of an EB virus producing cell line, c. culturing the EB virus infected cells on feeder cell layers in a nutrient medium containing at least one anti mycoplasma agent and either at least one anti-T cell immunosuppressant or at least one T cell mitogen to establish continuous cell lines. d. cloning the continuous cell lines by limiting dilution on feeder cell layers in a nutrient medium containing at least one anti mycoplasma agent, e. selecting for clones producing specific human mono-clonal antibodies to the antigen to hapten, f. growing the selected clones in a culture medium, and g. collecting the specific human monoclonal anti-bodies. As an alternative to the use of anti-T cell immunosuppressants or T cell mitogens, T lymphocytes may be removed from the cell population prior to the culture stage. By ensuring that the B cells are mycoplasma free during the culturing and cloning stages and also by suppressing the tendency of T lymphocytes to inhibit or kill the nascent EB virus transformed cell lines, it is possible to clone EB virus transformed lymphoblasts and thereby to achieve long term human monoclonal antibody production.

Description

SPECIFICATION Production of human monoclonal antibodies The production of human monoclonal antibodies by in vitro methods has wide ranging clinical potential. For example human monoclonal antibodies could replace human sera for the purposes of human HLA histocompatibility testing. Moreover such human antibodies could also be effective in the treatment of certain infectious (particularly acute viral) diseases, in infertility control and as immunosuppressive agents, to reduce the risk of graft rejection.

At present there are two methods of preparing human monoclonal antibody producing cell lines. In the first, the cell lines are produced by somatic cell hybridisation in a manner that is closely analogous to the production of mouse monoclonal antibodies. In the second, the cell lines are produced by the transformation of lymphocytes into B lymphoblas- toid cell lines by infection with Epstein-Barr (EB) virus.

Although the transformation method has been successfully used to produce specific antibody secreting cell lines, it has been found to suffer from two major drawbacks. These are a rapid decline of antibody levels when the cell lines are grown in vitro, together with a signal lack of success encountered by those attempting to clone the B lymphoblastoid cells.

It is the primary object of the present invention to provide a process for the production of human monoclonal antibodies via the transformation of lymphocytes with EB-virus in which the above disadvantages are substantially alleviated. Further objects of the present invention will become apparent from the following detailed description thereof.

According to the present invention there is provided a process for the production of specific human monocional antibodies to a predefined antigen or hapten which comprises immunising B lymphocyte cells against the antigen, or hapten, infecting the lymphocyte cells with Epstein-Barr (EB) virus derived from a mycoplasma free culture of an EB virus producing cell line, culturing the EB virus infected cells on feeder cell layers in a nutrient medium containing one or more antimycoplasma agents to establish continuous, antigen or hapten specific cell lines, cloning the antigen or hapten specific cells by limiting dilution on feeder cell layers in a nutrient medium containing one or more antimycoplasma agents, selecting for clones producing specific human monoclonal antibodies in the antigen or hapten, growing the selected clones in a culture medium, and collecting the specific human monoclonal antibodies.

The present inventors have found that the difficulties encountered by earlier workers in the cloning of EB virus transformed lymphob lasts were primarily caused by the presence of mycoplasma in the transformed B cells. By ensuring that the B cells are mycoplasma free during the infection, culturing and cloning stages of the present process, the present inventors have been able to clone the EB virus transformed lymphoblasts successfully and thereby to achieve long term human monoclo nal antibody production.

Immunisation of the B lymphocyte cells may take place in vivo or in vitro In the former case, immunisation may be by an invasive procedure, such as a blood transfu sion or a transplant, by a normal physiological process, such as pregnancy or contraction of a disease (eg rheumatoid arthritis), or by delibe rate immunisation, for example treatment of Rhesus D negative subjects with Rhesus D positive blood. In the latter case, lymphocytes from either immunised or non-immunised indi viduals may be primed by culturing them in the presence of the antigen or hapten to which sensitization is required.

Lymphocytes immunised in vivo or for in vitro immunisation may be der-ived from a variety of sources, for example spleen, tonsil, bone marrow, lymph node and peripheral blood.

