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. When used as medications, the generic name ends in -mab (see "Nomenclature of monoclonal antibodies").

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 ("epitopes") of the antigen. This natural mixture of antibodies is known as polyclonal antibodies.

To produce monoclonal antibodies, one removes B-cells from the spleen or lymph nodes 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), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of antibodies. The hybridomas are sufficiently diluted to ensure clonality and grown. The antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA) or immuno-dot blot, and the most sensitive one is picked out.

Monoclonal antibodies can be produced in cell culture or in live 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. Production in cell culture is usually preferred as the ascites technique proves to be very painful and if replacement techniques exist, it is rather unethical. Fermentation chambers have been used to produce antibodies on a larger scale.

In the above process, one uses myeloma cell lines that have lost their ability to produce their own antibodies or antibody chain, so as to not contaminate the target antibody. Furthermore, one employs 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). These cells are preselected by the use of 8-azaguanine media prior to the fusion. Cells that possess the HGPRT enzyme will be killed by the 8-azaguanine. During the fusion process many cells can fuse. Myeloma with myeloma, spleen cell with spleen cell, 3 cells of different types etc.... The desired fusions are between healthy B-cells producing antibodies against the antigen of interest and myeloma cells. These are relatively 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 the presence and quantity of this substance, for instance in a Western blot test (to detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a cell). There are also very useful in immunohistochemistry which detect antigen in fixed tissueu sections. Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.

Monoclonal antibodies for cancer treatment

One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified 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 with its Fc region. The illustration 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. Although murine antibodies are very similar to human ones there are differences. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.

A solution to this problem would be to generate human antibodies directly from humans. However, this is not easy primarily because it is clearly not ethical to challenge humans with antigen in order to produce antibody. Furthermore, it is not easy to generate human antibodies against human tissues.

Various approaches using recombinant DNA technology to overcome this problem have been tried since the late 1980s. 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 mice genetically engineered to produce more human-like antibodies.

Monoclonal antibodies Drugs Approved

This is a list adapted from information in a 2003 Nature Medicine article and organized according to Indication.

• Transplant rejection
  • Muronomab-CD3, approved 1986 (first ever approval)
  • Daclizumab, approved 1997
  • Basiliximab, approved 1998

• Cardiovascular disease
  • Abciximab, approved 1994

• Cancer
  • Rituximab, approved 1997
  • Trastuzumab (Herceptin®), approved 1998
  • Gemtuzumab ozogamicin, approved 2000
  • Alemtuzumab, approved 2001
  • Ibritumomab tiuxetan, approved 2002
  • Cetuximab, approved 2004
  • Bevacizumab, approved 2004
  • Tositumomab, approved 2003

• Viral infection
  • Palivizumab, approved 1998

• Inflammatory diseases
  • Infliximab, approved 1998
  • Eculizumab, approved 2000
  • Omalizumab, approved 2004
  • Efalizumab
  • Adalimumab

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 (a paraprotein). This was used to study the structure of antibodies, but it was not yet 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 1975; 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 1988 Greg Winter and his team pioneered the techniques to humanise monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.

See also

• Nomenclature of monoclonal antibodies

References

1. Waldmann, Thomas A. (2003). Immunotherapy: past, present and future. Nature Medicine 9, 269-277.
2. ORTHOCLONE OKT^®3 Information page at Ortho Biotech. Retrieved 2005-03-10.
3. ZENAPAX ® (daclizumab) Information page at Roche Pharmaceuticals. Retrieved 2005-03-10.
4. Novartis product page for Simulect (basiliximab for injection) . Retrieved 2005-03-09.
5. 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.
6. Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332:323-7. PMID 3127726.

External links

• Monoclonal Antibodies, from John W. Kimball's online biology textbook
• Production and Quality Control of Monoclonal Antibodies; European Commission directive, July 1995

Immune system | Biotechnology | Monoclonal antibodies

Monoklonaler Antikörper | anticuerpo monoclonal | モノクローナル抗体 | Przeciwciała monoklonalne | 单克隆抗体