Polyclonal antibodies

Polyclonal antibodies (or antisera) are antibodies that are derived from different B cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope.

Production

These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is polyclonal purified from the mammal’s serum.

By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

1. surfactants which promote concentration of protein antigens molecules over a large surface area, and
2. immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Animal selection

Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this purpose. Animal selection should be based upon:

1. the amount of antibody needed,
2. the relationship between the donor of the antigen and the recipient antibody producer (generally the more distant the phylogenetic relationship, the greater the potential for high titer antibody response) and
3. the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the antibodies to be made. Immunization and phlebotomies are stress associated and, at least when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such animals can dramatically reduce morbidity and mortality due to pathogenic organisms, especially Pasteurella multocida in rabbits.

Goats or horses are generally used when large quantities of antisera are required. Many investigators favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure. One week’s eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in immunoassays. Chicken IgY does not fix mammalian complement component C1 and it does not perform as a precipitating antibody using standard solutions.

Although mice are used most frequently for monoclonal antibody production, their small size usually prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.

When using rabbits, young adult animals (2.5–3.0 kg or 5.5-6.5lbs) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals.

Antigen preparation

The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10>

Keyhole limpet hemocyanin (KLH) and bovine serum albumin are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have been successfully used for delivery of small peptides and this technique is more efficient than delivery with oily emulsion adjuvants.

Antigen quantity

Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges:

1. rabbit, 50–1000 µg;
2. mouse, 10–200 µg;
3. guinea pig, 50–500 µg; and
4. goat, 250–5000 µg. Optimal “priming” doses are reported to be at the low end of each range.

The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.

Peptide Antibodies

When a peptide is being used to generate the antibody, it is extremely important to design the antigens properly. There are several resources that can aid in the design as well as companies that offer this service. Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that will enable you to score individual peptide sequences based upon a relation epitope mapping database of previous immunogens used to generate antibodies. Finally, as a general rule peptides should follow some basic criteria.

When examining peptides for synthesis and immunization, it is recommended that certain residues and sequences be avoided due to potential synthesis problems. This includes some of the more common characteristics:

• Extremely long repeats of the same amino acid (e.g. RRRR) • Serine (S), Threonine (T), Alanine (A), and Valine (V) doublets • Ending or starting a sequence with a proline (P) • Glutamine (Q) or Asparagine (N) at the n-terminus • Peptides over weighted with hydrophobic residues (e.g. V,A,L,I, etc…)

Reactivity

Investigators should also consider the status of nativity of protein antigens when used as immunogens and reaction with antibodies produced. Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins. If elicited antibodies are to be used on membrane blots (proteins subjected to denaturing conditions) then antibodies should be made against denatured proteins. On the other hand, if antibodies are to be used to react with a native protein or block a protein active site, then antibodies should be made against the native protein. Adjuvants can often alter the nativity of the protein. Generally, absorbed protein antigens in a preformed oil-in-water emulsion adjuvant, retain greater native protein structure than those in water-in-oil emulsions.

Asepticity

Antigens should always be prepared using techniques that ensure that they are free of microbial contamination. Most protein antigen preparations can be sterilized by passage through a 0.22u filter. Septic abscesses often occur at inoculation sites of animals when contaminated preparations are used. This can result in failure of immunization against the targeted antigen.

Adjuvants

There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types varies depending upon whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titer, high avidity antibodies. No single adjuvant is ideal for all purposes and all have advantages and disadvantages. Adjuvant use generally is accompanied by undesirable side effects of varying severity and duration. Research on new adjuvants focuses on substances which have minimal toxicity while retaining maximum immunostimulation. Investigators should always be aware of potential pain and distress associated with adjuvant use in laboratory animals.

The most frequently used adjuvants for antibody production are Freund’s, Alum, the Ribi Adjuvant System and Titermax.

Freund’s adjuvants

There are two basic types of Freund's adjuvants: Freund’s Complete Adjuvant (FCA) and Freund’s Incomplete Adjuvant (FIA). FCA is a water-in-oil emulsion that localizes antigen for release periods up to 6 months. It is formulated with mineral oil, the surfactant mannide monoleate and heat killed Mycobacterium tuberculosis, Mycobacterium butyricum or their extracts (for aggregation of macrophages at the inoculation site). This potent adjuvant stimulates both cell mediated and humoral immunity with preferential induction of antibody against epitopes of denatured proteins. Although FCA has historically been the most widely used adjuvant, it is one of the more toxic agents due to non-metabolizable mineral oil and it induces granulomatous reactions. Its use is limited to laboratory animals and it should be used only with weak antigens. It should not be used more than once in a single animal since multiple FCA inoculations can cause severe systemic reactions and decreased immune responses. Freund’s Incomplete Adjuvant has the same formulation as FCA but does not contain mycobacterium or its components. FIA usually is limited to booster doses of antigen since it normally much less effective than FCA for primary antibody induction. Freund’s adjuvants are normally mixed with equal parts of antigen preparations to form stable emulsions.

Ribi Adjuvant System

Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species oriented formulations of the adjuvant system are available. These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing and presentation. This adjuvant system is much less toxic and less potent than FCA but generally induces satisfactory amounts of high avidity antibodies against protein antigens.

Titermax

Titermax represents a newer generation of adjuvants that are less toxic and contain no biologically derived materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other surfactant materials and have potent adjuvant properties which favor chemotaxis, complement activation and antibody production. Titermax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major histocompatibility molecules on macrophages. Titermax presents antigen in a highly concentrated form to the immune system, which often results in antibody titers comparable to or higher than FCA.

