Monoclonal antibodies
Overview
Monoclonal antibodies are monospecific antibodies
Monospecific antibody
Monospecific antibodies are antibodies that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.-Hybridoma cell:...

 that are the same because they are made by identical immune cells
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

 that are all clones
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...

 of a unique parent cell.

Given almost any substance, it is possible to produce 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
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

, molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....

. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies
Nomenclature of monoclonal antibodies
The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary, names to monoclonal antibodies. An antibody is a protein that is produced in B cells and used by the immune system of humans and other vertebrate animals to identify a specific foreign object like...

").
The idea of a "magic bullet" was first proposed by Paul Ehrlich
Paul Ehrlich
Paul Ehrlich was a German scientist in the fields of hematology, immunology, and chemotherapy, and Nobel laureate. He is noted for curing syphilis and for his research in autoimmunity, calling it "horror autotoxicus"...

, 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.
Encyclopedia
Monoclonal antibodies are monospecific antibodies
Monospecific antibody
Monospecific antibodies are antibodies that all have affinity for the same antigen. Monoclonal antibodies are monospecific, but monospecific antibodies may also be produced by other means than producing them from a common germ cell.-Hybridoma cell:...

 that are the same because they are made by identical immune cells
White blood cell
White blood cells, or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a...

 that are all clones
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...

 of a unique parent cell.

Given almost any substance, it is possible to produce 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
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

, molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....

. When used as medications, the non-proprietary drug name ends in -mab (see "Nomenclature of monoclonal antibodies
Nomenclature of monoclonal antibodies
The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary, names to monoclonal antibodies. An antibody is a protein that is produced in B cells and used by the immune system of humans and other vertebrate animals to identify a specific foreign object like...

").

Discovery

The idea of a "magic bullet" was first proposed by Paul Ehrlich
Paul Ehrlich
Paul Ehrlich was a German scientist in the fields of hematology, immunology, and chemotherapy, and Nobel laureate. He is noted for curing syphilis and for his research in autoimmunity, calling it "horror autotoxicus"...

, 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. He and Élie Metchnikoff received the 1908 Nobel Prize for Physiology or Medicine for this work, which led to an effective syphilis treatment by 1910.

In the 1970s, the B-cell cancer multiple myeloma
Multiple myeloma
Multiple myeloma , also known as plasma cell myeloma or Kahler's disease , is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies...

 was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein
Paraprotein
A paraprotein is an immunoglobulin or immunoglobulin light-chain that is produced in excess by the clonal proliferation of plasma cells. Detection of paraproteins in the urine or blood is most often associated with benign MGUS , where they remain "silent", and multiple myeloma. An excess in the...

). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

.

Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973 and remains widely cited among those using human-derived hybridomas, 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. The invention was conceived by George Pieczenik, with John Sedat, Elizabeth Blackburn
Elizabeth Blackburn
Elizabeth Helen Blackburn, AC, FRS is an Australian-born American biological researcher at the University of California, San Francisco, who studies the telomere, a structure at the end of chromosomes that protects the chromosome. Blackburn co-discovered telomerase, the enzyme that replenishes the...

's husband, as a witness and reduced to practice by Cotton and Milstein, and then by Kohler and Milstein. Georges Köhler
Georges J. F. Köhler
-External links:* http://www.nobel.se/medicine/laureates/1984/...

, César Milstein
César Milstein
César Milstein FRS was an Argentine biochemist in the field of antibody research. Milstein shared the Nobel Prize in Physiology or Medicine in 1984 with Niels K. Jerne and Georges Köhler.-Biography:...

, and Niels Kaj Jerne
Niels Kaj Jerne
Niels Kaj Jerne, FRS was a Danish immunologist. He was awarded the Nobel Prize in Physiology or Medicine in 1984. The citation read "For theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies"....

 in 1975; who shared the Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...

 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
Greg Winter
Sir Gregory Winter FRS is a British pioneer of therapeutic monoclonal antibodies. He invented techniques to both humanise and, later, to fully humanise using phage display, antibodies for therapeutic uses...

 and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.

Production


Hybridoma cell production

Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma
Rabbit hybridoma
A rabbit hybridoma is a hybrid cell line formed by the fusion of an antibody producing rabbit B cell with a cancerous B-cell .- History :...

