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.