WO1986000927A1

WO1986000927A1 - Antibody production

Info

Publication number: WO1986000927A1
Application number: PCT/GB1985/000340
Authority: WO
Inventors: Selby John Starkie; Gwynfor Rees Williams
Original assignee: Axon Healthcare Ltd.
Priority date: 1984-07-31
Filing date: 1985-07-30
Publication date: 1986-02-13
Also published as: EP0191049A1; JPS61502798A; GB8419456D0

Abstract

A process for preparing a human monoclonal antibody, which comprises stimulating human B lymphocytes with a mitogen, and isolating and selecting clones of antibody-producing proliferating cells.

Description

ANTIBODY PRODUCTION

FIELD OF THE INVENTION

This invention relates to antibodies and their production, and particularly to the production of monoclonal antibodies.

BACKGROUND OF THE INVENTION

Monoclonal antibodies are of enormous potential in the diagnosis and treatment of disease. They may be^' produced by the now classical Milstein technique, which comprises fusing lymphocytes with immortal, e.g. myeloma, cells, and screening and cloning the resultant hybrid cells. Much work has been done in the production of mouse and rat monoclonal antibodies, although human monoclonal antibodies have also been investigated. There are various types of immortal cell, e.g. lymphoblastoid cells, but myeloma cells have generally been preferred where there is any suggestion that the growing cells, e.g. those induced by Epsteiή-Barr virus ("EBv" hereinafter) , might not be easy to control. EBv is tumour-associated.

EBv is an example of a wide variety of substances which can conveniently be classified as "mitogens", i.e. materials which cause proliferation of cells. It is known that EBv transforms and immortalises human B lymphocytes. During transformation, it is impossible to select clones because it is not possible to know which clone among the variety of transformed lymphocytes is producing anti-antigen. Antibody production ceases after about 6 weeks. PRIOR ART

Recently, as disclosed in GB-A-2127434, Dorothy Crawford has used EBv to transform human B lymphocytes and obtain a lymphoblastoid cell line capable of producing an IgG anti-Rhesus D monoclonal antibody. The procedure involves first immunising a subject with D antigen, isolating and rosetting specific B lymphocytes, infecting the isolated cells with EBv and cloning them by limiting dilution.

Crawford's procedure involves choosing anti-D- producing cells and then infecting them to cause proliferation. It depends on the separation of anti-D- producing cells by the rosetting technique, and therefore that antibody is produced by the rosetted cells; depending on the stage of their maturation, however, B lymphocytes may either fail to bind antigen or release antibody-antigen complex. The procedure requires that T lymphocytes are removed in an initial rosetting stage, in order to avoid them causing cytotoxicity of the EBv-infected B lymphocytes. After infection, single clones of proliferating cells are isolated from the many such clones arising from the specifically-selected (by rosetting) lymphocytes. STATEMENT OF THE INVENTION

A process according to the present invention, for preparing human monoclonal antibodies, comprises stimulating human B lymphocytes with a mitogen, and isolating and selecting clones of antibody-producing proliferating cells. DETAILED DESCRIPTION OF THE INVENTION The process of the invention is simpler and more flexible than Crawford's procedure. In particular, the novel process is not limited to producing antibodies to the Rhesus D blood group antigen. Because a mitogen is used before selection, selection can be used to find specific antibodies against antigens to various human cells or other material. Reasonable trial can lead to the discovery of various antibodies. Selection may be by limiting or progressive dilutions.

Examples of antigens on human cells are blood group (e.g. A, B, 0, Rhesus D, d, E, e, C or c, or p) antigens. HLA polymorphic antigens, auto-antibody-inducing antigens, tissue-specific antigens, tumour-specific antigens, tumour-associated antigens, onco-foetal antigens and cell-type-specific antigens. Other biological material may be derived from various organisms, including people, e.g. on cells or in body fluids. The material may be on malignant cells, as described above, or on normal cells (which may be tissue-specific) . Materials from body fluids may be proteins, glycoproteins, lipoproteins, carbohydrates, steroids or peptides, of any of the types described earlier in this paragraph, and including regulatory materials such as hormones, neuropeptides, complement components and pheromones. Materials from non-human and, indeed, non-mammalian species may be derived from fungi, bacteria (especially Gram-negative bacteria) which may be pathogenic, viruses, protozoa, various pathogenic micro-organisms and other, intermediate forms such as PPLO and mycoplasma. Viruses (and other organisms) may be living or dead or refractionated, e.g. into coats, cores or "viroid" particles. Such materials may alternatively be classified as pathogens, parasites, symbionts, commensals and non-pathogens. The mitogen used in this invention may be, for example, an antigen, virus, lectin or chemical material. Specific mitogens which may be used are plant lectins such as phytohaemagglutin and Pokeweed mitogen, lipopolysaccharides, interleukin, T lymphocyte factor and mitogenic inorganic chemicals such as barium sulphate and calcium ions. The preferred mitogen, in this invention, is EBv.