After immunisation the total B cell popula tion may be infected with EB virus. However, since even after immunisation in vivo, only a small fraction of the B iymphocytes produce the desired antibody, the establishment of specific cell lines is facilitated by preselection (after immunisation but prior to infection) of antigen or hapten specific cells.Several methods of selection are available, these in clude (a) binding fluorescence labelled antigen or hapten to the surface of the antigen or hapten specific cells and then separating the labelled cells on a fluorescence activated cell sorter, (b) removing cells which do not bind anti gen or hapten, or, which is preferred, (c) rosetting the antigen or hapten specific lymphocytes with antigen or hapten coupled erythrocytes and then separating the rosettes by differential centrifugation, for example on a percoll gradient.

Selection may be of either a particular im munoglobulin subclass or a particular antigen or hapten specificity.

The lymphocytes are infected by Epstein Barr virus derived from a mycoplasma free culture of an EB virus producing cell line.

Preferably the cell line is either a marmoset cell line (B 95/8) of the type described by G Miller et al, Proc. Natl. Acad. Sci. USA, 1972, 69, 383 or a cell line derived there from. In one preferred embodiment of the present invention the cell line is a myco plasma free B 95/8 variant developed, for example, by culturing a parent B 95/8 cell line under limiting dilutions in the presence of macrophage feeder layers and antimycoplasma agents.

When the EB virus producing cell line itself is mycoplasma free then the lymphocyte cells may be infected with EB virus simply by suspending the cells in the culture supernatant of the cell line. However, when, as is often the case, the EB virus producing cell line is contaminated with mycoplasma then, prior to the infection step, the mycoplasma should be removed in order to produce a mycoplasma free culture. Mycoplasma removal may be effected, for example, by cocultivation of contaminated cells with macrophages in the presence of antibiotics, such as Kanamycin, gentamycin or trobicin, or by low density passage in the presence of macrophages and antibiotics. Once the mycoplasma has been removed, the lymphocytes may then be infected in the normai manner.

The EB virus infected cells are then cultured on feeder cell layers in a nutrient medium containing one or more anti-mycoplasma agents to establish continuous, antigen or hapten specific cell lines. The feeder cells may be, for example, allogeneic peripheral blood mononuclear cells or, which is preferred, peritoneal macrophages, especially of mouse or human origin. Suitable anti-mycoplasma agents include trobicin, gentamycin and kanamycin.

Other suitable anti-mycoplasma agents will be well known to those skilled in this art. In addition to the anti-mycoplasma agents the nutrient medium may also contain antimicrobial (antibacterial and antifungicidal) agents, such as penicillin, streptomycin, kanamycin, mycostatin and gentamycin, specific anti T cell immunosuppressants, such as cyclosporin A, or, which have the same effect, T cell mitogens such as phytohaemagglutinin, and amino acids, such as arginine and glutamine.

The presence of anti-microbial agents in nutrient media is a well known feature of cell culture technology, and alternative agents to those listed will be immediately apparent to the skilled microbiologist. The addition of specific anti-T cell immunosuppressants or T cell mitogens to the nutrient medium suppresses the tendency of T lymphocytes to inhibit or kill the nascent EB virus transformed cell lines. (As an alternative to this chemical suppression the T cells may be removed from the cell population prior to the culture stage).

Finaliy-the presence of an arginine supplement in the nutrient medium is preferred to compensate for the loss of mycoplasma arginine.

The detection of established cell lines is most readily performed by observation of the various cultures for signs of cell proliferation.

Continuous lines are generally established after about 4 weeks.

Once continuous cell lines have been established they may immediately be cloned by limiting dilution on feeder cell layers in a nutrient medium containing one or more antimycoplasma agents. Alternatively and preferably, the established lines are first reselected either for a particular immunoglobulin subclass or a particular antigen or hapten specificity. The method of selection may be one of those outlined above, with rosetting being preferred. The reselected antigen or hapten specific cells are then cloned in the manner of the present process. The preferred feeder cells and antimycoplasma agents are listed above.

