Types and nomenclature of monoclonal antibodies. Therapeutic monoclonal antibodies may be murine (suffix: -omab), chimeric (suffix: -ximab), humanized (suffix: e.g. -zumab) or human (e.g. -umab). “Murine” parts of antibody are shown in red, “human” – in blue. Source: Buss et al., 2012.
Antibodies, or immunoglobulins, are protein molecules produced by B-lymphocyte immune cells (plasmacytes), when encountering foreign (and therefore potentially dangerous for the body) agents. Those include bacteria, viruses, snake venom, tumor cells, etc. Generally, they are called antigens (from antibody-generator) . A sum of different antibodies is produced by different clones of B-lymphocytes against the same antigen, therefore they are called “polyclonal” (pAbs). Such antibody “cocktail” increases efficacy of immune response, as numerous antibodies may bind to various parts of the pathogen. Different animal species have various types and classes of antibodies varying in their structure. Generally, antibodies contain variable and constant domains. Antibodies employ variable domains to recognize antigens, and constant ones to interact with the cells of host immune system. For example, the antibodies of the same class will have similar constant domains and different variable domains.
Due to antibodies exceptional specificity of binding to a pathogen, they have become a unique instrument for diagnosis and therapy of various diseases. However, the boom in immunotherapy took place after discovering the technology of producing specific monoclonal antibodies (mAbs) in 1970s. Monoclonal antibodies are produced by a single clone of В-lymphocytes; it is an individual component of polyclonal “cocktail”. All such mAb have distinct structure and characteristics and therefore “recognize” a strictly specific antigen site.
Technology of mAb production was jointly developed and published in 1975 by a young German scientist Georges Köhler and his mentor, an Argentine-British professor Cesar Milstein. In theory, it is not so difficult to obtain identical mAbs: only one proper clone of producing B-lymphocyte is required. However, in practice, the challenge is that B-lymphocytes are mortal and tend to die rapidly in laboratory setting. Köhler and Milstein invented a method of “immortalizing” antibody producers by coupling them with tumor cells. Therefore, such a hybrid inherits ability for antibody synthesis on one hand and immortality on the other hand. This technology of mAb production is called hybridoma. The development of this technology earned Köhler and Milstein the Nobel Prize in 1984. It is noteworthy that this technology was not patented, and the researchers who could have become billionaires opted for scientific development. Hybridoma technology enabled unlimited production of antibody products with known and constant composition. The use of mAbs is more acceptable for clinical practice compared to multicomponent pAbs combinations with their varying composition since identical “cocktail” of pAbs cannot be obtained.
Just one year after Köhler and Milstein Nobel Prize , FDA (Food and Drug Administration) approved and released Muromonab (Murine Monoclonal Antibody), the first product containing murine mAbs for prevention of rejection of transplanted organs. However, the product was soon recalled as numerous subjects experienced an allergic reaction to it. This way, a serious drawback of murine antibodies was discovered, i.e. their immunogenicity . Therefore, further development was aimed at elimination of murine protein from mAbs being allergic to humans. Thus, by means of gene engineering, chimeric and humanized antibodies were developed. In these antibodies, only the pathogen-recognizing sites remain murine, while human ones replaced the other components of the protein. Thus, human protein share in chimeric antibodies is around 65%, while it amounts to approximately 95% in humanized antibodies. The use of modified antibodies resulted in a significant reduction in the percentage of negative responses to therapy and an increase in its efficacy.
Soon, due to further development of gene- and bio- engineering, fully human monoclonal antibodies were obtained. For example, this became possible thanks to phage display technology. This mAbs technology does not require animal immunization. It utilizes only viruses (bacteriophages) and bacteria. Method is based on the hypermutation phenomenon in viruses resulting in variability of their surface proteins. To remain unrecognized, the viruses constantly change their “image”. Therefore, if human immunoglobulin gene fragments are built in the genes coding the surface phage proteins, an assortment of their variations may be obtained. Their protein product, i.e. antibody fragments, will further be displayed by a phage on its surface (hence the name). After that the most suitable virus variant can be chosen, which presents antibodies with best binding to the specific antigen, and then replicate it in bacteria. Due to physical conjugation of the gene and coded protein in the viral particle, DNA sequence of the relevant antibody fragment is easily determined and may further be produced in large amounts similar to any other protein. An American George Smith suggested phage display technology as early as in 1985, and antibody phage display was developed by an Englishman Gregory Winter in 1990. In 2018, these scientists were awarded the Nobel Prize in Chemistry. In 2002, FDA approved Adalimumab, the first mAb product created with phage display technology. It is intended for the treatment of rheumatoid arthritis, psoriasis, etc. and it has become one of the most commercially successful biopharmaceuticals.
In addition to display technologies, human immunoglobulin gene cloning enabled production of fully human antibodies using another method. Transgenic mouse lines with built-in human immunoglobulin genes and their own suppressed immunoglobulin genes were developed. A transgenic organism is a living organism with a gene that is artificially inserted into its genome and cannot be acquired using only natural crossing. Immunization of such mice (administration of a specific pathogen) results in production of human antibodies by murine immune system. This method has an advantage over display technology: it can be faster and easier as it generally does not require further antibody optimization. Panitumumab, the first product manufactured using this technique based on fully human mAbs, was released in 2006 for the treatment of colorectal cancer.
Due to highly selective effect, the modern mAbs generally are well tolerated by patients and have less adverse effects. mAbs are top-selling biopharmaceuticals and their market is being developed extensively. Last year alone, 13 novel mAbs products were approved in Europe/USA, while their total number exceeds 130, and hundreds of similar products are in various phases of clinical trials.
Currently, mAbs have the broadest application in oncology. These antibodies may exhibit targeted effect on tumor cells without affecting the healthy ones. In 2018, the Nobel Prize (this time in Physiology or Medicine) was awarded for a novel approach in cancer immunotherapy enabling to produce antibodies effective for the treatment of previously hopeless cases by inhibiting immunological checkpoints . mAbs are also effective for the treatment of diseases with autoimmune component such as rheumatoid arthritis, psoriasis, multiple sclerosis. Overall list of diseases and situations in which mAbs may be applied is relatively large. It includes rejection of transplanted organs, cardiovascular and infectious diseases, macular degeneration, asthma, and some rare (orphan) diseases and syndromes. Attempts are being made to produce mAbs capable of reaching the brain by crossing blood-brain barrier for the treatment of neurodegenerative diseases (e.g. Alzheimer’s disease).
Bioengineers are constantly seeking to improve mAbs. Several products containing conjugated mAbs, i.e. coupled with cytotoxic agents (radioactive particles, toxins), have been approved, thus enabling selective intoxication of tumor cells without affecting normal ones. There are also biospecific mAbs that recognize two targets at a time. The first such antibody (Catumaxomab) was marketed in 2009 as an antitumor product, reinforcing immune response and facilitating death of tumor cells.
mAb discovery suggested that the researchers have finally found a “magic bullet” (a medical concept), a strictly selective product affecting only its target without detrimental effect on the body. However, currently mAbs are more commonly used as adjuvant therapy as they are still costly, difficult to produce and have some efficacy and stability issues. Nevertheless, mAb containing products are top-selling biopharmaceuticals, while immunotherapy is one of the most fast-developing, successful and promising areas.