Nanoantibodies – "magic bullets" of Russian production
Magic nanopoules and camels from ChernogolovkaAlfiya Bulatova, STRF.ru
However, one injection of such a drug (and, as a rule, several are required) can cost from several tens to hundreds of thousands of rubles. However, scientists from the Institute of Gene Biology of the Russian Academy of Sciences assure that domestic cheaper analogues are just around the corner.
"Magic Bullets"In the 70s of the last century, a turning point came in the development of anti-cancer drugs – the so-called monoclonal antibodies (MAT) were created.
Due to the fact that these protein molecules have the properties to specifically recognize a certain antigen or protein of a certain structure, they can significantly more selectively affect the target cells of a malignant tumor than traditional chemotherapy drugs, while significantly less damaging normal cells of the body. It is not surprising that already in the early 80s, the MAT began to be called anti-cancer "magic bullets". However, despite the fact that pharmaceutical and biotech companies have since invested more than one billion dollars in research related to the development of this technology, and a number of new antitumor drugs, including antibodies "armed" with toxins and radionuclides, have shown high efficiency, progress in this area is quite slow. compared to expectations. Dreams of a "magic bullet" once again did not come true, but transformed into a more realistic idea of antibody-based chemotherapy drugs as an effective, highly specific and relatively low-toxic additional tool for fighting cancer.
This technology, like everything else in the world, has its disadvantages. Firstly, the MAT molecules are quite large and they are not able to penetrate inside the cell or deep into the tissues. Secondly, to achieve the desired result, their concentration should be 5-10 thousand times higher than the concentration of target molecules. And thirdly, mats are produced exclusively on cell cultures, which makes their production not cheap. And in the conditions of import in Russia, the prices for these drugs are generally sky-high. After all, for us, their cost is related not only to the production process, but also to the procedure of clinical trials.
But, as it turned out, these problems are solvable. At least, this is evidenced by the results of the project "Creation of nanoantibodies for effective control of pathological neoangiogenesis induced by vascular endothelial growth factor", supported in 2008 by Rosnauka under the federal target program "Research and Development in priority areas of development of the scientific and technological complex of Russia for 2007-2012".
Belgian nanobody in the Russian manner"The idea of our research arose relatively recently and is connected with the discovery of Belgian scientists," says Sergey Vladimirovich Tillib, PhD, head of the scientific group of the Institute of Gene Biology of the Russian Academy of Sciences, "In 1993 they found unique antibodies of a simplified structure in representatives of the Camel family, which did not consist of four chains of immunoglobulin (two heavy and two light), and from the dimer of only one shortened heavy chain.
That is, the recognizing, variable domain of these protein molecules was formed by only one polypeptide chain. And this meant that working with them greatly simplifies all currently existing recombinant technologies. In addition, these antibodies, capable of specifically recognizing certain antigens, are the smallest of all currently known proteins with similar properties."
Based on this discovery, a new technology consisting of two parts was developed. First, a representative of the camel family is immunized (in the Russian case, they were double-humped camels) in order to cause the formation of exactly those antibodies that recognize the antigens of interest to us. Then we work with ribonucleic acid (RNA) molecules, which, like instructions, carry information about the structure of the antibody. Further, after the isolation of RNA from the lymphocytes of an immunized animal, the actual genetic engineering work is carried out in the laboratory.
"The main result of the first stage is a library of all variable domains (antibody fragments) of this animal," explains Sergey Tillib, "Each clone is a functional unit that recognizes something and corresponds to a real antibody. We are making a library that repeatedly covers the list of all possible clones and is millions of times smaller compared to conventional recombinant libraries."
With this library, if it is stored under the right conditions, you can work indefinitely. From it, the selection of antigen-recognizing (variable) domains of antibodies capable of binding specifically to a given antigen similarly to a complete antibody is carried out. "The Belgians call this antigen-recognizing domain nanobody. We first dubbed it "mini-antibody", and now - "nanoantibody". On the one hand, this is a tribute to fashion, and on the other hand, it is a good equivalent to the generally accepted term," smiles Sergey Tillib, "We are actively calling them that now and it seems to suit everyone."
Specificity of nanoantibodiesLike conventional antibodies, for example, the same MAT, nanoantibodies have high specificity and low toxicity, but along with this they have several potential advantages over their canonical counterparts.
These advantages of nanoantibodies can be used in the future for various kinds of research and the creation of new biotechnological devices, as well as for the diagnosis and treatment of diseases.
Firstly, they have a much smaller size (2x4 nanometers) and new structural features – better penetration into the tissue and recognition of what has not been recognized until now. "Such antibodies do not exist in the human body, and therefore there is no adaptation to them. Thus, it becomes possible to bypass the tricks of abnormal, pathological cells and microorganisms that have managed to adapt to the human immune system. Maybe we can find a weak link in their defense," says Mr. Tillib.
Secondly, nanoantibodies are much easier and cheaper to produce in large quantities. The fact is that after synthesis they are already functional and do not require any additional modifications. This makes it possible to immediately develop them both in bacterial and yeast cells, which makes the way of creating these proteins significantly more economical.