The nutrient medium may also contain antimicrobial agents and amino acid supplements, especially arginine, as set out above.

The resulting clones are then tested for the presence of antibody to the predefined antigen or hapten. Clones that are positive for antibody production are grown in a culture medium, preferably containing antimycoplasma agents, and the human monoclonal antibodies are collected in accordance with known procedures. The monoclonal antibodies may be purified by such known techniques as electrophoresis, chromatography, etc.

By appropriate immunisation, human monoclonal antibodies to any hapten or antigen may be prepared by the present process. A list of antigens and haptens that have a particular significance in the medical field, especially the diagnostic and therapeutic areas, is given in European Patent Application No 44,722 and in US Patent No 4,193,983, and the contents of both of these publications is incorporated herein by reference. The present process allows specific antibodies to these and other ligands to be produced on a continuous and long term basis. Furthermore it is a particular advantage of the present process that antibodies of a given immunoglobulin sublass may be selected for production, either by the selection of appropriate antigen or hapten specific cells of by selection of appropriate clones.

The present process, and cell lines and antibodies produced thereby will now be described by way of example only.

1. Immunisation of B Lymphocytes A woman who was phenotype negative for the Rhesus D blood group was repeatedly immunised with Rhesus D positive cells over a period of years. She was known to be a high producer of anti-Rhesus D antibody. Approximately one week after receiving a booster dose of antigen a blood sample was taken and the lymphocytes separated using the Ficoll Hypaque floatation technique.

2. Selection and Isolation of Antigen Specific Cells Sterile lymphocytes, isolated as described in (1) above, were incubated with Rhesus D positive erythrocytes and centifuged to in crease contact between lymphocytes and red cells. The mixture was centrifuged over Ficoll Hypaque and the cell sediment, consisting of the "rosetted" cells and the non attached red cells, was removed.The sediment was treated briefly with an aqueous solution of ammonium chloride to lyse the erythrocytes and the lymphocytes were then repeatedly washed by a process of resuspension in a tissue culture medium consisting of RPMI 1640 medium, 10% (v/v) foetal calf serum (pre-heated to 57"C for 30 mins to destroy viruses and mycoplasma), glutamine (20 mmol), arginine (0.2mg/ml), gentamycin (25 jug/ml), kanamycin (25 yg/ml), trobicin (20 yg/ml), fungizone (20yg/ml), polymixin (251U/ml), streptomycin (50 ,ug/ml) and penicillin (1001U/ml) (all from Flow Labs, Irvine, Scotland), followed by centrifugation.

3. Production of Mycoplasma free B95/8 Variant Tissue culture supernatant derived from the B95/8 Marmoset cell line was used as the sourse of EB virus. However most preparations that were tested were found to be contaminated with mycoplasma. In order to develop a mycoplasma free B95/8 variant, a mycoplasma contaminated cell line was cloned under limiting dilutions in the presence of mouse macrophage feeder layers in a tissue culture medium containing RPMI-1640 medium, 10% (v/v) foetal calf serum (pre-heated to 57"C for 30 mins to destroy viruses and and mycoplasma), penicillin (1001U/ml) streptomycin (50 ,ug/ml), L-glutamine (20 mmol) arginine (0.2 mg/ml), gentamycin (25 yg/ml), kanamycin (25 ,ug/ml), trobicin (20 g/ml), fungizone (20 llg/ml) and polymixin (251U/ml). Cloned lines derived from the cells grown at the lowest cell density (an average of 0.3 cells per tissue culture well) were grown up into flasks and tested for mycoplasma contamination. A line that was mycoplasma negative and a high secretor of EB virus was chosen for use as a virus source.

4. Infection of B Lymphocytes with EB virus derived from the Mycoplasma free B95/8 Variant Lymphocytes prepared as described in (2) above, were incubated at 37"C for 60 mins with a 1 ml aliquot of mycoplasma free B95/8 variant (step(3) above) supernatant containing 1 % phytohaemagglutinin (Wellcome Labs).