Specol: Specol is a water in oil adjuvant made of purified mineral oil. It has been reported to induce immune response comparable to Freund's adjuvant in rabbit and other research animal while producing fewer histological lesions.

Humanized antibody

Humanized antibodies or chimeric antibodies are a type of monoclonal antibody that have been synthesized using recombinant DNA technology to circumvent the clinical problem of immune response to foreign antigens. The standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences, and the human immune system recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects.

Humanized antibodies are produced by merging the DNA that encodes the binding portion of a monoclonal mouse antibody with human antibody-producing DNA. One then uses mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies that are not as immunogenic as the murine variety.

Alternatives

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

Monoclonal antibody therapy

Each antibody binds only one specific antigen.

Monoclonal antibody therapy is the use of monoclonal antibodies (or mAb) to specifically target cells. The main objective is stimulating the patient's immune system to attack the malignant tumor cells and the prevention of tumor growth by blocking specific cell receptors. Variations exist within this treatment, e.g. radioimmunotherapy, where a radioactive dose localizes on target cell line, delivering lethal chemical doses to the target.

Structure and function of human and therapeutic antibodies

Immunoglobulin G (IgG) antibodies are large heterodimeric molecules, approximately 150 kDa and are composed of two different kinds of polypeptide chain, called the heavy (~50kDa) and the light chain (~25kDa). There are two types of light chains, kappa (κ) and lambda (λ). By cleavage with enzyme papain, the Fab (fragment-antigen binding) part can be separated from the Fc (fragment crystalline) part of the molecule (see image). The Fab fragments contain the variable domains, which consist of three hypervariable amino acid domains responsible for the antibody specificity embedded into constant regions. There are four known IgG subclasses all of which are involved in Antibody-dependent cellular cytotoxicity.

The immune system responds to the environmental factors it encounters on the basis of discrimination between self and non-self. Tumor cells are not specifically targeted by one's immune system since tumor cells are the patient's own cells. Tumor cells, however are highly abnormal, and many display unusual antigens that are either inappropriate for the cell type, its environment, or are only normally present during the organisms' development (e.g. fetal antigens).

Other tumor cells display cell surface receptors that are rare or absent on the surfaces of healthy cells, and which are responsible for activating cellular signal transduction pathways that cause the unregulated growth and division of the tumor cell. Examples include ErbB2, a constitutively active cell surface receptor that is produced at abnormally high levels on the surface of approximately 30% of breast cancer tumor cells. Such breast cancer is known a HER2 positive breast cancer.

Antibodies are a key component of the adaptive immune response, playing a central role in both in the recognition of foreign antigens and the stimulation of an immune response to them. The advent of monoclonal antibody technology has made it possible to raise antibodies against specific antigens presented on the surfaces of tumors.

Origins of monoclonal antibody therapy

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.

Immunotherapy developed as a technique with the discovery of the structure of antibodies and the development of hybridoma technology, which provided the first reliable source of monoclonal antibodies. These advances allowed for the specific targeting of tumors both in vitro and in vivo. Initial research on malignant neoplasms found MAb therapy of limited and generally short-lived success with malignancies of the blood. Furthermore treatment had to be specifically tailored to each individual patient, thus proving to be impracticable for the routine clinical setting.

Throughout the progression of monoclonal drug development there have been four major antibody types developed: murine, chimeric, humanised and human. Initial therapeutic antibodies were simple murine analogues, which contributed to the early lack of success. It has since been shown that these antibodies have: a short half-life in vivo (due to immune complex formation), limited penetration into tumour sites, and that they inadequately recruit host effector functions. To overcome these difficulties the technical issues initially experienced had to be surpassed. Chimeric and humanized antibodies have generally replaced murine antibodies in modern therapeutic antibody applications. Hybridoma technology has been replaced by recombinant DNA technology, transgenic mice and phage display. Understanding of proteomics has proven essential in identifying novel tumour targets.

Murine monoclonal antibodies

Initially, murine antibodies were obtained by hybridoma technology, for which Kohler and Milstein received a Nobel prize. However the dissimilarity between murine and human immune systems led to the clinical failure of these antibodies, except in some specific circumstances. Major problems associated with murine antibodies included reduced stimulation of cytotoxicity and the formation complexes after repeated administration, which resulted in mild allergic reactions and sometimes anaphylactic shock.

Chimeric and humanized monoclonal antibodies

To reduce murine antibody immunogenicity, murine molecules were engineered to remove immunogenic content and to increase their immunologic efficiency. This was initially achieved by the production of chimeric and humanized antibodies. Chimeric antibodies are composed of murine variable regions fused onto human constant regions. Human gene sequences, taken from the kappa light chain and the IgG1 heavy chain, results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increases serum half-life.

Humanised antibodies are produced by grafting murine hypervariable amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin. However it has been shown in several studies that humanised antibodies bind antigen much more weakly than the parent murine monoclonal antibody, with reported decreases in affinity of up to several hundredfold. Increases in antibody-antigen binding strength have been achieved by introducing mutations into the complementarity determining regions (CDR), using techniques such as chain-shuffling, randomization of complementarity determining regions and generation of antibody libraries with mutations within the variable regions by error-prone PCR, E-coli mutator strains, and site-specific mutagenesis.

Human monoclonal antibodies

Human monoclonal antibodies are produced using transgenic mice or phage display libraries. Human monoclonal antibodies are produced by transferring human immunoglobulin genes into the murine genome, after which the transgenic mouse is vaccinated against the desired antigen, leading to the production of monoclonal antibodies. Phage display libraries allow the transformation of murine antibodies in vitro into fully human antibodies.

