A general representation of the methods used to produce monoclonal antibodies.

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Monospecific antibodies are Antibodies that all have affinity for the same Antigen. Cloning in Biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as Bacteria, Insects 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. Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as Molecular biology is the study of Biology at a molecular level Medicine is the art and science of healing It encompasses a range of Health care practices evolved to maintain and restore Human Health by the When used as medications, the generic name ends in -mab (see "Nomenclature of monoclonal antibodies"). Components Infix for origin/source The infix preceding the -mab suffix denotes the animal origin of the antibodies

Discovery

The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated 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. Paul Ehrlich ( March 14, 1854 &ndash August 20, 1915) was a German Scientist who won the 1908 Nobel Paul Ehrlich ( March 14, 1854 &ndash August 20, 1915) was a German Scientist who won the 1908 Nobel

In the 1970s the B-cell cancer multiple myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This article is about the Decade 1970-1979 For the Year 1970 see 1970. Multiple myeloma (also known as MM, myeloma, plasma cell myeloma, or as Kahler's disease after Otto Kahler) is a type of A paraprotein is an abnormal Protein in the Urine or Blood, most often associated with benign MGUS (monoclonal gammopathy of undetermined significance This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

A process of producing monoclonal antibodies involving human-mouse hybrid cells was described by Jerrold Schwaber in 1973^[1] and remains widely cited among those using human-derived hybridomas. The year 1973 in Science and Technology involved some significant events listed below ^[2], but claims to priority have been controversial. A science history paper on the subject gave some credit to Schwaber for inventing a technique that was widely cited, but stopped short of suggesting that he had been cheated^[3]. The invention is generally accredited to Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975;^[4] who shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. Georges Jean Franz Köhler ( Munich, March 17 1946 &ndash March 1 1995 in Freiburg im Breisgau) was a German César Milstein ( October 8 1927 &ndash March 24 2002) was an Argentine Biochemist in the field of Antibody Niels Kaj Jerne, FRS ( December 23, 1911 &ndash October 7, 1994) was a Danish ( English -born Immunologist The year 1975 in Science and Technology involved some significant events listed below The Nobel Prize in Physiology or Medicine (Nobelpriset i fysiologi eller medicin is awarded once a year by the Swedish Karolinska Institute. The year 1984 in Science and Technology involved some significant events 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 humanize monoclonal antibodies,^[5] removing the reactions that many monoclonal antibodies caused in some patients. Sir Gregory Winter FRS is a British pioneer of therapeutic Monoclonal antibodies.

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.

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

Technician hand filling wells with a liquid for a research test. A technician is generally someone in a technological field who has a relatively practical understanding of the general theoretical principles of that field e 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). Multiple myeloma (also known as MM, myeloma, plasma cell myeloma, or as Kahler's disease after Otto Kahler) is a type of The House Mouse ( Mus musculus) is one of the most numerous species of the genus Mus commonly termed a Mouse. A lymphocyte is a type of White blood cell in the Vertebrate Immune system. Hybridoma are cells that have been engineered to produce a desired Antibody in large amounts

Lab technician bathing prepared slides in a solution. This technician prepares slides of monoclonal antibodies for researchers. The cells shown are labeling human breast cancer. Breast cancer is a Cancer that starts in the cells of the Breast in women and men

Hybridoma Cell Production

Monoclonal antibodies are typically made by fusing the spleen cells from a mouse that has been immunized with the desired antigen with myeloma cells. However, recent advances have allowed the use of rabbit B-cells. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium is used in which only fused cells can grow. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (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 biochemical pathway that they can use to synthesize purines. However, when cells are exposed to aminopterin (a folic acid analogue), they are unable to use this other, rescue, pathway and are now fully dependent on HGPRT for survival. The selective culture medium is called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine. Hypoxanthine is a naturally occurring Purine derivative It is occasionally found as a constituent of Nucleic acids where it is present in the Anticodon Aminopterin (4-aminopteroic acid a 4-amino analog of Folic acid, is an Antineoplastic drug with Immunosuppressive properties used in Chemotherapy Thymidine (more precisely called deoxythymidine; can also be labelled deoxyribosylthymine, and thymine deoxyriboside) is a Chemical compound This medium is selective for fused (hybridoma) cells because 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 are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal because it is a cancer cell. The fused hybrid cells are called hybridomas, and since they are derived from cancer cells, are immortal and can be grown indefinitely. Hybridoma are cells that have been engineered to produce a desired Antibody in large amounts

This mixture of cells is then diluted and clones are grown from single parent cells. The antibodies secreted by the different clones are then tested for their ability to bind to the antigen (for example with a test such as EIA or Antigen Microarray Assay) or immuno-dot blot, and the most productive and stable clone is then grown in culture medium to a high volume. A Dot blot (or Slot blot) is a technique in Molecular biology used to detect Biomolecules It represents a simplification of the Northern blot 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. A mouse (plural mice) is a small Animal that belongs to one In higher Vertebrates the peritoneum is the Serous membrane that forms the lining of the abdominal cavity &mdash it covers most of the intra-abdominal In Medicine ( Gastroenterology) ascites (also known as peritoneal cavity fluid, peritoneal fluid excess, hydroperitoneum or more

The medium must be enriched during selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Briclone is an IL-6 enriched Cloning medium for use in the stages following fusion in Hybridoma production Production in cell culture is usually preferred as the ascites technique is painful to the animal and if replacement techniques exist, this method is considered unethical. Ethics is a major branch of Philosophy, encompassing right conduct and good life