It may sometimes be desirable, before contacting the B lymphocytes and the mitogen in the first step of the novel process, to stimulate or sensitise the lymphocytes. Such a procedure is described in an International Patent Application entitled "Cell Treatment" filed in the names of Axon Healthcare Ltd., Starkie and Williams on 30th July 1985, the description of which appears below under the heading Addendum, and which is incorporated herein by reference.

In the process of the invention, human B lymphocytes are first induced to replicate, in culture, e.g. by infecting them with EBv. The cells may then be transferred to a nutrient medium. For example, they are immersed in a nutrient solution.

Human B lymphocytes may be obtained from human blood, human lymph nodes or human tissues, e.g. malignant tissues. Cells may be separated by density gradient sedimentation. Whole blood, heparinised blood or defibrinated blood may also be separated by density gradient sedimentation. The cells may then be harvested by a conventional technique, e.g. from the interface between the medium in which the cells have been suspended and, say, a mixture of Ficoll and Triosil. After washing, the cells are treated with a mitogen and grown on a suitable medium.

At this stage, T lymphocytes may be removed from the nutrient medium, e.g. suspension, by various methods. For example, cytotoxic or affinity-binding antisera or antibodies, affinity removal with sheep erythrocytes (which may be AET-treated) , or Cyclosporin A, may be used. Replication of the B lymphocytes may then be estimated microscopically. Human B lymphocytes replicating in vitro synthesise and release immunoglobulin. Human immunoglobulin may be detected and measured by subjecting a sample of the spent growth medium to an enzyme immunoassay or other standard technique. Human B lymphocytes which replicate ^n vitro can be separated and isolated so that single cells replicate into single clones of cells. The antibody released by single clone of cells is a monoclonal antibody: all the molecules have the same heavy and light chain isotypes, allotypes and idiotypes; the antigen binding sites of all the molecules have the same specificity and affinity for antigen, and the same binding site idiotype.

The next stage in the procedure is to isolate clones. Firstly, a suspension of feeder cells may be placed in a multi-well culture plate. Feeder cells may be any viable cells which do not grow rapidly in culture, but which support the growth of isolated human B lymphoblastoid cells. Examples include human fibroblasts, endothelial cells from humans or other species, human B lymphocytes or human B lymphoblastoid cells, replication of which has been _.prevented by irradiation or a chemical such as a cytostatic or chelating compound, or human or other macrophages obtained from blood or body spaces such as the peritoneum.

Replicating B lymphocytes are diluted with growth medium and introduced into the wells, such that a statistically-predictable number of B lymphocytes, e.g. 0.3, 1 or 5, is present in a given volume. The various cultures are then incubated, and fresh growth medium is added to each well as required, e.g. daily.

Isolation of the B lymphocytes, and their replication, are estimated microscopically. Their release of human immunoglobulin and its antibody activity are detected and measured by conventional enzyme immunoassay or other known method. A procedure for antibody detection is described in an International Patent Application entitled "Antibody Detection" filed in the names of Axon Healthcare Ltd., Starkie and Williams on 30th July 1985. The process of the invention can be used to prepare valuable monoclonal antibodies. The products can be used, for example, for HLA tissue-typing, therapy, blood- typing and diagnosis. The process has been used to produce, first a human monoclonal antibody which binds with the cells of an oligodendroglioma, and second, one or more human monoclonal antibodies which bind with antigens of one or more bacteria, including type organisms of Pseudomonas. The following Examples illustrate the invention. Example 1

Human B lymphocytes were obtained from a human oligodendroglioma. The tissue was diced and teased with scalpel blades or needles and washed with fluid tissue culture growth medium. Many cells, including human B lymphocytes, were suspended in the medium. The medium was collected and allowed to stand for some minutes (usually about 15) , so that particles sedimented in the tube. This process was repeated up to four times. Each sediment was resuspended in fresh medium and the cells were separated by density gradient sedimentation. 10 ml of the suspension of cells were carefully layered over 20 ml of Hypaque, and centrifuged at 400 g for 20 minutes. The required cells were harvested from the interface of the medium and the Hypaque.