FDA approved therapeutic antibodies

The first FDA-approved therapeutic monoclonal antibody was a murine IgG2a CD3 specific transplant rejection drug, Muromonab (OKT-3), in 1986. This drug found use in solid organ transplant recipients who became steroid resistant. Currently, twenty-one FDA-approved therapies exist, and hundreds of therapies are undergoing clinical trials. Most are concerned with immunological and oncological targets.

FDA approved monoclonal antibodies

Antibody Brand name Approval date Type Target Approved treatment(s) Abciximab
ReoPro 1994 chimeric inhibition of glycoprotein IIb/IIIa Cardiovascular disease
Adalimumab
Humira 2002 human inhibition of TNF-a signalling Inflammatory diseases (mostly auto-immune disorders) Alemtuzumab
Campath 2001 humanized CD52
Chronic lymphocytic leukemia
Basiliximab
Simulect 1998 chimeric IL-2 receptor a Transplant rejection
Bevacizumab
Avastin 2004 humanized vascular endothelial growth factor
Colorectal cancer
Cetuximab
Erbitux 2004 chimeric epidermal growth factor receptor
Colorectal cancer Daclizumab
Zenapax 1997 humanized IL-2 receptor a Transplant rejection Eculizumab
Soliris 2007 humanized complement system protein C5 Inflammatory diseases including paroxysmal nocturnal hemoglobinuria Efalizumab
Raptiva 2002 humanized CD11a
Inflammatory diseases (psoriasis) Ibritumomab tiuxetan
Zevalin 2002 murine CD20
Non-Hodgkin lymphoma (with yttrium-90 or indium-111) Infliximab
Remicade 1998 chimeric inhibition of TNF-a signalling Inflammatory diseases (mostly auto-immune disorders) Muromonab-CD3
Orthoclone OKT3 1986 murine T cell CD3 Receptor Transplant rejection Natalizumab
Tysabri 2006 humanized T cell VLA4 receptor Inflammatory diseases (mainly autoimmune-related multiple sclerosis therapy) Omalizumab
Xolair 2004 humanized immunoglobulin E (IgE) Inflammatory diseases (mainly allergy-related asthma therapy) Palivizumab
Synagis 1998 humanized an epitope of the F protein of RSV Viral infection (especially Respiratory Syncytial Virus (RSV) Panitumumab
Vectibix 2006 human epidermal growth factor receptor Colorectal cancer Ranibizumab
Lucentis 2006 humanized vascular endothelial growth factor
Macular degeneration
Gemtuzumab ozogamicin
Mylotarg 2000 humanized CD33
Acute myelogenous leukemia (with calicheamicin) Rituximab
Rituxan, Mabthera 1997 chimeric CD20
Non-Hodgkin lymphoma
Tositumomab
Bexxar 2003 murine CD20
Non-Hodgkin lymphoma Trastuzumab
Herceptin 1998 humanized ErbB2
Breast cancer
Radioimmunotherapy

Radioimmunotherapy involves the use of radioactively conjugated murine antibodies against cellular antigens. Most research currently involved their application to lymphomas, as these are highly radio-sensitive malignancies. To limit radiation exposure, murine antibodies were especially chosen, as their high immunogenicity promotes rapid clearance from the body. Tositumomab is an exemplar used for non-Hodgkins lymphoma.

Antibody-directed enzyme prodrug therapy (ADEPT)

ADEPT involves the application of cancer associated monoclonal antibodies which are linked to a drug-activating enzyme. Subsequent systemic administration of a non-toxic agent results in its conversion to a toxic drug, and resulting in a cytotoxic effect which can be targeted at malignant cells. The clinical success of ADEPT treatments has been limited to date. However it holds great promise, and recent reports suggest that it will have a role in future oncological treatment.

Drug and gene therapy: Immuno-liposomes

Immunoliposomes are antibody-conjugated liposomes. Liposomes can carry drugs or therapeutic nucleotides and when conjugated with monoclonal antibodies, may be directed against malignant cells. Although this technique is still in its infancy, significant advances have been made. Immunoliposomes have been successfully used in vivo to achieve targeted delivery of tumour-suppressing genes into tumours, using an antibody fragment against the human transferrin receptor. Tissue-specific gene delivery using immunoliposomes has also been achieved in brain, and breast cancer tissue.

Therapeutic Monoclonal Antibody Market Future

Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially. The current “big 5” therapeutic antibodies on the market: Avastin, Herceptin (both oncology), Humira, Remicade (both Autoimmune and Infectious Disease ‘AIID’) and Rituxan (oncology and AIID) accounted for 80% of revenues in 2006.

In the immediate future, it is likely that Genentech/Roche will retain their control over the market (due to ownership of 3 of the “big 5” products), oncology and AIID will remain the mAb segment therapeutic focus (because these are the disease areas addressed by the big 5) and the three most commercially important ‘targets’ for the mAb class will be VEGF (Avastin), TNF-alpha (Remicade and Humira) and CD20 (Rituxan). Experts forecast that the therapeutic antibody market will continue to be dominated by Oncology and AIID segments (82-84 percent) from 2004 to 2011. Furthermore, experts note a potential for change in the balance between Oncology and AIID in the coming years. While Oncology therapeutics dominated the market in 2004, AIID is expected to dominate by 2011.

Monoclonal antibodies

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. 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").

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.

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 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 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 is generally accredited to Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; who 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 humanize monoclonal antibodies, 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.

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. 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 prepares slides of monoclonal antibodies for researchers. The cells shown are labeling human breast cancer.