Recombinant

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant DNA is a form of synthetic DNA that is engineered through the combination or insertion of one or more DNA strands thereby combining DNA sequences Cloning in Biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as Bacteria, Insects Phage display is a method for the study of protein-protein protein-peptide and protein-DNA interactions that utilizes Bacteriophage to connect proteins with the genetic information Yeast display (or yeast surface display) is a technique used in the field of Protein engineering. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. A virus (from the Latin virus meaning Toxin or Poison) is a sub-microscopic infectious agent that is unable Yeasts are a growth form of eukaryotic Microorganisms classified in the kingdom Fungi, with about 1500 Species currently described These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. In Chemistry, an amino acid is a Molecule containing both Amine and Carboxyl Functional groups In Biochemistry, this ^[6] These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. ^[7] Fermentation chambers have been used to produce these antibodies on a large scale.

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). The western blot (alternatively immunoblot) is an Analytical technique used to detect specific Proteins in a given sample of tissue homogenate or Immunofluorescence is the labeling of antibodies or Antigens with fluorescent Dyes This technique is often used to visualize the subcellular 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. Immunoprecipitation (IP is the technique of precipitating a protein Antigen out of solution using an Antibody that specifically binds to that particular Affinity chromatography is a chromatographic method of separating biochemical mixtures based on a highly specific biologic interaction such as that between Antigen

Monoclonal antibodies for cancer treatment

Main article: Monoclonal antibody therapy

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. Monoclonal antibody therapy is the use of Monoclonal antibodies (or mAb to specifically target cells Cancer (medical term Malignant Neoplasm) is a class of Diseases in which a group of cells display uncontrolled An antigen (from antibody-generating) or immunogen is a substance that prompts the generation of Antibodies and can cause an immune response 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. A toxin ( Greek:, toxikon, lit (poison for use on arrows is a Poisonous substance produced by living cells or organisms that is active at very low A radionuclide is an Atom with an unstable nucleus, which is a nucleus characterized by excess energy which is available to be imparted either to a newly-created Cytokines are a category of signalling Proteins and Glycoproteins that like Hormones and Neurotransmitters, are used extensively in cellular A bispecific antibody is a Monoclonal antibodies which is designed to bind to two different Antigens. The fragment antigen binding (Fab fragment is a region on an Antibody which binds to Antigens. In fact, every intact antibody can bind to cell receptors or other proteins with its Fc region. The fragment crystallizable region (Fc region is the tail region of an Antibody that interacts with cell surface receptors called Fc receptors and some proteins

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. An adept is an individual identified as having attained a specific level of knowledge skill or aptitude in doctrines relevant to a particular author or organization Antibody-Dependent Cell-Mediated Cytotoxicity ( ADCC) is a mechanism of cell-mediated immunity whereby an effector cell of the Immune system actively lyses ^[8]

The illustration below shows all these possibilities:

Chimeric and humanized antibodies

Main article: humanized antibody

One problem in medical applications is that the standard procedure of producing monoclonal antibodies yields mouse antibodies. Humanized antibodies or chimeric antibodies are a type of Monoclonal antibody that have been synthesized using Recombinant DNA technology to circumvent 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. An immune system is a collection of mechanisms within an Organism that protects against Disease by identifying and killing Pathogens and Tumor

A solution to this problem would be to generate human antibodies directly from humans. However, this is not easy, primarily because it is generally not seen as ethical to challenge humans with antigen in order to produce antibody; the ethics of doing the same to non-humans is a matter of debate. 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. Mammals ( class Mammalia) are a class of Vertebrate Animals characterized by the presence of Sweat glands, including sweat glands Cell culture is the process by which prokaryotic, or eukaryotic cells are grown under controlled conditions (Bacteria cannot be used for this purpose, since they cannot produce this kind of glycoprotein. Not to be confused with Peptidoglycan. Glycoproteins are proteins that contain Oligosaccharide chains ( Glycans) covalently attached ) 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. This article is about organisms which have been genetically modified Monoclonal antibodies have been generated and approved to treat: cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection (see monoclonal antibody therapy). Cancer (medical term Malignant Neoplasm) is a class of Diseases in which a group of cells display uncontrolled Cardiovascular disease or cardiovascular diseases refers to the class of diseases that involve the Heart or Blood vessels ( arteries and Inflammation ( Latin, inflamatio, to set on fire is the complex biological response of vascular tissues to harmful stimuli such as Pathogens Macular degeneration is a medical condition usually of older adults which results in a loss of vision in the center of the visual field (the Macula) because Transplant rejection occurs when a transplanted organ or tissue fails to be accepted by the body of the transplant recipient Multiple sclerosis (abbreviated MS also known as disseminated sclerosis or encephalomyelitis disseminata) is an autoimmune condition in which the An infection is the detrimental Colonization of a host Organism by a foreign Species. Monoclonal antibody therapy is the use of Monoclonal antibodies (or mAb to specifically target cells

In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U. Pharmaceutical Research and Manufacturers of America (PhRMA is an industry trade group representing the Pharmaceutical research and Biotechnology companies in the S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration. ^[9]

Examples

See also

• List of monoclonal antibodies
• Monoclonal antibody therapy
• Nomenclature of monoclonal antibodies
• Polyclonal antibodies
• Nanobodies

References

External links

• Monoclonal Antibodies, from John W. Kimball's online biology textbook
• MeSH Monoclonal+antibodies