Monoclonal Antibody Technology
The clonal selection theory for antibody production means that all of the antibody molecules produced by a single B-cell are identical and recognise and bind to a single antigen (or, rather, antigenic determinant). Such an antibody is called a monoclonal antibody.
A polyclonal antibody, on the other hand, is really a mixture of antibody molecules that recognise different antigenic determinants. (It may also contain different antibodies that recognise the same antigenic determinant.)
If an antigen is injected into an animal, many different antibodies to the antigen are frequently produced, some binding to the same region (antigenic determinant) of the antigen molecule, others binding to different regions. Serum from such an animal would therefore contain a mixture of antibodies to the same antigen: it would be a polyclonal antiserum.
Polyclonal antibodies are useful in many ways, but because they are mixtures of different antibody molecules, they pose certain problems to biochemists who study antibodies. With polyclonal antibodies, it is impossible to study the molecular properties of a single type of antibody molecule in isolation from the others.
Ideally, one would wish to look at one antibody that is produced by a single immune cell (that is, a monoclonal antibody), rather than a mixture of antibodies produced by many different immune cells. Before 1975 the only major source of monoclonal antibodies were myeloma cells. Myelomas are cancers of the B-cells of the immune system.
It is well known that when a cancer arises it is almost always derived from a single normal cell whose growth control mechanisms have gone haywire. A cancer of the B-cells, therefore, consists of cells derived from a single, antibody-producing cell.
Since each antibody-producing cell makes antibody molecules, all of which are identical, having the same antigen specificity, then the antibody produced by a myeloma is a monoclonal antibody.
Myelomas are rapidly growing cancers and can be isolated from the rest of the (normal) antibody-producing cells of an animal and grown in a test-tube. Myelomas were therefore valuable for studies aimed at understanding the basic structure and properties of antibody molecules.
However, because myelomas, like all cancers, arise randomly within a normal B-cell population, any one of the millions of antibody-producing cells in the body could become a myeloma.
The monoclonal antibodies produced by myelomas therefore tend to lack any known antigen specificity: the chances of identifying specific antigens that they would recognise are very slim, and one would need to screen hundreds of thousands of different antigens to be sure to identify their specificities. This is impracticable.
What was needed was a method for producing large amounts of monoclonal antibodies to known antigens at will. This would allow detailed studies of how antibodies bind to their specific antigens to be carried out, and it would eliminate any complications caused by the mixtures of antibodies that occur in polyclonal antibody preparations. B-cells are, unfortunately, very difficult to grow outside the body.
If they are transferred from their bodily environment to special nutrient broths they usually die, in contrast to myeloma cells, which can readily be grown in isolation from the body. However, Kohler and Milstein hit upon a way of solving this problem and allowing B-cells to grow and produce their monoclonal antibodies to defined antigens in test-tubes.
Kohler obtained his doctoral degree from the University of Freibourg in Germany in 1984, after which he immediately joined Milstein's research group in Cambridge to carry out his postdoctoral research project. Milstein's team had been working for many years on the mechanisms by which B-cells generate antibody diversity.
One approach that they were using at the time Kohler joined the laboratory was to fuse two different myeloma cells together to produce a hybrid cell. Such hybrid cells, like their unfused myeloma parent cells, were able to grow in test-tubes in the presence of appropriate nutrients.
When pairs of myelomas were fused, the hybrids were found to express the specific antibodies of both parent myeloma cells. From these studies of myeloma hybrid cells, Milstein and his colleagues learned more about the mechanisms of antibody diversification in B-cells.
Kohler began to work on the antibodies produced by myeloma cells and on hybrids between two different myelomas, but he and Milstein realised that these studies would be much more informative if they could obtain a myeloma that produces an antibody to a known specific antigen.
Unfortunately, the antigens recognised by the antibodies produced by almost all of the myelomas studied at that time had not been identified: they were monoclonal antibodies without any known antigen identity.
A few myelomas were available whose antigens had been found, by chance, but these proved not to be useful to Kohler and Milstein, since they failed to grow well under laboratory conditions.
Notwithstanding these problems, the two scientists continued with their studies, and one approach they considered was that of screening many antigens with some of the myeloma-derived monoclonal antibodies, in order to determine whether or not they could find, by a process equivalent to searching for a needle in a haystack, the particular antigens that these antibodies recognised.
This would be a laborious task, since many hundreds of thousands of antigens would need to be screened to have a reasonable chance of finding the antigens in question.
Kohler and Milstein had the simple idea of fusing B-cells, obtained from an animal immunised with a particular antigen, with myeloma cells. If these hybrid cells could be obtained and grown in culture, they thought, it might be that they would produce not only the monoclonal antibody of the myeloma parent cell (against an unspecified antigen), but also the monoclonal antibody from the B-cell parent (against the specified antigen).
In other words, if an antigen is injected into an animal, that animal will produce antibodies to the antigen. If the B-cells from these animals could be grown as hybrids with myeloma cells, then some of these hybrids should produce antibodies to the injected antigen, and they could readily be detected by their ability to recognise the antigen that was injected.
B-cells fail to grow outside of an animal, but they might grow if they were fused to myeloma cells, which often grow prolifically in test-tubes.
Theoretical considerations suggested that the chances of production of specific monoclonal antibodies by B-cell/myeloma hybrids by the method proposed by Kohler and Milstein were slim and that the work involved would be very time-consuming. However, Milstein and Kohler decided to go ahead anyway, just in case their theoretical considerations were not entirely correct.