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. 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), an enzyme necessary for the salvage synthesis of nucleic acids.

This enzyme enables cells to synthesize purines by the salvage pathway, here 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 already existing biochemical pathway, the de novo pathway that they can use to synthesize purines. However, when cells are exposed to Aminopterin (a folic acid analogue, which inhibits Dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and are now fully dependent on the salvage pathway for survival.

The selective culture medium is called HAT medium because it contains Hypoxanthine, Aminopterin, and Thymidine. This medium is selective for fused (hybridoma) cells, because unfused myeloma cells cannot grow because they lack HGPRT. The unfused normal spleen cells cannot grow indefinitely because of their limited life span. Therefore, only hybridoma cells are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner gives it immortality (as 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.

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 tested for their ability to bind to the antigen (for example with a test such as ELISA 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.

The hybridomas are grown indefinitely in asuitable cell culture media, or they are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid. 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. 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.

Recombinant

The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast 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 sequences from which antibodies with desired specificities can be selected. 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.

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.

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.

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 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. (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, multiple sclerosis, 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.

Examples

Monoclonal antibodies

Type	Application	Mechanism	Mode
infliximab	rheumatoid arthritis Crohn's disease	inhibits TNF-α	chimeric
basiliximab	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	chimeric
abciximab	Prevent coagulation in coronary angioplasty	inhibits the receptor GpIIb/IIIa on platelets	chimeric
daclizumab	Acute rejection of kidney transplants	inhibits IL-2 on activated T cells	humanized
gemtuzumab	relapsed acute myeloid leukaemia	targets an antigen on leukemia cells	humanized
alemtuzumab	B cell leukemia	targets an antigen CD52 on T- and B-lymphocytes	humanized
rituximab	non-Hodgkin's lymphoma	targets phosphoprotein CD20 on B lymphocytes	chimeric
palivizumab	RSV infections in children	inhibits an RSV protein	humanized
trastuzumab	anti-cancer therapy for a specific kind of breast cancer	targets the HER2/neu (erbB2) receptor	humanized
etanercept	rheumatoid arthritis	contains TNF receptor	fusion protein
adalimumab	rheumatoid arthritis	inhibits TNF-α	humanized

Intravenous immunoglobulin

Intravenous immunoglobulin (IVIG) is a blood product administered intravenously. It contains the pooled IgG immunoglobulins (antibodies) extracted from the plasma of over one thousand blood donors. IVIG's effects last between 2 weeks and 3 months. It is mainly used as treatment in three major categories:

• Immune deficiencies - Immune deficiencies such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immune deficiencies), and acquired compromised immunity conditions ([secondary immune deficiencies), featuring low antibody levels.
• Inflammatory and autoimmune diseases.
• Acute infections.

Mechanism of action

IVIG is given as a plasma protein replacement therapy (IgG) for immune deficient patients which have decreased or abolished antibody production capabilities. In these immune deficient patients, IVIG is administered to maintain adequate antibodies levels to prevent infections and confers a passive immunity. Treatment is given every 3-4 weeks. In the case of patients with autoimmune disease, IVIG is administered at a high dose (generally 1-2 grams IVIG per kg body weight) to attempt to decrease the severity of the autoimmune disease.

The precise mechanism by which IVIG suppresses harmful inflammation has not been definitively established but is believed to involve the inhibitory Fc receptor. The actual primary target(s) of IVIG in autoimmune disease are still unclear, however. IVIG may work via a multi-step model where the injected IVIG first forms a type of immune complex in the patient. Once these immune complexes are formed, they interact with activating Fc receptors on dendritic cellswhich then mediate anti-inflammatory effects helping to reduce the severity of the autoimmune disease or inflammatory state.

Additionally, the donor antibody may bind directly with the abnormal host antibody, stimulating its removal. Alternatively, the massive quantity of antibody may stimulate the host's complement system, leading to enhanced removal of all antibodies, including the harmful ones. IVIG also blocks the antibody receptors on immune cells (macrophages), leading to decreased damage by these cells, or regulation of macrophage phagocytosis.

IVIG may also regulate the immune response by reacting with a number of membrane receptors on T cells, B cells, and monocytes that are pertinent to autoreactivity and induction of tolerance to self.

A recent report stated that IVIG application to activated T cells leads to their decreased ability to engage microglia. As a result of IVIG treatment of T cells, the findings showed reduced levels of tumor necrosis factor-alpha and interleukin-10 in T cell-microglia co-culture. The results add to the understanding of how IVIG may affect inflammation of the central nervous system in autoimmune inflammatory diseases.

IVIG is useful in some acute infection cases such as in Kawasaki's Disease and pediatric HIV infection.

IVIG notes

• IVIG is an infusion of IgG antibodies only. Therefore, peripheral tissues that are defended mainly by IgA antibodies, such as the eyes, lungs, gut and urinary tract are not fully protected by the IVIG treatment.
• XLA patients are immune to the most virulent adverse effect, anaphylactic shock, as they do not have the antibodies to react against the treatment. Anaphylactic shock has a higher chance to occur in IgA deficient patients which do have other antibody types.
• In case of recurring side effects, it is recommended to slow the pace of the IVIG administration and to reduce the dosage. It is also advisable to change IVIG brand, as some people react against to a specific brand.
• If the patient is diabetic, he should take into consideration the medium in which the antibodies are solubilized in the IVIG treatment, as some brand solubilize antibodies with high concentrated sugars (such as sucrose and maltose).
• FDA guidelines for IVIG state the product should be:
  • Prepared out of at least 1,000 different human donors.
  • All four IgG subgroups (1-4) should be present.
  • The IgG should maintain biological activity and lifetime of at least 21 days.
  • Does not contain samples which are HIV, hepatitis B, hepatitis C positive.
  • Screened and treated in a manner that destroys viruses.
• IVIG is also considered a modulator of the immune system and was shown to be beneficial in treating numerous autoimmune diseases such as relapsing and remitting multiple sclerosis (MS), myasthenia gravis, pemphigus, polymyositis (PM), dermatomyositis (DM), Wegener's granulomatosis (WG), Churg-Strauss syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) and more.
• IVIG can be given to pregnant women.
• IVIG is also used as a treatment for unexplained recurring miscarriages. The effectiveness of the therapy is controversial.
• IVIG cost is climbing and well over $50/g. ($10,000 for a 220lbs person at 2g/kg)