.
Polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...

 is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which only fused cells can grow is used. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis
Salvage pathway
A salvage pathway is a pathway in which nucleotides are synthesized from intermediates in the degradative pathway for nucleotides....

 of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis
De novo synthesis
De novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as...

 pathway is also disrupted. By exposing cells to aminopterin
Aminopterin
Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...

 (a folic acid analogue, which inhibits dihydrofolate reductase
Dihydrofolate reductase
- Function :Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids...

, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring supplementation to survive.

The selective culture medium is called HAT medium because it contains hypoxanthine
Hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,...

, aminopterin, and thymidine
Thymidine
Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...

. This medium is selective for fused (hybridoma
Hybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...

) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (as it is a cancer cell).

This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...

 or Antigen Microarray Assay) or immuno-dot blot
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...

. The most productive and stable clone is then selected for future use.

The hybridomas can be grown indefinitely in a suitable cell culture medium.They can also be injected into mice
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...

 (in the peritoneal cavity
Peritoneum
The peritoneum is the serous membrane that forms the lining of the abdominal cavity or the coelom — it covers most of the intra-abdominal organs — in amniotes and some invertebrates...

, surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites
Ascites
Ascites is a gastroenterological term for an accumulation of fluid in the peritoneal cavity.The medical condition is also known as peritoneal cavity fluid, peritoneal fluid excess, hydroperitoneum or more archaically as abdominal dropsy. Although most commonly due to cirrhosis and severe liver...

 fluid.

The medium must be enriched during in-vitro 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. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical
Ethics
Ethics, also known as moral philosophy, is a branch of philosophy that addresses questions about morality—that is, concepts such as good and evil, right and wrong, virtue and vice, justice and crime, etc.Major branches of ethics include:...

.

Purification of monoclonal antibodies

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration
Filtration
Filtration is commonly the mechanical or physical operation which is used for the separation of solids from fluids by interposing a medium through which only the fluid can pass...

 with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling
Membrane fouling
Membrane fouling is a process where solute or particles deposit onto a membrane surface or into membrane pores in a way that degrades the membrane's performance. It is a major obstacle to the widespread use of this technology. Membrane fouling can cause severe flux decline and affect the quality of...

 in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultrafiltration
Ultrafiltration
Ultrafiltration is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane...

 or dialysis
Dialysis (biochemistry)
In biochemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing....

.

Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography
Ion exchange chromatography
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the...

. Either cation exchange chromatography is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point
Isoelectric point
The isoelectric point , sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge....

 (pI). For example, albumin
Albumin
Albumin refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat denaturation. They are commonly found in blood plasma, and are unique to other blood proteins in that they are not glycosylated...

 has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrin
Transferrin
Transferrins are iron-binding blood plasma glycoproteins that control the level of free iron in biological fluids. In humans, it is encoded by the TF gene.Transferrin is a glycoprotein that binds iron very tightly but reversibly...

, on the other hand, has a pI of 5.9, so it cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation.

Transferrin can instead be removed by size exclusion chromatography
Size exclusion chromatography
Size-exclusion chromatography is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight . It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers...

. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times.

A much quicker, single-step method of separation is Protein A/G
Protein A/G
Protein A/G is a recombinant fusion protein that combines IgG binding domains of both Protein A and Protein G. Protein A/G contains four Fc binding domains from Protein A and two from Protein G, yielding a final mass of 50,460 daltons...

 affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...

. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine
Cysteine
Cysteine is an α-amino acid with the chemical formula HO2CCHCH2SH. It is a non-essential amino acid, which means that it is biosynthesized in humans. Its codons are UGU and UGC. The side chain on cysteine is thiol, which is polar and thus cysteine is usually classified as a hydrophilic amino acid...

, which allows selective attachment to a carrier protein, such as KLH
Keyhole limpet hemocyanin
Keyhole limpet hemocyanin is a large, multisubunit, oxygen-carrying, metalloprotein found in the hemolymph of the giant keyhole limpet, Megathura crenulata, that lives off the coast of California from Monterey Bay to Isla Asuncion off Baja California.-Protein properties:Keyhole limpet hemocyanin...

 during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

To further select for antibodies, the antibodies can be precipitated out
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...

 using sodium sulfate
Sodium sulfate
Sodium sulfate is the sodium salt of sulfuric acid. When anhydrous, it is a white crystalline solid of formula Na2SO4 known as the mineral thenardite; the decahydrate Na2SO4·10H2O has been known as Glauber's salt or, historically, sal mirabilis since the 17th century. Another solid is the...

 or ammonium sulfate
Ammonium sulfate
Ammonium sulfate , 2SO4, is an inorganic salt with a number of commercial uses. The most common use is as a soil fertilizer. It contains 21% nitrogen as ammonium cations, and 24% sulfur as sulfate anions...

. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis
Dialysis
In medicine, dialysis is a process for removing waste and excess water from the blood, and is primarily used to provide an artificial replacement for lost kidney function in people with renal failure...

.

The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

 and capillary electrophoresis
Capillary electrophoresis
Capillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...

 can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.

Recombinant

The production of recombinant
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...

 monoclonal antibodies involves technologies, referred to as repertoire cloning
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...

or phage display
Phage display
Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P...

/yeast display
Yeast display
Yeast display is a technique used in the field of protein engineering. The yeast display technique was first published by the laboratory of Professor K. Dane Wittrup. The technology was sold to Abbott Laboratories in 2001....

. Recombinant antibody engineering involves the use of virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

es or yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...

 to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 sequences from which antibodies with desired specificities can be selected. The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik http://www2.mrc-lmb.cam.ac.uk/archive/g_pieczenik.html 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. Fermentation chambers have been used to produce these antibodies on a large scale.

Chimeric antibodies

Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two are sufficient to invoke an immune response occurred when murine monoclonal antibodies were injected into humans and resulted in their rapid removal from the blood, systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).

In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells, and the expression of this chimeric
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...

 DNA through cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...

 yielded partially-mouse, partially-human monoclonal antibody. For this product, the descriptive terms "chimeric" and "humanised" monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.

'Fully' human monoclonal antibodies

Ever since the discovery that monoclonal antibodies could be generated in-vitro, scientists have targeted the creation of 'fully' human antibodies to avoid some of the side effects of humanised and chimeric antibodies. Two successful approaches were identified — phage display-generated antibodies and mice genetically engineered
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...

 to produce more human-like antibodies.

One of the most successful commercial organisations behind therapeutic monoclonal antibodies was Cambridge Antibody Technology
Cambridge Antibody Technology
Cambridge Antibody Technology was a biotechnology company headquartered in Cambridge, United Kingdom...

 (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage antibodies. This was reported in a key Nature
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...

 publication.

Other significant publications include:

CAT developed their display technologies further into several, patented antibody discovery/functional genomics tools, which were named ProximolTM and ProAbTM. ProAb was announced in December 1997 and involved highthroughput screening of antibody libraries against diseased and non-diseased tissue, whilst Proximol used a free radical enzymatic reaction to label molecules in proximity to a given protein.

Genetically engineered mice, so called transgenic mice, can be modified to produce human antibodies, and this has been exploited by a number of commercial organisations:
  • Medarex
    Medarex
    Medarex is an American biotechnology company owned by Bristol Myers Squibb and based in Princeton, New Jersey. The company has manufacturing facilities in Bloomsbury and Annandale, New Jersey, and research facilities in Milpitas and Sunnyvale, California. Medarex specializes in therapeutic...

     — who market their UltiMab platform
  • Abgenix — who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen
    Amgen
    Amgen Inc. is an international biotechnology company headquartered in Thousand Oaks, California. Located in the Conejo Valley, Amgen is the world's largest independent biotech firm. The company employs approximately 17,000 staff members. Its products include Epogen, Aranesp, Enbrel, Kineret,...

    .
  • Regeneron
    Regeneron
    Regeneron is a biotechnology company headquartered in Tarrytown, in New York, USA. The company was founded in 1988. Originally focused on neurotrophic factors and their regenerative capabilities , it branched out into the study of both cytokine and tyrosine kinase receptors - leading to the three...

    's VelocImmune technology.


Monoclonal antibodies have been generated and approved to treat: cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

, cardiovascular disease
Cardiovascular disease
Heart disease or cardiovascular disease are the class of diseases that involve the heart or blood vessels . While the term technically refers to any disease that affects the cardiovascular system , it is usually used to refer to those related to atherosclerosis...

, inflammatory diseases, macular degeneration
Macular degeneration
Age-related macular degeneration is a medical condition which usually affects older adults and results in a loss of vision in the center of the visual field because of damage to the retina. It occurs in “dry” and “wet” forms. It is a major cause of blindness and visual impairment in older adults...

