Monoclonal antibody

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine.

Production

If a foreign substance (an antigen) is injected into a vertebrate such as a mouse or a human, some of the immune system's B-cells will turn into plasma cells and start to produce antibodies that bind to that antigen. Each B-cell produces only one kind of antibody, but different B-cells will produce structurally different antibodies that bind to different parts of the antigen. This mixture of antibodies is known as polyclonal antibodies.

To produce monoclonal antibodies, one removes B-cells from the spleen of an animal that has been challenged with the antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is done by making the cell membranes more permeable. The fused hybrid cells (called hybridomas) will multiply rapidly and indefinitely (since they are cancer cells after all) and will produce large amounts of antibodies. The hybridomas are sufficiently diluted and grown, thus obtaining a number of different colonies, each producing only one type of antibody. The antibodies from the different colonies are then tested for their ability to bind to the antigen (for example with a test such as ELISA), and the most effective one is picked out.

Monoclonal antibodies can be produced in cell culture or in animals. When the hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid.

In the above process, myeloma cell lines are used that have lost their ability to produce their own antibodies, so as to not dilute the target antibody. Furthermore, one uses only myeloma cells that have lost a specific enzyme (hypoxanthine-guanine phosphoribosyltransferase, HGPRT) and therefore cannot grow under certain conditions (namely in the presence of HAT medium). Fusions between healthy B-cells and myeloma cells are rare, but when one succeeds, then the healthy partner supplies the needed enzyme and the fused cell can survive in HAT medium. This is the trick to detect the successfully fused cells.

Applications

Once monoclonal antibodies for a given substance have been produced, they can be used to detect for the presence and quantity of this substance, for instance in a Western blot test (to detect a substance in a solution) or an immunofluorescence test (to detect a substance in a whole cell). Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

In medicinal treatments, the small variation (if any) in recognizing the antigen helps to reduce side effects. However, there are drawbacks to using monoclonal antibodies as opposed to polyclonals. Each B-lymphocyte produces antibodies that are specific not to an antigen, but to an epitope of that antigen. An epitope is a small piece of the antigen to which the antibody binds. Polyclonal antibodies bind to many epitopes of a given antigen, while monoclonals bind to a single epitope. In the processing of antibodies, certain binding capabilities are degraded. If the monoclonal antibody is susceptible to such degradation, it is useless. Polyclonals will still be useful even if certain epitope-binding species are degraded.

Monoclonal antibodies for cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cells specific antigen and induce an immunological response on the target cancer cell. Such mAb could also be modificated for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact every intact antibody can bind to cell receptors or other proteins, however with their Fc region. The picture below shows all these possibilities:

Chimeric and humanized antibodies

One problem in medical applications is that the standard procedure of producing monoclonal antibodies yields mouse antibodies, and these are rejected by the human immune system. Various approaches to overcome this problem have been tried. In one approach, one takes the DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies. (Bacteria cannot be used for this purpose, since they cannot produce this kind of glycoprotein.) Depending on how big a part of the mouse antibody is used, one talks about chimeric antibodies or humanized antibodies. Another approach involves genetically engineered mice that produce more human-like antibodies.

FDA approved monoclonal antibodies

This is a list adapted from information in a 2003 Nature Medicine articleTemplate:Ref and organized according to indication.

• Transplant rejection
  • Muronomab-CD3, an Ortho Biotech productTemplate:Ref, approved 1986 (first ever approval)
  • Daclizumab, a Roche Pharmaceuticals productTemplate:Ref, approved 1997
  • Basiliximab, a Novartis Pharmaceuticals productTemplate:Ref, approved 1998

• Cardiovascular disease
  • Abciximab, approved 1994

• Cancer
  • Rituximab, approved 1997
  • Trastuzumab, approved 1998
  • Gemtuzumab ozogamicin, approved 2000
  • Alemzutumab, approved 2001
  • Ibritumomab, approved 2002

• Viral infection
  • Palivizumab, approved 1998

• Inflammatory diseases
  • Infliximab, approved 1998

Discovery

The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century figured that if a compound could be made that selectively targeted a disease causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.

In the 1970s, the B-cell cancer myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody. This was used to study the structure of antibodies, but it was not possible to produce identical antibodies specific to a given antigen.

The process of producing monoclonal antibodies described above was invented by Georges Köhler and César Milstein in 1975Template:Ref; they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody producing B-cells, and be able to select for the successfully fused cells.

In the 1980s Greg Winter pioneered the techniques to humanise monoclonal antibodies, while in the 1990s he began work on domain antibodies.

References

note for drug-related information sites: please take into account warnings on pages that might indicate "for US audiences only" or some other "intended audience"

1. Template:Note Waldman, Thomas A. (2003). Immunotherapy: past, present and future (http://www.nature.com/cgi-taf/DynaPage.taf?file=/nm/journal/v9/n3/full/nm0303-269.html&filetype=pdf). Nature Medicine 9, 269-277.
2. Template:Note ORTHOCLONE OKT^®3 Information page at Ortho Biotech (http://www.orthobiotech.com/orthoclone.html). Retrieved 2005-03-10.
3. Template:Note ZENAPAX ® (daclizumab) Information page at Roche Pharmaceuticals (http://www.rocheusa.com/products/zenapax/). Retrieved 2005-03-10.
4. Template:Note Novartis product page for Simulect (basiliximab for injection)  (http://www.pharma.us.novartis.com/products/name/simulect.jsp). Retrieved 2005-03-09.
5. Template:Note Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7. PMID 1172191. Reproduced in J Immunol 2005;174:2453-5. PMID 15728446.de:Monoklonaler Antikörper

ja:モノクローナル抗体 zh:单克隆抗体