Uses of IVIG

Dosage of IVIG is dependent on indication.

For primary immune dysfunction 100 to 400 mg/kg of body weight every 3 to 4 weeks is implemented.

For neurological and autoimmune diseases 2 grams per kilogram of body weight is implemented for three to six months over a five day course once a month. Then maintenance therapy of 100 to 400 mg/kg of body weight every 3 to 4 weeks follows.

FDA-approved indications

• Allogeneic bone marrow transplant
  
• Chronic lymphocytic leukemia
• Idiopathic thrombocytopenic purpura
• Pediatric HIV
• Primary immunodeficiencies
• Kawasaki disease
  
• Alzheimer's Disease
• Kidney transplant with a high antibody recipient or with an ABO incompatible donor
• Common Variable Immune Deficiency

In 2004 the FDA approved the Cedars-Sinai IVIG Protocol which has been 90-95% successful in removing antibodies from the blood of kidney transplant recipients so that they can accept a living

Off-label Uses

• Chronic fatigue syndrome
• Chronic inflammatory demyelinating polyneuropathy (CIDP)
• Clostridium difficile colitis
• Dermatomyositis and polymyositis
• Graves' ophthalmopathy
  
• Guillain-Barré syndrome
• Kawasaki disease
• Muscular Dystrophy
• Inclusion body myositis
• Lambert-Eaton syndrome
• Lupus erythematosus
• Multifocal motor neuropathy
• Multiple sclerosis
• Myasthenia gravis
• Neonatal alloimmune thrombocytopenia
• Parvovirus B19
• Pemphigus
  
• Post-transfusion purpura
• Renal transplant rejection
• Spontaneous Abortion/Miscarriage
• Stiff person syndrome
• Severe sepsis and septic shock in critically ill adults
• Toxic epidermal necrolysis
• In chronic lymphocytic leukemia and multiple myeloma, as well as various rare deficiencies of immunoglobulin synthesis (e.g. X-linked agammaglobulinemia, hypogammaglobulinemia), IVIG is administered to maintain adequate immunoglobulin levels to prevent infections.

Complications and side effects

Complications of IVIG therapy include:

anaphylactic shock, especially in IgA deficient patients, who by definition can still produce IgG antibodies. IgA deficient patients are more likely to produce IgG against the IVIG administration than normal patients.

• headache
• dermatitis - usually peeling of the skin of the palms and soles
• infection (such as HIV or viral hepatitis) by contaminated blood product; there is also an as yet unknown risk of contracting variant CJD (vCJD).
• pulmonary edema from fluid overload, due to the high colloid oncotic pressure of IVIG
• allergic/anaphylactic reactions
• damage such as hepatitis caused directly by antibodies contained in the pooled IVIG
• acute renal failure
  
• venous thrombosis
• aseptic meningitis

Interferon

Interferons (IFNs) are natural proteins produced by the cells of the immune system of most vertebrates in response to challenges by foreign agents such as viruses, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferons are produced by a wide variety of cells in response to the presence of double-stranded RNA, a key indicator of viral infection. Interferons assist the immune response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection.

Types of interferon

There are three major classes of interferons that have been described for humans according to the type of receptor through which they signal:

• Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β and IFN-ω.

• Interferon type II: Binds to IFNGR. In humans this is IFN-γ.

• Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12)

Signaling pathway

While there is evidence to suggest other signaling mechanisms exist, the JAK-STAT signaling pathway is the best-characterised and commonly accepted IFN signaling pathway.

Natural function and synthesis

Interferons in general have several effects in common. They are antiviral and possess antioncogenic properties, macrophage and natural killer lymphocyte activation, and enhancement of major histocompatibility complex glycoprotein classes I and II, and thus presentation of foreign (microbial) peptides to T cells. In a majority of cases, the production of interferons is induced in response to microbes such as viruses and bacteria and their products (viral glycoproteins, viral RNA, bacterial endotoxin, bacterial flagella, CpG sites), as well as mitogens and other cytokines, for example interleukin 1, interleukin 2, interleukin-12, tumor necrosis factor and colony-stimulating factor, that are synthesised in the response to the appearance of various antigens in the body. Their metabolism and excretion take place mainly in the liver and kidneys. They rarely pass the placenta but they can cross the blood-brain barrier.

The therapeutically used forms are denoted by Greek letters indicating their origin: leukocytes, fibroblasts, and lymphocytes for interferon-alpha, -beta and -gamma, respectively.

Viral induction of interferons

All classes of interferon are very important in fighting RNA virus infections. However, their presence also accounts for some of the host symptoms, such as sore muscles and fever. They are secreted when abnormally large amounts of dsRNA are found in a cell. dsRNA is normally present in very low quantities. The dsRNA acts like a trigger for the production of interferon (via Toll Like Receptor 3 (TLR 3), a pattern recognition receptor of the innate immune system which leads to activation of the transcription factor IRF3 and late phase NF kappa Beta). The gene that codes for this cytokine is switched on in an infected cell, and the interferon synthesized and secreted to surrounding cells.