,
transplant rejection
Transplant rejection
Transplant rejection occurs when transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be lessened by determining the molecular similitude between donor and recipient and by use of immunosuppressant drugs after...

, multiple sclerosis
Multiple sclerosis
Multiple sclerosis is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms...

, and viral infection
Infection
An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...

 (see monoclonal antibody therapy
Monoclonal antibody therapy
Monoclonal antibody therapy is the use of monoclonal antibodies to specifically bind to target cells or proteins. This may then stimulate the patient's immune system to attack those cells...

).

In August 2006 the Pharmaceutical Research and Manufacturers of America
Pharmaceutical Research and Manufacturers of America
Pharmaceutical Research and Manufacturers of America , founded in 1958, is a trade group representing the pharmaceutical research and biopharmaceutical companies in the United States. PhRMA's stated mission is advocacy for public policies that encourage the discovery of new medicines for patients...

 reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...

.

Diagnostic tests

Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...

 test and immuno dot blot
Dot blot
right|thumb|350px|Schematic of the use of two [[Allele specific oligonucleotide|ASO]] probes on duplicate Dot-blot filters.A Dot blot is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods...

 tests detect the protein on a membrane. They are also very useful in immunohistochemistry
Immunohistochemistry
Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...

, which detect antigen in fixed tissue sections and immunofluorescence
Immunofluorescence
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows...

 test, which detect the substance in a frozen tissue section or in live cells...

Cancer treatment

One possible treatment for cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

 involves monoclonal antibodies that bind only to cancer cell-specific antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

s and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...

, radioisotope
Radionuclide
A radionuclide is an atom with an unstable nucleus, which is a nucleus characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or to an atomic electron. The radionuclide, in this process, undergoes radioactive decay, and emits gamma...

, cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...

 or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions
Fragment antigen binding
The fragment antigen-binding is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope — the antigen-binding site — at the amino terminal end of the monomer...

 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
Fragment crystallizable region
The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system...

.
The illustration below shows all these possibilities:

MAbs approved by the FDA include
  • Bevacizumab
    Bevacizumab
    Bevacizumab is a drug that blocks angiogenesis, the growth of new blood vessels. It is commonly used to treat various cancers, including colorectal, lung, breast, kidney, and glioblastomas....

  • Cetuximab
    Cetuximab
    Cetuximab is a chimeric monoclonal antibody, an epidermal growth factor receptor inhibitor, given by intravenous infusion for treatment of metastatic colorectal cancer and head and neck cancer.- Distribution :Cetuximab is manufactured and distributed in North America by ImClone and Bristol-Myers...

  • Panitumumab
    Panitumumab
    Panitumumab , formerly ABX-EGF, is a fully human monoclonal antibody specific to the epidermal growth factor receptor ....

  • Trastuzumab
    Trastuzumab
    Trastuzumab is a monoclonal antibody that interferes with the HER2/neu receptor.The HER receptors are proteins that are embedded in the cell membrane and communicate molecular signals from outside the cell to inside the cell, and turn genes on and off...


Autoimmune diseases

Monoclonal antibodies used for autoimmune disease
Autoimmune disease
Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body actually attacks its own cells. The immune system mistakes some part of the body as a pathogen and attacks it. This may be restricted to...

s include infliximab
Infliximab
Infliximab is a monoclonal antibody against tumour necrosis factor alpha . It is used to treat autoimmune diseases. Remicade is marketed by Janssen Biotech, Inc...

 and adalimumab
Adalimumab
Adalimumab is the third TNF inhibitor, after infliximab and etanercept, to be approved in the United States. Like infliximab and etanercept, adalimumab binds to TNFα, preventing it from activating TNF receptors; adalimumab was constructed from a fully human monoclonal antibody, while infliximab...

, which are effective in rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...

