Monoclonal Antibodies
Humans (and mice) have the ability to make antibodies able to
- recognize (by binding to) virtually any antigenic determinant (epitope)
- to discriminate between even similar epitopes.
Not only does this provide the basis for protection against disease organisms, but it makes antibodies attractive candidates to target other types of molecules found in the body, such as:
- receptors or other proteins present on the surface of normal cells
- molecules present uniquely on the surface of cancer cells.
Thus the remarkable specificity of antibodies makes them promising agents for human therapy. Imagine, for example, being able to
- make an antibody that will bind only to the cancer cells in a patient
- coupling a cytotoxic agent (e.g. a strong radioactive isotope) to that antibody, and then
- giving the complex to the patient so it can seek out and destroy the cancer cells (and no normal cells).
But there are problems to be solved before antibodies can be used in human therapy.
1. The response of the immune system to any antigen, even the simplest, is polyclonal. That is, the system manufactures antibodies of a great range of structures both in their binding regions as well as in their effector regions.
2. Even if one were to isolate a single antibody-secreting cell, and place it in culture, it would die out after a few generations because of the limited growth potential of all normal somatic cells.
What is needed is a way to make "monoclonal antibodies":
- antibodies of a single specificity that are
- all built alike because they are being manufactured by a single clone of plasma cells
- that can be grown indefinitely.
This problem was solved for mice in 1975 with a technique devised by Köhler and Milstein (for which they were awarded a Nobel Prize).
An antibody-secreting B cell, like any other cell, can become cancerous. The unchecked proliferation of such a cell is called a myeloma.
Köhler and Milstein found a way to combine
- the unlimited growth potential of myeloma cells with
- the predetermined antibody specificity of normal immune spleen cells.
They did this by literally fusing myeloma cells with antibody-secreting cells from an immunized mouse. The technique is called somatic cell hybridization. The result is a hybridoma.
The procedure
Mix
- spleen cells from a mouse that has been immunized with the desired antigen with
- myeloma cells.
Use an agent to facilitate fusion of adjacent plasma membranes.
Even so, the success rate is so low that there must be a way to select for the rare successful fusions. So,
use myeloma cells that have:
- lost the ability to synthesize hypoxanthine-guanine-
phosphoribosyltransferase (HGPRT).
This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate pathway that they can use to synthesize purines.
However, when cells are exposed to aminopterin (a folic acid analog), they are unable to use this other pathway and are now fully dependent on HGPRT for survival.
- lost the ability to synthesize any antibody molecules of their own (so as not to produce a hybridoma producing two kinds of antibody molecules).
1. The first property is exploited by transferring the cell fusion mixture to a culture medium - called HAT medium because it contains:
- hypoxanthine
- aminopterin
- the pyrimidine thymidine
The logic:
- Unfused myeloma cells cannot grow because they lack HGPRT.
- Unfused normal spleen cells cannot grow indefinitely because of their limited life span. However,
- Hybridoma cells (produced by successful fusions) are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal.
2. Test the supernatants from each culture to find those producing the desired antibody.
3. Because the original cultures may have been started with more than one hybridoma cell, you must now isolate single cells from each antibody-positive culture and subculture them.
4. Again, test each supernatant for the desired antibodies. Each positive subculture - having been started from a single cell - represents a clone and its antibodies are monoclonal. That is, each culture secretes a single kind of antibody molecule directed against a single determinant on a preselected antigen.
5. Scale up the size of the cultures of the successful clones.
Hybridoma cultures can be maintained indefinitely:
- in vitro; that is, in culture vessels. The yield runs from 10-60 µg/ml.
- in vivo; i.e., growing in mice. Here the antibody concentration in the serum and other body fluids can reach 1-10 mg/ml. However, animal welfare activists in Europe and in the U.S. are trying to limit the use of mice for the production of monoclonals.
Uses for monoclonal antibodies
Monoclonal antibodies are widely used as diagnostic and research reagents. Their introduction into human therapy has been much slower.
In some in vivo applications, the antibody itself is sufficient. Once bound to its target, it triggers the normal effector mechanisms of the body.
In other cases, the monoclonal antibody is coupled to another molecule, for example
- a fluorescent molecule to aid in imaging the target
- a strongly-radioactive atom, such as Iodine-131 to aid in killing the target.
- OKT3. Binds to a molecule on the surface of T cells. Used to prevent acute rejection of organ, e.g., kidney, transplants.
- LymphoCide. Binds to CD22, a molecule found on some B-cell leukemias.
- Rituximab (trade name = Rituxan). Binds to the CD20 molecule found on most B-cell lymphomas but not on normal B cells.
- Lym-1 (trade name = Oncolym). Binds to the HLA-DR-encoded histocompatibility antigen that can be expressed at high levels on lymphoma cells.
- Daclizumab (trade name = Zenopax). Binds to part of the IL-2 receptor produced at the surface of activated T cells. Used to prevent acute rejection of transplanted kidneys.
- Infliximab. Binds to tumor necrosis factor-alpha (TNF-alpha). Shows promise against some inflammatory diseases such as rheumatoid arthritis.
- Herceptin. Binds HER-2/neu, a growth factor receptor found on some tumor cells (some breast cancers, lymphomas). The only monoclonal so far that seems to be effective against solid tumors.
- CMA-676.A conjugate of
- a monoclonal antibody that binds CD33, a cell-surface molecule expressed by the cancerous cells in acute myelogenous leukemia (AML) but not found on the normal stem cells needed to repopulate the bone marrow.
- calicheamicin, an oligosaccharide that blocks the binding of transcription factors (proteins) to DNA and thus inhibits transcription.
CMA-676 is the first immunotoxin that shows promise in the fight against cancer.
- Vitaxin. Binds to a vascular integrin (anb3) found on the blood vessels of tumors but not on the blood vessels supplying normal tissues. In Phase II clinical trials, Vitaxin has shown some promise in shrinking solid tumors without harmful side effects.
Problems with monoclonal therapy
Why are there so few monoclonals being used in human therapy a quarter century after their discovery? The main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies"). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.
(Monoclonal antibodies raised in humans would lessen the problem, but few people would want to be immunized in an attempt to make them and most of the attempts that have been made have been unsuccessful.)
Two approaches have been used in an attempt to reduce the problem of HAMA.
- Chimeric antibodies. The antigen-binding part (variable regions) of the mouse antibody is fused to the effector part (constant region) of a human antibody using genetic engineering. Infliximab is one of these.
- "Humanized" antibodies. The amino acids responsible for making the antigen binding site (the hypervariable regions) are inserted into a human antibody molecule replacing its own hypervariable regions. Daclizumab, Vitaxin, and herceptin are examples.
In both cases, the new gene is expressed in mammalian cells grown in tissue culture (E. coli cannot add the sugars that are a necessary part of these glycoproteins).
Looking ahead
Although still in the experimental stage, other ways of solving the problem of HAMA are being studied. One of these is to exploit transgenic technology to make transgenic mice that:
- have had human antibody gene loci inserted into their bodies (using the embryonic stem cell method).
- have had their own genes for making antibodies "knocked out".
The result is a mouse that
- can be immunized with the desired antigen
- produces human, not mouse, antibodies against the antigen
- can yield cells to be fused with myeloma cells to manufacture all-human monoclonal antibodies.
20 September 1999