As the original cell dies from the cytolytic RNA virus, these thousands of viruses will infect nearby cells. However, these cells have received interferon, which essentially warns these other cells of the virus. They then start producing large amounts of a protein known as protein kinase R (or PKR). If a virus infects a cell that has been “pre-warned” by interferon, the PKR is indirectly activated by the dsRNA (actually by 2'-5' oligoadenylate produced by the 2'-5' oligoadenylate-synthetase which is produced due to TLR3 activation), and begins transferring phosphate groups (phosphorylating) to a protein known as eIF-2, a eukaryotic translation initiation factor. After phosphorylation, eIF2 has a reduced ability to initiate translation, the production of proteins coded by cellular mRNA. This prevents viral replication and inhibits normal cell ribosome function, killing both the virus and the host cell if the response is active for a sufficient amount of time. All RNA within the cell is also degraded, preventing the mRNA from being translated by eIF2 if some of the eIF2 failed to be phosphorylated.

Furthermore, interferon leads to upregulation of MHC I and therefore to increased presentation of viral peptides to cytotoxic CD8 T cells, as well as to a change in the proteasome (exchange of some beta subunits by b1i, b2i, b5i - then known as the immunoproteasome) which leads to increased production of MHC I compatible peptides.

Interferon can cause increased p53 activity in virus infected cells. It acts as an inducer and causes increased production of the p53 gene product. This promotes apoptosis, limiting the ability of the virus to spread. Increased levels of transcription are observed even in cells which are not infected, but only infected cells show increased apoptosis. This increased transcription may serve to prepare susceptible cells so they can respond quickly in the case of infection. When p53 is induced by viral presence, it behaves differently than it usually does. Some p53 target genes are expressed under viral load, but others, especially those that respond to DNA damage, aren’t. One of the genes that is not activated is p21, which can promote cell survival. Leaving this gene inactive would help promote the apoptotic effect. Interferon enhances the apoptotic effects of p53, but it is not strictly required. Normal cells exhibit a stronger apoptotic response than cells without p53.

Additionally, interferon has been shown to have therapeutic effect against certain cancers. It is probable that one mechanism of this effect is p53 induction. This could be useful clinically: Interferons could supplement or replace chemotherapy drugs that activate p53 but also cause unwanted side effects. Some of these side effects can be serious, severe and permanent.

Virus resistance to interferons

In a study of the blocking of interferon (IFN) by the Japanese Encephalitis Virus (JEV), a group of researchers infected human recombinant IFN-alpha with JEV, DEN-2, and PL406, which are all viruses, and found that some viruses have manifested methods that give them a way around the IFN-alpha/beta response. The viruses need to master these methods so they can have the ability to carry on viral replication and production of new viruses. The ways that viruses find a way around the IFN response is through the inhibition of interferon signaling, production, and the blocking of the functions of IFN-induced proteins.

It is not unusual to find viruses encoding for a multiple number of mechanisms to allow them to elude the IFN response at many different levels. While doing the study with JEV, Lin and his coworkers found that IFN-alpha's inability to block JEV means that JEV may be able to block IFN-alpha signaling which in turn would prevent IFN from having STAT1, STAT2, ISGF3, and IRF-9 signaling. DEN-2 also significantly reduces interferon ability to active JAK-STAT. Some other viral gene products that have been found to have an effect on IFN signaling include EBNA-2, Polyomavirus large T antigen, EBV EBNA1, HPV E7, HCMV, and HHV8. Several poxviruses encode a soluble IFN receptor homologue that acts as a decoy to inhibit the biological activity of IFN, and that activity is for IFN to bind to their cognate receptors on the cell surface to initiate a signaling cascade, known as the Janus kinase(JAK)-signal transducer and activation of transcription(Stat) pathways. For example, a group of researchers found that the B18R protein, which acts as a type 1 IFN receptor and is produced by the vaccinia virus, inhibited IFN's ability to begin the phosphorylation of JAK1 which reduced the antiviral effect of IFN.

Some viruses can encode proteins that bind to dsRNA. In a study where the researchers infected Human U cells with reovirus-sigma3 protein and then, using the Western blot test, they found that reovirus-sigma3 protein does bind to dsRNA. Along with that, another study in which the researchers infected mouse L cells with vaccinia virus E3L found that E3L encodes the p25 protein that binds to dsRNA. Without double stranded RNA (dsRNA), because it is bound to by the proteins, it is not able to create IFN-induced PKR and 2'-5' oligoadenylate-synthetase making IFN ineffective. It was also found that JEV was able to inhibit IFN-alpha's ability to activate or create ISGs such as PKR. PKR was not able to be found in the JEV infected cells and PKR RNA levels were found to be lower in those same infected cells, and this disruption of PKR can occur, for example, in cells infected with flavaviruses.

The H5N1 influenza virus, also known as bird flu, has been shown to have resistance to interferon and other anti-viral cytokines. This is part of the reason for its high mortality rates in humans. It is resistant due to a single amino acid mutation in Non-Structual protein 1 (NS1), the precise mechanism of how this confers immunity is unclear (reference is Lethal H5N1 influenza viruses escape host anti-viral cytokine responses, Sang Heui Seo, Nature Med, 2002).

Pharmaceutical uses

Three vials filled with human leukocyte interferon.