After this time the cells were washed and resuspended in tissue culture medium containing RPM1 1 640 medium, 10% (v/v) foetal calf serum (pre-heated to 57"C for 30 mins to destroy viruses and mycoplasma), penicillin (100 lU/ml), streptomycin (50 g/ml), Lglutamine (20 mmol), arginine (0.2 mg/ml), gentamycin (25 yg/ml), kanamycin (25 yg/ml), trobicin (20 g/ml), fungizone (20 jtg/ml) and polymixin (25 lU/ml).

5. Infection of B Lymphocytes with EB virus derived from Mycoplasma contaminated B95/8 Lymphocytes prepared as described in (2) above were incubated (at 37"C for 60 mins) with a Iml aliquot of B95/8 cell line supernatant containing 1% phytohaemagglutinin.

After this time the cells were washed and resuspended in tissue culture medium as described in (4) above.

6. Culturing Technique Tissue culture medium containing the EB virus infected lymphocytes was pipetted onto monolayers consisting of mouse peritoneal macrophages. 1% phytohaemagglutinin was added and the mixture was cultured at 37"C in a humidified atmosphere containing 5% carbon dioxide in air and fed weekly by replacement of half the tissue culture medium without disturbance of the cell layer. Cultures were observed at regular intervals for signs of cell proliferation. Culture supernatants from those wells which contained proliferating foci of cells after 4 weeks culture were removed and tested for production of Rhesus D antibody.

7. Detection and Isolation of Antigen Specific Cells Once established the polyclonal cell line was grown in the absence of feeder cells.

After a period it was noted that, although antibody to the Rhesus D antigen was detectable in the supernatant of the culture the level of specific antibody fell off indicating that the culture had become overgrown with non-antibody producing clones.

In order to correct for the tendency of nonantibody producing clones to overgrow the culture the specific antibody producing clones were enriched by the method described in (2) above. Subsequent culture of the "rosetted" cells in tissue culture medium as described in (4) above, was accompanied by a burst of antibody activity which returned to high levels.

The selection and isolation of specific antibody producing clones was repeated at approximately monthly intervals over a period of 1 6 months.

8. Cloning Technique After approximately 9 months an aliquot of the polyclonal cell line was taken and diluted so as to give a range of cell densities in the wells of a 96 well tissue culture plate (flat bottomed and pre-seeded with mouse peritoneal macrophages). The cell densities ranged from 20 to 0.3 cells per well. The plates were then cultured, using tissue culture medium as described in (4) above, for 4 to 6 weeks with repeated changes of the tissue culture medium. After approximately 4 weeks positive wells could be identified visually and the cells contained therein were transferred to larger tissue culture wells (2ml volume) containing no feeder cells.

9. Selection of Antibody Producing Clones The supernatants of the positive clones (from (8) above) were tested in a microhaemagglutination assay (in the presence and absence of albumin) for the presence of anti Rhesus D antibody. The supernatants were also subjected to a Coombs test, again in the presence and absence of albumin, in which the developing agent was either an anti-immunoglobulin or an anti-C3d.

No antibody was found which was capable of haemagglutination in saline alone. However, after treatment of the culture supernatants with the oxidising agent, dithiothreitol, most became "saline agglutinators".

10. Growth of Positive Clones The lines were found to be stable on further growth after transfer to larger culture vessels (250 ml sealed flasks). They were also capable of survival and growth after transfer to serum free (Iscoves') medium.

11. Collection of Monoclonal Antibody Purified antibody was obtained by passing the supernatant, in a buffer pH 7.0, over a column containing Staphylococal protein A linked, by the cyanogen bromide technique, to Sepharose beads. Subsequent elution, at pH 3.5, of the bound antibody yielded a purified material in relatively concentrated form (between 15 iLg/ml and 200 ,ug/ml).

12. Test for Antibody to anti Rhesus D Anti body The tests outlined in (9) above were used to identify the specificity of the antibody. The quantity of the antibody was measured by calculating the protein concentration on the assumption that all the antibody present will be specific antibody. Monoclonality was co nfirmed by electrophoresis and isoelectric focussing.

