Monoclonal antibodies are antibodies that are the same because they are made by identical B-lymphocytes that are all clones of a unique parent cell.They are prepared by lymphocyte hybridization. Nowadays scientists try to join monoclonal antibodies with radioactive isotope or toxic chemical substance, which destroy a tumor cell or cell infected with virus. Modern opinions concerning structure and functions of antibodies, mechanisms of producing monoclonal antibodies, their experimental and clinical using in Immunology are described by the author.
Key words:monoclonal antibodies, lymphocyte hybridization, magic bullets.
36 years ago Cambridge researchers Drs. Cesar Milstein and George Köhler found a way how to make any required antibody in the test tube. It was a breakthrough because scientists invented a «magic bullet» — a tailored weapon that can home in and destroy the causes of disease. Antibodies, the complex glycoprotein molecules produced by the immune system, are the first line of defense against disease. The human immune system can make more than two million different antibodies, each of which recognizes, binds itself to and begins the process of destroying just one target, a protein antigen on a bacterium or virus. Antibodies are a crucial component of the immune system, circulating in the blood and lymphatic system, and binding to foreign antigens expressed on cells. Once bound, the foreign cells are marked for destruction by macrophages and complement. They are produced by white blood cells called plasma cells. Before Milstein and Köhler's achievement it was only possible to make antibodies in tiny quantities and messy mixtures. But they found a way how to stimulate cells for production antibodies, and then make those cells into «immortal» cell cultures, that could be grown to any required size and are able to produce just the one antibody for as long as it was required [4].
An antibody also known as an «immunoglobulin» is a large Y-shaped protein circulating in the blood stream. Antibody recognizes a unique part of the foreign target, called an antigen. In 1959 Rodney Porter tried to hydrolyse rabbit’s immunoglobulin IgG with the help of enzyme papain. He got three fragments. Two were the same and saved the ability of connecting with antigens. That is why the author termed them as Fab-fragments (Fragment, Antigen Binding). The third one could crystallize and was called as Fc-fragment (Fragment, Crystallizable). An organism has the same Fc-fragments of all his antibodies. They don’t lash antigens but can react with macrophages, lymphocytes and complement factors. In 1961 Jerald Edelman and Powlik dissociated the whole molecules of antibodies. They understood that the chains of an immunoglobulin are connected with the help of disulfide bonds [3]. Each antibody is composed of two hard (H, 53–75 kDa) and two light (L, 23 kDa) identical polypeptide chains. Each heavy and light chain can be divided into domains consisting of 110 amino acids. There are five heavy chain classes: mu (µ), delta (δ), gamma (γ), alpha (α) and epsilon (ε). There are two light chain types: kappa (κ) and lambda (λ). The light chain consists of two domains, indicated as VL and CL. VL is a variable region of the light chain and is a region participating in antigen binding. CL is a constant region which is invariant for a given light chain type. Each light chain is a product of three structural genes. The heavy chain has the same domains. They are called VH (antigen binding) and CH (constant). They are the product of at list four various genes. In addition the heavy chains of most immunoglobulins have a region known as the hinge (between CH1 and CH2) with the help of which Fab-fragment can move in relation to the Fc-fragment. Disulfide bonds are important for maintaining the tertiary structure of the immunoglobulin subunit and the quaternary structure of the whole antibody as well. Light chain has two and heavy chain — four disulfide bonds. Finally, immunoglobulins are glycoproteins. Different heavy chain classes have different types of carbohydrate groups and different locations of carbohydrate attachment. It is important for correct immunoglobulin folding and is transported during synthesis. The immune system acts to identify and remove «foreign» agents. The role of the immunoglobulin molecule is to provide a means for linking the recognition of «foreign» to the mechanisms that can act on the «foreign» agent. Variable regions bind antigenic determinants, while constant regions interact with other molecules and cells of the immune system [1,7].
Production of desired specificity antibodies in quantity and with reproducible characteristics had always been a challenge. These goals were achieved by the introduction of hybridoma technology by Köhler and Milstein in the 1970s. Since that, monoclonal antibodies have been playing a great role in biological research and are of great interest for clinical purposes. Monoclonal antibodies are typically made by fusing immortal myeloma cells which with the spleen cells from a mouse immunized with the desired antigen. Polyethylene glycol or a strong electric field is used to fuse plasma membranes. To get only fused hybridomas special selective medium is used. It is called HAT-medium because it contains hypoxanthine, aminopterin and thymidine. Unfused myeloma cells cannot grow because they lack hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the synthesis of nucleic acids, and thus cannot replicate their DNA. Unfused spleen cells die because of their limited life cycle. Only fused hybrid cells, are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits making it immortal (as it is a cancer cell). Aminopterin poisons the de novo synthesis of purines. The myeloma cells, mutagenized and selected to be HGPRT-negative, are killed by HAT-containing medium unless they have fused and therefore contain the enzymes of the spleen cell. Thus for several days after a fusion there is extensive cell death. Periodically, the myeloma cells should be cycled through selective medium such as 8-azaguanine, to assure that they have not reverted to a drug-resistant cells. Then mixture of cells is diluted and clones are grown from single parent cells. The antibodies secreted by the different clones are assayed for their ability to bind to the antigen (with a test such as ELISA, Antigen Microarray Assay or immuno-dot blot). The most productive and stable clone is selected for future use. The hybridomas can be grown indefinitely in a suitable cell culture media, or they can be injected into mice (in the peritoneal cavity), they produce tumors containing antibody-rich fluid called ascites fluid. One mouse can produce 50 mg of antibodies. Antibodies large quantity production is beside the purpose and needs a lot of animals. The medium must be enriched during selection to further favourable hybridoma growth. This can be achieved by the use of a feeder fibrocyte cells layer. 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 and if replacement techniques exist, this method is considered to be unethical [7].