Uses

Just as their natural function, interferons have antiviral, antiseptic and antioncogenic properties when administered as drugs.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for many cancers.

More than half of hepatitis C patients treated with interferon respond with viral elimination (sustained virological response), better blood tests and better liver histology (detected on biopsy). There is some evidence that giving interferon immediately following infection can prevent chronic hepatitis C. However, people infected by HCV often do not display symptoms of HCV infection until months or years later making early treatment difficult.

Interferons (interferon beta-1a and interferon beta-1b ) are also used in the treatment and control of multiple sclerosis, an autoimmune disorder.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However, mechanisms of such action of interferon are not well understood; it is thought that doses must be larger by several orders of magnitude to have any effect on the virus. Consequently, most Western scientists are skeptical of any claims of good efficacy.

Route of administration

When used in the systemic therapy, IFN-α and IFN-γ are mostly administered by an intramuscular injection. The injection of interferons in the muscle, in the vein, or under skin is generally well tolerated.

Interferon alpha can also be induced with small imidazoquinoline molecules by activation of TLR7 receptor. Aldara (Imiquimod) cream works with this mechanism to induce IFN alpha and IL12 and approved by FDA to treat Actinic keratosis, Superficial Basal Cell Carcinoma, and External Genital Warts.

Adverse effects

The most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression. Erythema, pain and hardness on the spot of injection are also frequently observed. Interferon therapy causes immunosuppression, in particular though neutropenia and can result in some infections manifesting in unusual ways.

All known adverse effects are usually reversible and disappear a few days after the therapy has been finished.

Types

Several different types of interferon are now approved for use in humans.

More recently, the FDA approved pegylated interferon-alpha, in which polyethylene glycol is added to make the interferon last longer in the body. (Pegylated interferon-alpha-2b was approved in January 2001; pegylated interferon-alpha-2a was approved in October 2002.) The pegylated form is injected once weekly, rather than three times per week for conventional interferon-alpha. Used in combination with the antiviral drug ribavirin, pegylated interferon produces sustained cure rates of 75% or better in people with genotype 2 or 3 hepatitis C (which is easier to treat) but still less than 50% in people with genotype 1 (which is most common in the U.S. and Western Europe).

Interferon-beta (Interferon beta-1a and Interferon beta-1b) is used in the treatment and control of multiple sclerosis. By an as-yet-unknown mechanism, interferon-beta inhibits the production of Th1 cytokines and the activation of monocytes.

History

While aiming to develop an improved vaccine for smallpox, two Japanese virologists, Yasu-ichi Nagano and Yasuhiko Kojima working at the Institute for Infectious Diseases at the University of Tokyo, noticed that rabbit-skin or testis previously inoculated with UV-inactivated virus exhibited inhibition of viral growth when re-infected at the same site with live virus. They hypothesised that this was due to some inhibitory factor, and began to characterise it by fractionation of the UV-irradiated viral homogenates using an ultracentrifuge. They published these findings in 1954 in the French journal now known as “Journal de la Société de Biologie”. While this paper demonstrated that the activity could be separated from the virus particles, it could not reconcile the antiviral activity demonstrated in the rabbit skin experiments, with the observation that the same supernatant led to the production of antiviral antibodies in mice. A further paper in 1958, involving triple-ultracentrifugation of the homogenate demonstrated that the inhibitory factor was distinct from the virus particles, leading to trace contamination being ascribed to the 1954 observations.

Meanwhile, the British virologist Alick Isaacs and the Swiss researcher Jean Lindenmann, at the National Institute for Medical Research in London, noticed an interference effect caused by heat-inactivated influenza virus on the growth of live influenza virus in chicken egg membranes in a nutritive solution chorioallantoic membrane. They published their results in 1957; in this paper they coined the term ‘interferon’, and today that specific interfering agent is known as a ‘Type I interferon’.

Nagano’s work was never fully appreciated in the scientific community; possibly because it was printed in French, but also because his in vivo system was perhaps too complex to provide clear results in the characterisation and purification of interferon. As time passed, Nagano became aware that his work had not been widely recognised, yet did not actively seek revaluation of his status in field of interferon research. As such, the majority of the credit for discovery of the interferon goes to Isaacs and Lindenmann, with whom there is no record of Nagano ever having made personal contact.

As a drug

Interferon was scarce and expensive until 1980 when the interferon gene was inserted into bacteria using recombinant DNA technology, allowing mass cultivation and purification from bacterial cultures or derived from yeast (e.g. Reiferon Retard is the first yeast derived interferon-alpha 2a).

Global sales ~ 5 billion US $. The second most successful pharmaceutical ever to come from genetic engineering.