1 3. Tests for Ig classes Sheep red cells were coated with mouse monoclonal antibody directed to human im munoglobulin classes and subclasses (IgM, Lug1, Lug2, IgG3 and lgG4) using the chromic chloride technique.

Coated red cells were added to dilutions of culture supernatants and the trays were subse quently examined for haemagglutination.

14. Production of Human Monoclonal Anti bodies directed to Lymphocyte Membrane An tigens.

In other experiments clones have been de rived from a post prenancy blood sample taken from a woman who was producing an anti HLA-DR antibody. Enrichment was effected by erythrocytes coated with specific antigen.

Preliminary screening of culture supernatants allowed lymphocytotoxic (ie complement binding) antibodies directed to human B cells to be identified. This is a strong indication of HLA specificity.

Claims (14)

1. A process for the production of specific human monoclonal antibodies to a predefined antigen or hapten which comprises: (a) immunising B lymphocyte cells against the antigen or hapten to form immunised B lymphocyte cells, (b) infecting the immunised B lymphocyte cells with Epstein-Barr (EB) virus derived from a culture of an EB virus producing cell line to form EB virus infected cells, (c) culturing the EB virus infected cells on feeder cell layers in a nutrient medium containing at least one anti-mycoplasma agent and either at least one anti-T cell immunosuppressant or at least one T cell mitogen to establish continuous cell lines, (d) cloning the continuous cell lines by limiting dilution on feeder cell layers in a nutrient medium containing at least one antimycoplasma agent, (e) selecting for clones producing specific human monoclonal antibodies to the antigen or hapten, (f) growing the selected clones in a culture medium, and (g) collecting the specific human monoclonal antibodies.

2. A process according to claim 1 further comprising, after immunisation but prior to infection, preselecting antigen or hapten specific cells from the immunised B lymphocyte cells.

3. A process according to claim 2 wherein the antigen or hapten specific cells are prese lected for an immunoglobulin subclass.

4. A process according to any one of claims 1 to 3 further comprising, after estab lishment of continuous cell lines but prior to cloning, reslecting antigen or hapten specific cells from the continuous cell lines.

5. A process according to claim 4 wherein the antigen or hapten specific cells are rese lected for an immunoglobulin subclass.

6. A process according to any one of claims 1 to 5 wherein the culture of the EB virus producing cell line is mycoplasma free.

7. A process according to any one of claims 1 to 6 wherein the EB virus producing cell line is mycoplasma negative.

8. A process according to any one of claims 1 to 7 wherein the immunised B lymphocyte cells are infected with EB virus in the presence of either at least one anti-T cell immunosuppressant or at least one T cell mitogen to form EB virus infected cells.

9. A process according to any one of claims 1 to 8 wherein the nutrient medium contains arginine.

10. A process according to any one of claims 1 to 9 wherein the culture medium contains at least one anti-mycoplasma agent.

11. A process according to any one of claims 1 to 10 wherein the nutrient medium and the culture medium each contains trobicin, gentamycin and kanamycin.

1 2. A process for the production of specific human monoclonal antibodies to a preferred antigen or hapten which comprises: (a) immunising lymphocyte cells against the antigen or hapten to form immunised lymphocyte cells, (b) removing immunised T lymphocyte cells, (c) infecting the immunised B lymphocyte cells with Epstein-Barr (EB) virus derived from a culture of an EB virus producing cell line to form EB virus infected cells, (d) culturing the EB virus infected cells on feeder cell layers in a nutrient medium containing at least one anti-mycoplasma agent to establish continuous cell lines, (e) cloning the continuous cell lines by limiting dilution on feeder cell layers in a nutrient medium containing at least one antimycoplasma agent, (f) selecting for clones producing specific human monoclonal antibodies to the antigen or hapten, (g) growing the selected clones in a culture medium, and (h) collecting the specific human monoclonal antibodies.

1 3. A process for the production of specific human monoclonal antibodies to a predefined antigen or hapten according to claim 1 substantially as hereinbefore described with particular reference to any one of the Examples.

14. A process for the production of specific human monocional antibodies to a predefined antigen or hapten according to claim 1 2 substantially as hereinbefore described.