, Crohn's disease
Crohn's disease
Crohn's disease, also known as regional enteritis, is a type of inflammatory bowel disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms...

 and ulcerative Colitis
Ulcerative colitis
Ulcerative colitis is a form of inflammatory bowel disease . Ulcerative colitis is a form of colitis, a disease of the colon , that includes characteristic ulcers, or open sores. The main symptom of active disease is usually constant diarrhea mixed with blood, of gradual onset...

 by their ability to bind to and inhibit TNF-α. Basiliximab
Basiliximab
Basiliximab is a chimeric mouse-human monoclonal antibody to the α chain of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants...

 and daclizumab
Daclizumab
Daclizumab is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants....

 inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants. Omalizumab
Omalizumab
Omalizumab is a humanized antibody drug approved for patients with moderate-to-severe or severe allergic asthma, which is caused by hypersensitivity reactions to certain harmless environmental substances...

 inhibits human immunoglobulin E
Immunoglobulin E
Immunoglobulin E is a class of antibody that has been found only in mammals. IgE is a monomeric antibody with 4 Ig-like domains...

 (IgE) and is useful in moderate-to-severe allergic asthma
Asthma
Asthma is the common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include wheezing, coughing, chest tightness, and shortness of breath...

.

Examples

Below are examples of clinically important monoclonal antibodies.
Main category Type Application Mechanism/Target Mode
Anti-
inflammatory
infliximab
Infliximab
Infliximab is a monoclonal antibody against tumour necrosis factor alpha . It is used to treat autoimmune diseases. Remicade is marketed by Janssen Biotech, Inc...

  • rheumatoid arthritis
    Rheumatoid arthritis
    Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...

  • Crohn's disease
    Crohn's disease
    Crohn's disease, also known as regional enteritis, is a type of inflammatory bowel disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms...

  • Ulcerative Colitis
    Ulcerative colitis
    Ulcerative colitis is a form of inflammatory bowel disease . Ulcerative colitis is a form of colitis, a disease of the colon , that includes characteristic ulcers, or open sores. The main symptom of active disease is usually constant diarrhea mixed with blood, of gradual onset...

inhibits TNF-α  chimeric
adalimumab
Adalimumab
Adalimumab is the third TNF inhibitor, after infliximab and etanercept, to be approved in the United States. Like infliximab and etanercept, adalimumab binds to TNFα, preventing it from activating TNF receptors; adalimumab was constructed from a fully human monoclonal antibody, while infliximab...

  • rheumatoid arthritis
    Rheumatoid arthritis
    Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...

  • Crohn's disease
    Crohn's disease
    Crohn's disease, also known as regional enteritis, is a type of inflammatory bowel disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms...

  • Ulcerative Colitis
    Ulcerative colitis
    Ulcerative colitis is a form of inflammatory bowel disease . Ulcerative colitis is a form of colitis, a disease of the colon , that includes characteristic ulcers, or open sores. The main symptom of active disease is usually constant diarrhea mixed with blood, of gradual onset...

  • inhibits TNF-α  human
    basiliximab
    Basiliximab
    Basiliximab is a chimeric mouse-human monoclonal antibody to the α chain of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants...

  • Acute rejection of kidney transplants
  • inhibits IL-2 on activated T cells  chimeric
    daclizumab
    Daclizumab
    Daclizumab is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants....

  • Acute rejection of kidney transplants
  • inhibits IL-2 on activated T cells  humanized
    omalizumab
    Omalizumab
    Omalizumab is a humanized antibody drug approved for patients with moderate-to-severe or severe allergic asthma, which is caused by hypersensitivity reactions to certain harmless environmental substances...

  • moderate-to-severe allergic asthma
    Asthma
    Asthma is the common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include wheezing, coughing, chest tightness, and shortness of breath...

  • inhibits human immunoglobulin E
    Immunoglobulin E
    Immunoglobulin E is a class of antibody that has been found only in mammals. IgE is a monomeric antibody with 4 Ig-like domains...

     (IgE)
    humanized
    Anti-cancer gemtuzumab
  • relapsed acute myeloid leukaemia
  • targets myeloid cell surface antigen CD33
    CD33
    CD33 or Siglec-3 is a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells.It binds sialic acids, therefore is a member of the SIGLEC family of lectins....

     on leukemia
    Leukemia
    Leukemia or leukaemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called "blasts". Leukemia is a broad term covering a spectrum of diseases...

     cells
    humanized
    alemtuzumab
    Alemtuzumab
    Alemtuzumab is a monoclonal antibody used in the treatment of chronic lymphocytic leukemia , cutaneous T-cell lymphoma and T-cell lymphoma...

  • B cell leukemia
  • targets an antigen CD52
    CD52
    CD52 is a protein present on the surface of mature lymphocytes, but not on the stem cells from which these lymphocytes were derived.It also is found in monocytes and dendritic cells....

     on T- and B-lymphocytes 
    humanized
    rituximab
    Rituximab
    Rituximab, sold under the trade names Rituxan and MabThera, is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells...