Their perseverance was rewarded: when B-cells obtained from the spleens of mice injected with a chosen antigen were fused with myeloma cells, not only did the B-cell/myeloma hybrid cells grow well in test-tubes, but also some of the hybrid cells were found to produce antibodies specific to the antigen that was injected. Subsequent developments allowed such B-cell/ myeloma hybrids, which are called hybridomas, to be obtained at ease.
Hybridomas can now be obtained that produce antibodies to virtually any antigen. Kohler and Milstein had developed a much-needed method for making monoclonal antibodies at will to a chosen antigen.
An outline of the method for monoclonal antibody production is shown in Diagram 1. An animal is injected with the antigen against which monoclonal antibodies are required. The animal produces antibodies to this antigen, and the antibodies can be detected in the serum of these animals.
When the animal is producing sufficiently high levels of serum (polyclonal) antibodies to the antigen, its spleen is removed and its spleen cells, which are a rich source of the antibody-producing B-cells, are fused with myeloma cells (grown in test-tubes).
Parent spleen B-cells fail to survive because they cannot grow in the nutrient medium, and special drugs are used to selectively kill the parent myeloma cells that do not fuse.
In this way, only hybrid B-cell/myeloma cells (hybridomas) survive. The myelomas used nowadays do not produce their own antibody, and so the resultant hybridomas produce only the specific antibody that was made by the spleen B-cell partner. Hybridomas are separated from each other and allowed to multiply and grow in a special nutrient medium.
This produces many clones of hybridomas; each clone consists of a population of identical cells derived from a single original hybridoma cell. Hybridoma clones are then screened for their ability to produce antibodies that recognise the injected antigen.
Those that do make such antibodies are derived from a single B-cell in the animal that was injected with antigen: they produce monoclonal antibodies to the defined antigen. The spleen cell contributes the specific antibody producing property to the hybridoma, whereas the myeloma cell provides the important feature of being able to multiply and grow indefinitely in nutrient media in isolation from an animal's body.
Hybridomas producing monoclonal antibodies to a particular antigen can be grown in special nutrients in test-tubes forever, if that were necessary. They can also be frozen, stored for many years, then thawed and revived, when they continue to produce their antibodies. This means that monoclonal antibodies can be produced in limitless quantities, allowing their continued use in a standardised way.
Polyclonal antisera, in addition to containing many different antibodies, are not available in limitless supply, since they are usually obtained from the blood of an animal and when the animal dies the supply of the polyclonal antibodies becomes exhausted.
It is very difficult to produce exactly the same polyclonal antiserum by injecting another animal with the same antigen, and this means that polyclonal antibodies cannot be standardised as readily as monoclonal antibodies.
Since Kohler and Milstein made their discovery, monoclonal antibody technology has advanced in many ways. For example, two monoclonal antibodies can be joined together to produce an antibody that recognises not one, but two antigens. Another approach that is being taken to exploit the method is that of linking monoclonal antibodies to poisons.
Some poisons, such as ricin, which is a protein obtained from castor beans, are very potent in their ability to kill living cells. It would be highly desirable in some medical circumstances to have a poison as powerful as ricin. For example, in order to kill cancer cells, a very potent toxin would be ideal.
Unfortunately, ricin kills normal cells as well as cancer cells, so it is of no use by itself as an anti-cancer drug. However, the ricin molecule is made up from two different subunits: one of them (the B subunit) binds to living cells, whilst the other (the A subunit) enters cells and kills them.
Without the A subunit, the B subunit binds to cells but causes them no harm. Without the B subunit, the A subunit will not bind to cells and cannot enter them. The actual killing property of ricin therefore lies with the A subunit, which is completely harmless on its own.
Nevertheless, when the A subunit is joined to an antibody that binds to living cells, the A subunit will enter those cells and kill them almost as powerfully as it kills them when the A subunit is joined to the B subunit of ricin.
In other words, when the B subunit of ricin is replaced with an antibody, the new antibodyA subunit complex is a potent poison. However, unlike the ricin B subunit, which binds to most cell types, antibodies can be produced that bind to specific kinds of cells and this means that antibodies can direct the A subunit to specific types of cell and selectively kill them.
If now one obtains a monoclonal antibody that binds to an antigen on cancer cells, and if this antigen does not occur on normal cells, then the antibody-A subunit complex will kill cancer cells and not normal cells (Diagram 2).
The exciting possibility of destroying specific living cells, whilst leaving intact those cells one desires, is an area of intense research and one of its benefits may be the development of 'magic bullets', or antibody-poison conjugates, in which the antibody seeks out antigens on the surface of specific cancer cells (or other cells), and the poison kills those cells.
Diagnoses of diseases and other conditions, such as pregnancy, are also promising applications of monoclonal antibodies. Their high specificity for individual antigens, their long-term availability and their ease of production and standardisation, make them well suited for diagnosis.
If a disease or physiological situation is associated with changes in levels of a specific antigen, monoclonal antibodies that bind to the antigen can potentially be used to detect these altered levels and therefore to aid diagnosis.
Pregnancy, for example, results in increased levels of the hormone, chorionic gonadotropin, in urine. If a plastic stick, which has antibody that binds to the hormone attached to it, is dipped into the urine, the hormone will stick to the antibody.
The amount of hormone antigen 'captured' by the antibody on the stick can, be determined by linking the process to a colour-producing reaction. For instance, in some commercial kits the stick turns blue, indicating that chorionic gonadotropin levels in urine are high and pregnancy is confirmed.
Monoclonal antibodies have opened up new avenues and increased reproducibility and accuracy of such diagnostic tests.
Monoclonal antibodies will be used increasingly in biomedical research, medical diagnosis and disease treatment, and many more applications will undoubtedly be forthcoming.