Anti-cancer antibodies in the garden
How to make antibodies in plants
Kirill Stasevich, "Science and Life"We know that antibodies are called special immune proteins that help us get rid of infection.
The point of their work is to specifically recognize a foreign molecule (which may be either a bacterial toxin, or a protein forming the envelope of a virus, or something else). The "bad" molecules associated with antibodies and their owners, pathogens, are neutralized and destroyed by immune cells.
Immunity in its usual form – with special organs, with a huge number of different cells and proteins – is considered a characteristic feature of animals, and even then not all, but mainly vertebrates (although some elements of the immune system can be found in invertebrates). Therefore, we would be very surprised if someone told us about plant antibodies. Nevertheless, there are such, and they even have a special name in English: "plantibody" – "plant", plant + "antibody", antibody (in Russian it will turn out something like "plantbody"). However, we are not talking here about the plants' own antibodies, but about animal proteins-immunoglobulins, whose genes were inserted into the plant organism.
Why was it necessary? It would not be an exaggeration to say that without a variety of methods based on the use of antibodies, modern biotechnology and medicine would be like without hands. Immunoglobulins are used everywhere, from cleaning the necessary molecules from the accompanying "garbage" to diagnostics: for example, if we need to detect a parasite in a biological sample, then with the help of highly specific and strongly binding antibodies to the parasite, even a negligible amount of it can be determined. A simple enumeration of all analytical and preparative methods with antibodies would be enough for a full-fledged article, even if we do not mention immunotherapy, when antibodies are used to fight tumors or parasites.
But where to get them? The obvious answer is from immunized animals: we inject a rat, or a goat, or a rabbit with some kind of antigen (for example, bacterial protein), and then we purify the immunoglobulins we need from the animal's blood. However, the immune system in response to the antigen produces many varieties of antibodies, and then you have to separate the right variety with the right characteristics from others. There is also the technology of monoclonal antibodies, when we get a clone of immune cells synthesizing only one variant of immunoglobulins. However, this method itself is also not simple, suffice it to say that it is based on the fusion of two cells into one: a B-lymphocyte, carrying information about the desired antibody, combines with a cancerous myeloma cell. The resulting hybrid receives immortality and the ability to divide infinitely from myeloma, and at the same time it produces antibodies. But if we want to get a lot of antibodies – for example, for clinical treatment – then a new problem arises: if we make them using hybrid technology, it will take quite a long time.
In general, conventional methods of obtaining antibodies with the desired characteristics are quite time-consuming and relatively difficult. Therefore, at some point, the researchers came up with the idea to use the achievements of genetic engineering here. That is, take the DNA encoding the immunoglobulin with the desired characteristics and insert it into the plant. Practice has shown that it will be much easier, cheaper and safer to obtain antibodies in plants: a large number of immunoglobulins can be obtained without resorting to immunization, without using pathogens and vaccines – plant cells themselves synthesize the necessary protein. And it is much easier to get transgenic plants than transgenic animals.
Last year, plant antibodies were mentioned in connection with Ebola. The experimental drug ZMapp, which saved the lives of several patients, is a humanized immunoglobulin against infection obtained from plants. First, mice were infected with the virus, after which mouse B cells, which began the synthesis of immunoglobulins against Ebola, were carried out through a hybridomic method and received many clones that produced many types of different-quality antibodies against the same virus. Among them, the best were chosen, and the immunoglobulin gene was extracted from the cells that synthesized them. Since it belonged to an animal, some fragments were changed into human ones in it – that's why such antibodies are called humanized, because they represent an animal-human chimera. They do this so that immunoglobulins originating from someone else's body do not themselves provoke the human immune system to an aggressive response. Finally, the resulting gene was injected into tobacco plants that produced large amounts of antibodies.
How to supply a plant cell with a foreign gene? First, the sequence encoding the immunoglobulin is inserted into the DNA of the bacterium Agrobacterium tumefaciens. By itself, it is a pathogen that causes the appearance of crown galls – tumor formations on the plant. At the same time, it has a remarkable feature from the point of view of genetic engineering: a bacterium can transfer part of its genome to a plant cell, and bacterial DNA is embedded in a plant chromosome. That is, A.tumefaciens is a natural genetic engineering tool adapted for working with plants. All that is required of a person is to make sure that the desired gene (for example, the immunoglobulin gene) is in that piece of bacterial DNA that will be embedded in the DNA of a plant cell.
It is equally important that the bacterium can successfully penetrate into the plant, infect it. Plants have their own ways of resisting an infectious invasion. Therefore, studies devoted to the general problems of interaction between plants and bacteria turn out to be very useful for such a seemingly purely applied task as obtaining plant antibodies. So it is not surprising that the project of the staff of the Institute of Physico-Chemical Biology named after Tatiana Komarova and Ekaterina Sheshukova Belozersky, which is called "Fundamental and Biotechnological aspects of Plant-pathogen Interaction", recently received the Moscow Government Prize for Young scientists. The project has already managed to obtain some important results: for example, it turned out that the gaseous methanol that plants emit when damaged is necessary to protect against bacteria. Moreover, methanol serves as a signal by which the damaged plant warns neighbors about the danger. That is, if you want to introduce a bacterium with an immunoglobulin gene into plant tissues, you need to take into account the methanol protection and warning system.
New data on how the plant protects itself from the invasion of the pathogen has allowed the development of a biotechnological platform for the production of vaccines and antibodies in plant cells. In 2011, Tatiana Komarova and her colleagues from the A.N.Belozersky Institute of Physico-Chemical Biology, the N.I. Vavilov Institute of Genetics and the N.N. Blokhin Cancer Center published an article in PLoS ONE describing the production of plant antibodies used in the treatment of breast cancer (Komarova et al., Plant-Made Trastuzumab (Herceptin) Inhibits HER2/Neu+ Cell Proliferation and Retards Tumor Growth). By itself, such a drug has existed for a long time under the name trastuzumab, or "Herceptin" – by binding to the cancer protein of one of the varieties of tumors, immunoglobulins suppress the growth of cancer. The authors obtained transgenic tobacco plants that synthesized these antibodies. Tests have confirmed that herbal trastuzumab also stops the division of tumor cells and stops the development of the disease.
Growth of ovarian cancer xenograft (SK-BR-3 cells) in control group mice (with saline injection),
under the action of plant antibodies (Plant-Made Trastuzumab, PMT)
and "pharmacy" trastuzumab (graph from the article in PLoS ONE) – VM.
The researchers went further and "taught" plants the synthesis of three more types of antibodies: the first one blocks the development of blood vessels in the tumor, thereby inhibiting its growth, the other two are directed against breast cancer cells themselves. Animal experiments have shown that the new immunoglobulins have higher anti-cancer activity than commercial Herceptin. After clinical trials, they can be recommended as diagnostic and therapeutic agents in the treatment of a malignant tumor (of course, if the test results are positive).
Plant antibodies can become a cheap and effective alternative to conventional clinical drugs, not only for cancer, but also in the case of infections, such as seasonal flu. Although, of course, the use of plant antibodies in oncology is of the greatest interest. The authors of the project claim that with the help of their technology, it is possible to provide personalized immunotherapy, that is, in a fairly short time (about 6 weeks), it is possible to create a vaccine aimed against an individual variant of cancer. It remains only to wish the researchers good luck in further experiments.
Portal "Eternal youth" http://vechnayamolodost.ru03.03.2015