  • non-Hodgkin's lymphoma
  • targets phosphoprotein CD20
    CD20
    B-lymphocyte antigen CD20 or CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells beginning at the pro-B phase and progressively increasing in concentration until maturity....

     on B lymphocytes 
    chimeric
    trastuzumab
    Trastuzumab
    Trastuzumab is a monoclonal antibody that interferes with the HER2/neu receptor.The HER receptors are proteins that are embedded in the cell membrane and communicate molecular signals from outside the cell to inside the cell, and turn genes on and off...

  • breast cancer with HER2/neu overexpression
  • targets the HER2/neu (erbB2) receptor humanized
    nimotuzumab
    Nimotuzumab
    Nimotuzumab is a humanized monoclonal antibody used to treat squamous cell carcinomas of the head and neck, recurrent or refractory high grade malignant glioma, anaplastic astrocytomas, glioblastomas and diffuse...

  • Approved in squamous cell carcinomas, Glioma
  • Clinical trials for other indications underway
  • EGFR
    Epidermal growth factor receptor
    The epidermal growth factor receptor is the cell-surface receptor for members of the epidermal growth factor family of extracellular protein ligands...

     inhibitor
    Humanized
    cetuximab
    Cetuximab
    Cetuximab is a chimeric monoclonal antibody, an epidermal growth factor receptor inhibitor, given by intravenous infusion for treatment of metastatic colorectal cancer and head and neck cancer.- Distribution :Cetuximab is manufactured and distributed in North America by ImClone and Bristol-Myers...

  • Approved in squamous cell carcinomas, colorectal carcinoma
  • EGFR
    Epidermal growth factor receptor
    The epidermal growth factor receptor is the cell-surface receptor for members of the epidermal growth factor family of extracellular protein ligands...

     inhibitor
    Chimeric
    bevacizumab
    Bevacizumab
    Bevacizumab is a drug that blocks angiogenesis, the growth of new blood vessels. It is commonly used to treat various cancers, including colorectal, lung, breast, kidney, and glioblastomas....

  • Anti-angiogenic cancer therapy
  • inhibits VEGF
    Vascular endothelial growth factor
    Vascular endothelial growth factor is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate....

     
    humanized
    Other palivizumab
    Palivizumab
    Palivizumab is a monoclonal antibody produced by recombinant DNA technology. It is used in the prevention of respiratory syncytial virus infections...

  • RSV infections in children
  • inhibits an RSV fusion (F) protein humanized
    abciximab
  • Prevent coagulation
    Coagulation
    Coagulation is a complex process by which blood forms clots. It is an important part of hemostasis, the cessation of blood loss from a damaged vessel, wherein a damaged blood vessel wall is covered by a platelet and fibrin-containing clot to stop bleeding and begin repair of the damaged vessel...

     in coronary angioplasty
  • inhibits the receptor GpIIb/IIIa on platelets  chimeric

    See also

    • Antibody mimetic
      Antibody mimetic
      Antibody mimetics are organic compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa...

    • Immunotoxin
      Immunotoxin
      An immunotoxin is a human-made protein that consists of a targeting portion linked to a toxin. When the protein binds to that cell, it is taken in through endocytosis, and the toxin kills the cell...

      s, which sometimes use monoclonal antibodies as the targeting mechanism
    • List of monoclonal antibodies
    • Monoclonal antibody therapy
      Monoclonal antibody therapy
      Monoclonal antibody therapy is the use of monoclonal antibodies to specifically bind to target cells or proteins. This may then stimulate the patient's immune system to attack those cells...

    • Nomenclature of monoclonal antibodies
      Nomenclature of monoclonal antibodies
      The nomenclature of monoclonal antibodies is a naming scheme for assigning generic, or nonproprietary, names to monoclonal antibodies. An antibody is a protein that is produced in B cells and used by the immune system of humans and other vertebrate animals to identify a specific foreign object like...

    • Polyclonal antibodies
    • Queen Mab
      Queen Mab
      Queen Mab is a fairy referred to in Shakespeare's play Romeo and Juliet. She also appears in other 17th century literature, and in various guises in later poetry, drama and cinema...

      Small mythological figure symbolizing hope (popular culture, used as biotech pun).

    External links

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