Misc. facts

• Interferon is species-specific: the substance prepared from infected eggs protected only chicken cells from virus infection, while the similar substance prepared from mice protected only mouse cells.
• Produced by many cells in the human body by a receptor dependent feedback mechanism.
• Interferons are part of the "first-wave" immune response of the innate immune system, acting within hours, whereas antibody production takes days.
• A book was written about it: Toine Pieters, Interferon: The Science and Selling of a Miracle Drug (London: Routledge, 2005), xiv+264 pp., ISBN 0-415-34246-5. This book charts the beginnings, history and fate of interferon. The story of its development and use is one of survival in the face of remarkable cycles of promise, hope and disappointment as a miracle drug. The book demonstrates how research on interferon led to new clinical definitions of cancer and a new rational for therapeutic use of the drug. Moreover, through the lens of interferon's voyage the author explores the interaction between the laboratories of science, medicine and society: from the post-penicillin era to the genetics revolution in medicine.
• There are two types of IFNs: Type I (binding to IFN-aR1 and IFN-aR2c receptors; IFNAR1 chain is not the major ligand-binding chain), and type II (binding to IFN-gammaR1 and IFN-gammaR2 receptors).
• In general, exposure of human cells to viruses or double stranded RNAs induces the production of IFN-a, IFN-b, and IFN-o species.
• For the most part, the IFN-alpha species are not glycosylated, although some contain carbohydrates.
• The IFN-alpha family represents a family of related and homologous proteins, each exhibiting a unique activity profile. Each IFN-a species seems to exhibit a distinct profile of activities [antiviral, antiproliferative, and stimulation of cytotoxic activities of natural killer (NK) cells and T cells]
• The IFNs and IFN-like molecules signal through the Jak-Stat pathway. The receptor for the Type I IFNs consists of two chains, IFN-aR1 and IFN-aR2c. The ligand INF-alpha is a monomer that binds to the two-chain complex of IFN-aR1 and INF-aR2c.
• Within each subtype of mammalian Type I IFN, there is additional variability in gene duplication. The IFN-a genes are duplicated to a much greater extent than any other subtype of Type I IFN. This observation in conjunction with the observation that the IFN-a subtypes generally possess the highest specific antiviral activity imply that physiologically, the body likely uses IFN-a as the primary antiviral defense protein and that the major function of IFN-a is defense.
• STRUCTURE: The Type I IFNs consist of five a-helices (labeled A–E) which are linked by one overhand loop (AB loop) and three shorter segments (BC, CD, and DE loops). Helices A, B, C, and E are arranged in an antiparallel fashion to form a left-handed four-helix bundle. The AB loop contains short segments of 3_10 helix and is best described in three segments labeled AB1, AB2, and AB3. In all Type I IFNs, the AB1 loop encircles and is linked to helix E by a disulfide bond. An additional disulfide bond is observed in most IFN-a subtypes but not IFN-b, which connects the N-terminus of the molecule to helix C. The AB loop is critical for high-affinity IFNAR2 binding and suggest that sequence differences in this region may hold the key to differences in biological activity between the different IFN-a subtypes.
• The NMR structure of IFNAR2 has been determined and exhibits the same general structure as IFN-gammaR1. However, the interdomain angle is approximately 90 degrees rather than 120 degrees. Only loops in N-terminal domain (L2–L4) have been shown to be important for IFN-a2 binding.
• The IFNs were the first of the proteins we now recognize as members of the Class II cytokine family.
• IFNa2 contain 165 amino acids; according to circular dichroism measurements ~68% of the residues adopt helical conformation.INFa2 is composed of five a-helices, labeled A–E, linked by one long overhand connection (AB loop) and three short segments (BC, CD and DE loops). The topology of the molecule resembles the classical up-up-down-down four-helixbundle motif; helices A, B, C, and E comprise the helix bundle.
• Type I IFNs are stable at acidic pH (pH 2) and are represented by two major subtypes, the fibroblast or beta interferon (IFN-b) and the leukocyte or alpha family of interferons (IFN-a).The only known interferon of type II is IFN-g, which is produced exclusively by lymphocytes.

Pharmaceutical forms of interferons in the market

Generic name	Trade name
Interferon alpha 2a	Roferon A
Interferon alpha 2b§	Intron A
Human leukocyte Interferon-alpha (HuIFN-alpha-Le)	Multiferon
Interferon beta 1a, liquid form	Rebif
Interferon beta 1a, lyophilized	Avonex
Interferon beta 1a, biogeneric (Iran)	Cinnovex
Interferon beta 1b	Betaseron / Betaferon
Pegylated interferon alpha 2a	Pegasys
Pegylated interferon alpha 2a (Egypt)	Reiferon Retard
Pegylated interferon alpha 2b	PegIntron
Pegylated interferon alpha 2b plus ribavirin (Canada)	Pegetron

^§ also marketed in India as Reliferon, a product of Reliance Biopharmaceuticals.

Cremophor EL

Cremophor EL is the registered trademark of BASF Corp. for its version of polyethoxylated castor oil. It is prepared by reacting 35 moles of ethylene oxide with each mole of castor oil. The resulting product is a mixture (CAS number 61791-12-6): the major component is the material in which the hydroxyl groups of the castor oil triglyceride have ethoxylated with ethylene oxide to form polyethylene glycol ethers. Minor components are the polyethyelene glycol esters of ricinoleic acid, polyethyelene glycols and polyethyelene glycol ethers of glycerol. Cremophor EL is a synthetic, nonionic surfactant. Its utility comes from its ability to stabilize emulsions of nonpolar materials in aqueous systems.

Cremophor EL is an excipient or additive in drugs. Therapeutically, modern drugs are rarely given in a pure chemical state, so most active ingredients are combined with excipients or additives such as Cremophor EL.

Uses

• Miconazole, anti-fungal
• Paclitaxel, anti-cancer

(Cremophor EL is used in Taxol (paclitaxel) and has been called as a dose limiting agent because of its toxicities. (A formulation of paclitaxel that uses nanoparticle albumin instead of Cremophor EL is marketed as an alternative under the trade name of Abraxane.))

• Aci-Jel (acetic acid / oxyquinoline / ricinoleic acid - vaginal)
• Sandimmune (cyclosporine injection, USP)
• Nelfinavir mesylate, HIV protese inhibitor
• Propofol, intravenous anaesthetic agent, originally presented in Cremophor for trials; now presented in a lipid emulsion
• Diazepam injection; superseded by lipid emulsion alternative (Diazemuls)
• Vitamin K injection