The cells were washed twice in growth medium, and then exposed to 1 ml of a suspension (solution) of EBv in growth medium to a concentration of 5 x 10 cells/ml and incubated at 37 C, 100% rh, in 5% carbon dioxide. T lymphocytes were effectively removed from the suspension by adding 1 μl cyclosporin A (1 mg/ml in absolute ethanol) to each ml of the growth medium with which the EBv infected cells were diluted.

Feeder cells were obtained by injecting 10 ml growth medium, at 37 C, into the peritoneal cavity of a freshly- killed mouse, and then withdrawing the growth medium. 0.1 ml of a suspension of feeder cells was placed into each well of a 96-well culture plate. 0.1 ml of a suspension of the replicating B lymphocytes was added to each well, the suspension being diluted with growth medium such that a statistically-predictable number of B ' lymphocytes was present in each well. The cultures, each 0.2 ml in volume, were incubated at 37 C, 100% rh, in 5% CO_?. 0.01 ml of foetal calf serum with glutamine, pyruvate and antibiotics, was added to each well, daily, until the volume in each reached 0.35 ml.

The B lymphocyte replication was estimated microscopically. Clones were isolated, to obtain clones making antibody against glioma. The release of human immunoglobulin and its antibody activity were detected by a conventional enzyme immunoassay. Example 2

-. Example 1 was repeated, but using human B lymphocytes obtained from the mesenteric lymph nodes of a patient with colon carcinoma. Example 3

Example 1 was repeated, but using human B lymphocytes obtained from the peripheral blood of a patient with ovarian carcinoma. Example 4

Example 1 was repeated, but using human B lymphocytes obtained from the draining lymph nodes of a human breast cancer, and separately from a human breast cancer. Example 5 Example 1 was repeated, but using human B lymphocytes obtained from the peripheral blood of normal people who have circulating antibodies reactive with the antigens of

Pseudomonas aeruginosa serotype 2, P. aeruginosa serotype

6, P_. aeruginosa serotype 7, P. aeruginosa serotype 11, Shigella sonnii. Salmonella paratyphi and Candida albicans.

Rabbit monoclonal antibodies may be produced by procedures as have been described above for human monoclonal antibodies.

ADDENDUM CELL TREATMENT FIELD OF THE INVENTION

This invention relates to cell treatment, with the intention that treatment should stimulate the growth of cells making a particular antibody or induce cells to make a particular antibody. BACKGROUND OF THE INVENTION

B lymphocytes proliferate in vivo, on stimulation caused by the presence of an antigen. The proliferating cells in a subject exposed to the antigen yield both plasma cells which actively synthesise and secrete antibody against the specific antigen and also memory cells which are effectively inert until stimulated by the presence of the antigen. Memory cells thus confer on a subject protection against reinfection by the specific antigen. OBJECT OF THE INVENTION

It is an object of the invention to produce antibodies in vitro.

STATEMENT OF THE INVENTION

According to the present invention, B lymphocyte cells are sensitised by exposure to an antigen in vitro, under conditions such that the B lymphocytes are induced to make antibody specific for the antigen. DETAILED DESCRIPTION OF THE INVENTION

The cells may be virgin B lymphocytes, or the sensitisation may be a secondary effect, or stimulation, with respect to already-sensitised B lymphocytes. Thus, in the invention, B lymphocytes (which may or may not be making immunoglobulin) and antigen are contacted (in circumstances under which the B lymphocytes are able to grow) in such a way that the B lymphocytes are induced to make antibody specific for the sensitising antigen, or B lymphocytes which are sensitised to the antigen (B memory cells) are induced to proliferate. If possible, isolated B memory cells may be used for specific purposes; such cells have the advantage that proliferation and antibody production are immediately consequent on exposure to the antigen.

