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Monoclonal antibodies

Drugs & Medication

Monoclonal antibodies

Nomenclature of monoclonal antibodies

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Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell and are 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").

Contents

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, César Milstein, and Niels Kaj Jerne in 1975[1]; 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[2], removing the reactions that many monoclonal antibodies caused in some patients.

Production

Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them. Researchers looking at slides of cultures of cells that make monoclonal antibodies. These are grown in a lab and the researchers are analyzing the products to select the most promising of them.

Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture. Monoclonal antibodies can be grown in unlimited quantities in the bottles shown in this picture.

Technician's hand filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma). Technician's hand filling wells with a liquid for a research test. This test involves preparation of cultures in which hybrids are grown in large quantities to produce desired antibody. This is effected by fusing myeloma cell and mouse lymphocyte to form a hybrid cell (hybridoma).

Lab technician bathing prepared slides in a solution. This technician is involved in the preparation of slides of monoclonal antibodies for researchers. These highly specific cells shown are those labeling human breast cancer. Lab technician bathing prepared slides in a solution. This technician is involved in the preparation of slides of monoclonal antibodies for researchers. These highly specific cells shown are those labeling human breast cancer.

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 recognize 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 found in serum 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 several times with the antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer) and that have lost the ability to produce antibodies. This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol, electroporation or, of historical importance, infection with some virus. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. 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.

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. The medium must be enriched during selection to favour hybridoma growth. This can be achieved by the use of a layer of feeder cells or supplement media such as briclone.

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 may be very painful to the animal and if replacement techniques exist, may be considered unethical. Fermentation chambers have been used to produce antibodies on a larger scale. Nowadays, bioengineering allow production of antibodies in plants.

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). They are also very useful in immunohistochemistry which detect antigen in fixed tissue 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:

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment.
Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. [3]

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 have been generated and approved to treat;cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, and viral infection (see monoclonal antibody therapy).

In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration.[4]

See also

References

  1. ^ 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.
  2. ^ Riechmann L, Clark M, Waldmann H, Winter G. Reshaping human antibodies for therapy. Nature 1988;332:323-7. PMID 3127726.
  3. ^ Modified from Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129
  4. ^ PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006. Retrieved 2006-09-04.

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This guide is licensed under the GNU Free Documentation License. It uses material from the Wikipedia.


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