Monoclonal antibodies: research and application
Ekaterina Sheshukova, "PostNauka"
When a foreign protein or bacterium enters the body, the defense system is triggered – the immune response. These are B-lymphocytes that begin to synthesize proteins that recognize any part of a foreign protein. And monoclonal antibodies are antibodies produced by immune cells belonging to the same cell clone. They recognize one specific site of the pathogen known to us and act on it. That is, it is not a mixture of antibodies that the body produces by a multitude of B cells in response to a pathogen, but only one specific species capable of recognizing one specific epitope, for example, a section of the domain of a certain cellular receptor.
Discovery of monoclonal antibodies
Monoclonal antibodies were discovered in 1975 by Georges Koehler and Cesar Milstein. They published an article on the development of a hybrid method. Scientists were able to merge B-lymphocytes synthesizing antibodies with myeloma cells (cancer cells) using polyethylene glycol or Sendai virus. Thus, scientists have obtained a hybridoma that has the properties of both a cancer cell (the ability to divide a large number of times) and B-lymphocytes (antibody synthesis). And then, by certain multi-stage stages of selection, they separated the cells synthesizing the necessary antibody.
The basis for the creation of therapeutic monoclonal antibodies are human immunoglobulins of the G1 subclass, since this type of antibodies has a number of advantages, one of which is the longest half–life from the blood. After introduction into the body, immunoglobulins of the G1 subclass live up to three weeks. This is very important, because the most important thing for therapy is that the medicine is in the patient's body as long as possible.
Accordingly, all antibodies are humanized so that the human body does not reject them. Despite the existence of common treatment systems, there are situations when scientists need to look for a new approach to the treatment of pathology. Everything is individual. For example, for a particular person, the resulting antibody may be an allergic agent or resistance to it occurs in the body. With such problems, systems for obtaining antibodies in plants can come to the rescue, since in a week or two you can get the desired antibodies, which will take much longer on systems with an animal cell. We know the sequence encoding a certain antibody, then we create a construct and infiltrate it into the plant with the help of an agrobacterium.
Tobacco and antibodies
We conduct our research on the plant Nicotiana benthamiana (Australian tobacco). Since a large number of plant pathogens can infect it, it is widely used in virology, thanks to which various vector systems for the production of target proteins based on plant viruses have been well studied and developed.
In our research, we use vectors based on plant viruses. One of them is the tobacco mosaic virus (TMV), widely known and fully studied, and the other is the potato X virus (HVK). We use them to deliver antibody-coding genes to a plant cell. After that, the plant cell synthesizes proteins from these vectors, which are collected in the plant into full-sized antibodies. Then we isolate them on special columns that are used in all antibody purification methods.
It is important to understand that the stage of purification of antibodies obtained in plants does not differ from those used on animal cells. In principle, the production of monoclonal antibodies in animal cells and plants is comparable in time – the difference in biosafety. Drugs that are obtained in animal cells can infect humans with animal viruses or prions.
A person cannot get infected from plants. Firstly, their viruses are not dangerous for us. Secondly, plants do not need nutrient media that have a risk of contamination by various agents that are dangerous to humans. These are the main theses that we use to defend our theory that plants are perfect for the synthesis of antibodies. But even if we take into account that the use of monoclonal antibodies will not reach therapy, they are still widely used in various types of diagnostics. In addition to medicine, antibodies are also used in molecular biology as a way to detect a target protein and in chemistry as a way to purify or isolate a protein or polysaccharide as well.
The use of monoclonal antibodies in the fight against cancer
Until recently, we only worked with an analogue of trastuzumab, an antibody designed to treat HER2–positive breast cancer. It recognizes the fourth domain of this receptor and interacts only with it, stopping cell proliferation.
However, there are well–known cases of acquiring resistance to trastuzumab (commercial name - herceptin) in patients. It is sold in pharmacies and is used in breast cancer therapy worldwide. Solving this problem, scientists have developed another antibody to this receptor, but interacting with the second domain – the dimerization domain. It was called pertuzumab (the commercial name is perjeta). And we set ourselves the task of synthesizing this antibody. The essence of the therapy was that perjeta was added to herceptin and their use helped to avoid cancer resistance only to herceptin. This was the only way they slowed down the growth of cancer formation and cell proliferation.
We were puzzled by the topic of obtaining pertuzumab in plants. To begin with, we went to a public service called Drug Bank. It contains a database of all known drugs, amino acid sequences of those proteins that are used in various medical fields. Including in the Drug Bank, we found sequences of pertuzumab (perjets). It remains only to adapt it to the synthesis in the plant, using an Internet resource to optimize the codon composition. Of course, the nucleotide sequence will be different for a plant cell and for an animal, but as a result we get the same amino acid sequence. After we obtained the optimized nucleotide sequence, we synthesized the nucleotide code and inserted it into vectors, which were later used for the synthesis of antibodies in the plant.
Prospects for the study of monoclonal antibodies
Sometimes there are situations such as, for example, with the Ebola virus: the pathogen is rapidly spreading, infecting people and leading them to death. As for testing drugs on humans, it is known that during the outbreak of Ebola hemorrhagic fever in West Africa, there was an urgent need to defeat the disease as early as possible. And in this case, antibodies that were partially obtained in plants worked. This is ZMapp. It was able to be used as a drug and was administered to seven patients, although its safety had not previously been tested in human testing. In five patients, the condition improved significantly, and the disease receded. Now, after successful emergency care for patients, systematic studies of biosafety and effectiveness of treatment (phase I/II) with ZMapp have been initiated. It would be desirable that the opportunity to use antibodies obtained in plants did not arise under similar circumstances. Therefore, we will continue to work to completely get rid of doubts about the use of antibodies synthesized in a plant cell.
The author is a candidate of Biological Sciences, researcher at the Laboratory of Genetic Control of Stress Resistance at the N. I. Vavilov Institute of General Genetics of the Russian Academy of Sciences.
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