The sensitised cells are treated so that they proliferate in culture and synthesise antibody which binds with epitopes on the antigen, e.g. as described above. The conditions suitable for conducting the method of the invention comprise any suitable B lymphocyte culture medium. This may be supplemented, as necessary or desired, with one or more agents which induce proliferation, e.g. feeder cells such as mouse macrophages.

The following Examples illustrate the invention. ("Sepharose" is a registered Trade Mark) . Example 1

30 ml whole blood were taken from a healthy adult man, and defibrinated. 10 ml aliquots of the defibrinated blood were layered over 15 ml aliquots of Hypaque and centrifuged at 400 g for 20 minutes. Serum was removed from the upper layer, and stored at 4 C. White blood cells were removed from the interface of serum and Hypaque. They were washed three times in RPMI 1640 growth medium (to which 10% foetal calf serum had been added) and resuspended in 30 ml growth medium containing 24 ml RPMI 1640, 2.4 ml foetal calf serum, 0.48 ml L-glutamine, 0.48 ml of penicillin and streptomycin, 0.24 ml sodium pyruvate and 2.4 ml of the serurn from the healthy adult man. One ml of cell suspension was placed in each of 3 rows of 10 culture wells.

Mouse macrophages were obtained by injecting 10 ml growth medium (at 37 C) into the peritoneal cavities of three freshly killed mice, and then withdrawing the suspensions of mouse macrophages in growth medium. One ml of the suspension obtained was added to each of the 3 rows of 10 culture wells described above. Tumour cells from the oligodendroglioma of a human subject were diced and macerated with scalpel blades. The tissue debris was homogenised in a Potter-Elvehjelm homogeniser. The antigenic material was centrifuged at 600 x g for 20 minutes, and then resuspended in sterile saline (0.85 g/1) to 2 g/1.

Doubling dilutions were prepared of the tumour antigen resuspension, and 0.1 ml of each dilution was added to one well of each of the 3 rows of culture wells containing mouse macrophages; in each row, therefore, each well received twice as much antigen as the well immediately to the right, and so that the 3 rows were 3 replicates of 10 culture wells. The cultures were incubated at 37 C, 100% rh in 5% carbon dioxide for 9 days. At intervals of 48 hours, 0.1 ml of growth medium (containing 40 μl foetal calf serum, 40 μl of serum from the healthy adult man, 8 μl of penicillin and streptomycin, 8 μl of L-glutamine and 4 μl of sodium pyruvate) was added to each culture well.

The culture obtained after 9 days may be treated by transformation with Epstein-Barr virus as described above. Example 2

The procedure of Example 1 was repeated, except that tissue was obtained from ovarian carcinoma tissue from a human subject. Example 3

The antigen Pseudomonas aeruginosa (serotype 11) was cultured by a standard bacteriological method. The organisms were harvested, and suspended in sterile saline. They were washed three times, by centrifugation and sterile aspiration, in sterile saline, and

7 resuspended in sterile saline to a concentration of 10 organisms per millilitre. The organisms were killed by repeatedly freezing to the temperature of liquid nitrogen and warming to 37 C, until a test sample of the suspension contained no live organisms. Six cycles of freezing and thawing were usually enough.

Dilutions were prepared of the killed organism, in culture medium, and 0.1 ml of each dilution was added to one well of each of three rows of culture wells containing mouse macrophages, prepared as in Example 1.

The further procedure of Example 1 was then followed.

Example 4

The procedure of Example 3 was repeated, but using

Branha ella catarrhalis as the antigen. Example 5

The procedure of Example 3 was repeated, but using

Shigella sonnii as the antigen. Example 6

Mouse IgG was obtained by affinity chromatography of mouse serum using a goat antibody against mouse gamma chain attached to Sepharose CL6B.

The mouse IgG was used as the antigen in the procedure of Example 1.

Claims

1. A process for preparing a human monoclonal antibody, which comprises stimulating human B lymphocytes with a mitogen, and isolating and selecting clones of antibody- producing proliferating cells.

2. A process according to claim 1, in which the mitogen is Epstein-Barr virus.

3. A process according to claim 1 or claim 2, in which the lymphocytes are induced to replicate in culture by infection with the mitogen, and are then transferred to a nutrient medium.

4. A process according to any preceding claim, in which T cells are removed after stimulation.

5. A process according to any preceding claim, which comprises the additional step of sensitising the lymphocytes before contacting them with the mitogen.