Plants are potential biopharming factories because they are capable to produce unlimited numbers and amounts of recombinant proteins safely and inexpensively. Plants have several advantages, which include the lack of animal pathogens and low cost of production. Two general methods are used to introduce transgenes encoding a suitable antibody into plants. They are agrobacterium-mediated transformation and particle bombardment. Agrobacterium-mediated transformation is used to transfer forein genes into the genome of the plant nucleus where individual genes encoding the heavy and light chains are co-expressed to produce full-size monoclonal antibody. Chloroplast transgenic plants have been obtained for stable expression of antibodies in the chloroplast genome. Chloroplasts can process foreign proteins with disulfide bridges but they lack the glycosylation processing machinery required for the proper functionality of the antibody. Viral vector systems can be used for the expression of both heavy and light chain genes and the assembly of full-size monoclonal antibody in tobacco plants. A large number of different crops can be used to produce antibodies including tobacco (Nicotiana tobaccum and N. benthamiana), cereals (rice, wheat, maize), legumes (pea, soybean) and fruit and root crops (tomato, potato) [5,6].
Monoclonal antibody therapy is the use of monoclonal antibodies to specifically bind to target cells. This may then stimulate the patient's immune system to attack those cells. It is possible to create a monoclonal antibody specific to almost any extracellular/cell surface target, and thus there is a large amount of research and development currently being undergone to create monoclonals for numerous serious diseases (such as rheumatoid arthritis, multiple sclerosis and different types of cancers). Infliximab and Adalimumab bind to tumor necrosis factor-alpha and show promise against some inflammatory diseases such as rheumatoid arthritis. There is a number of ways for monoclonal antibodies to be used in therapy. For example: they can be used to destroy tumor cells and prevent tumor growth by blocking specific cell receptors. Monoclonal antibodies can be modified in many ways depending on their intended use. They may be conjugated with specific markers (enzymes, radioisotopes, toxins, chemotherapies etc.) but it is also possible to produce bispecific antibodies with two different antigen binding sites by biochemical or cell fusion techniques. One of the most successful ways they having been used, has been in the field of diagnosis. If a monoclonal is «tagged» with a fluorescent dye, a doctor can see immediately when it has attached to a specific antigen [4]. Among 150 diagnostic monoclonal antibodies a third is used to detect pregnancy. No sooner than embryo obtains the womb, it fastens to its side. This process is called implantation. Afterwards with the help of chorionic gonadotropin signal goes to woman’s ovary to product hormones, which countenance womb’s endometrium. Menses are stopped and embryo is kept. By the time of the next menses the level of chorionic gonadotropin is so high that it can be found out in urine. Antibodies to this hormone can be produced. These sets are sold and one is able to get result in 5 minutes. It is possible to set pregnancy earlier. In this way the blood is used for the test. The main problem of organs transplantation is patient’s system immune response. The only way is to suppress it. Monoclonal antibodies interact with antigens on the surface of all T-cells rejecting transplanted organs. For example, Muromonab-CD3 binds to the CD3 molecule on the surface of T-cells, used to prevent acute rejection of kidney transplants. Daclizumab has also showed promise against T-cell lymphoma. Monoclonal antibodies are much more effective than medicine used to quell immune system. They quell only T-cells and not the whole organism like drugs do. Afterwards the patient is able to resist various infections [2]. Passive immunotherapy with monoclonal antibodies has important advantages over active immunization. Firstly, significantly larger doses of antibodies can be administered, and protection is immediate. Secondly, the duration of action is more predictable than antibody generated by active immunization. Thirdly, unlike active immunizations, there is no immunological memory of the abused drug and the possibility of unexpected cross reactivity with endogenous ligands is less likely with monoclonal antibodies.
Although monoclonal antibodies have significant promise as therapeutic agents, they are not without problems. The 3 major problems are the high cost, the risk of toxicity, and the potential for allergic-type reactions. The current cost for monoclonal antibody medications for treating cancer and other health problems is thousands of dollars per month. Nonspecific toxicity may occur, including infusion reactions, cytokine release, and hypersensitivity to foreign immunoglobulins. For example, infliximab and adalimumab can convert a latent case of tuberculosis into active disease and induce the formation of autoantibodies. Hypersensitivity reactions to the xenogeneic component of chimeric and humanized antibodies can occur upon the first dose of antibody and following repeated exposure. Those two types of antibodies were invented by Dr. Greg Winter. Chimeric antibodies combine the antigen-binding part (variable region) of a mouse antibody with the effector parts (constant regions) of a human antibody. Humanized antibodies combine only amino acids responsible for making the antigen binding site (the hypervariable regions) of the mouse with the rest of the human antibody molecule [4].
Nowadays with the development of bioengineering and progress in Immunology it is possible to create «magic bullets» for treating a large number of serious diseases. These bullets are called monoclonal antibodies. Though it is rather difficult to produce them and that is why they are so expensive we believe that in future it will be possible to save lives with the help of such drugs all over the world. At least forty different antibodies are awaiting approval for use in medicine. Dr Milstein thinks the next big breakthrough may come in the development of antibodies able to penetrate the surfaces of cancer cells and target the abnormal proteins inside. That could lead to a revolution in cancer therapy.
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