Thirdly, nanoantibodies, compared with the recognition domains of traditional antibodies, dissolve much better in the human body and are much more resistant to significant fluctuations in temperature and acidity (pH). According to Sergey Tillib, these antibodies are often extremely stable. It is assumed that many of them can withstand the passage of the gastrointestinal tract, which makes it possible to take them orally in the form of ordinary tablets. By the way, drugs developed on the basis of MAT are still injected into the human body in the form of injections.
Fourthly, it is quite easy to carry out all kinds of genetic engineering manipulations with nanoantibodies (for example, to create more effective combined structures involving two or more nanoantibodies, as well as other protein domains or functional groups). Also, compared to conventional antibodies, they are easier to humanize, that is, to adapt for use in humans.
All these advantages open up a wide field of application of nanoantibodies as the basis of new generation drugs for a wide range of diseases.
Double–humped camels are our everythingIn nature, such nanoantibodies are normally found only in representatives of the Camel family and some shark species.
"But, as you know, it is very problematic to work with sharks," Sergey Tillib grins, "So we decided to stop at camels."
It turned out that in Russian conditions, the most affordable animal is a double–humped camel, which is used in agriculture in the Astrakhan region and in Kazakhstan. There are no smaller and, apparently, more convenient representatives of the Camel family, alpacas, in Russia as a class, and it is too expensive to bring them here.
"Double–humped camels are our everything," the researcher laughs, "Moreover, they are suitable for our climate. Their wool in length in winter reaches about 40 centimeters, and they calmly withstand up to 30 degrees of frost in the open air."
Two-humped beauties were placed near Chernogolovka at the experimental base of the Institute of Ecology and Evolution named after A.N. Severtsova, where they are cared for by professional zoologists. By the way, the organization of work with animals, according to scientists, has become the most difficult part of the study. First of all, because the camels had to be placed in such a way that, on the one hand, they would be able to control all procedures from the beginning of the animal's immunization to the final blood collection, and on the other hand, to provide our smaller brothers with comfortable accommodation. "We have been able to do this for six years," Mr. Tillib says with pride, "In fact, the complexity of organizing work with animals stops a lot of researchers who could compete with us in this field. That's probably why we don't have any competitors in Russia yet."
"Magic nanopoules" of Russian productionThere is also no great competition abroad in this completely new field.
The main patent holder of this technology is the Belgian biopharmaceutical company Ablynx, which conducts large-scale work together with a number of well-known pharmaceutical companies to obtain and clinical trials of new drugs based on nanoantibodies. However, there is no such drug on sale yet. "The nanoantibody technology is clearly working, which is also confirmed by the growing interest in this technology of the largest pharmaceutical manufacturers (Boehringer Ingelheim, Merck Serono, Novartis, Wyeth) observed in the last couple of years, which are expanding joint programs with Ablynx," notes Sergey Tillib.
One of the many specific ways of applying this technology is to obtain nanoantibodies that block neoangiogenesis – the pathological growth of new blood vessels in the absence of an obvious need on the part of the body, which causes many serious diseases. For example, the uncontrolled growth of new blood vessels in the retina of the eye causes a violation of visual signals, which leads to blindness. Another example of pathological neoangiogenesis is the formation of new blood vessels in malignant tumors. If you block the growth of blood vessels, then the development of the tumor stops. And the most effective strategy for controlling undesirable neoangiogenesis in various pathologies (vascular degeneration of the retina, endometriosis and malignant tumors) is the creation of vascular endothelial growth factor blockers (FRES).
Today, two pharmacological drugs are presented on the Russian market, the effect of which is based on the suppression of the activity of the FES. Avastin (bevacizumab), manufactured by the Swiss company Roche, is a humanized monoclonal antibody to FES and is used to block the growth of blood vessels in a tumor, thereby inhibiting its development. Lucentis (ranibizumab) manufactured by Novartis is a fragment of the same humanized monoclonal antibody and is used in the treatment of the vascular form of age-related retinal dystrophy. However, the main disadvantage of these drugs, which reduces the possibility of their use in our country, is the high price.
"It is better to have our own analogues, which can be cheaper, and therefore more accessible to wider segments of the population," says Mr. Tillib, "Including this task we solved in the framework of our project "Creation of nanoantibodies for effective control of pathological neoangiogenesis induced by vascular endothelial growth factor." The aim of this study was to obtain nanoantibodies that block the physiological functions of FRES in order to create a pharmacological substance and a drug based on them in the future for the treatment of vascular retinal dystrophy and to prevent the growth of new blood vessels in cancerous tumors.
The project was carried out under the general supervision of Academician Georgy Georgiev. In addition to Sergey Tillib's group, scientists from other departments of the Institute of Gene Biology of the Russian Academy of Sciences took part in the work – employees of the laboratories of Igor Korobko and Sergey Larin, as well as employees of the Russian State Medical University, which acted as a co-executing organization. If Tillib's research group was responsible for the technology of nanoantibody generation itself, then in the laboratories of Korobko and Larin, a recombinant FRES protein was procloned, which was used both for immunization of camels and for subsequent selection and analysis of nanoantibodies, and test systems were set up and used to test the specific biological activity as a recombinant FRES protein, and the selected nanoantibodies. The result of almost two years of work was two patented nanoantibodies, which, according to the resulting activity, proved to be the most successful.
"Now that the funding of our project through Rosnauki has ended, we continue to work – we want to further increase the activity of the obtained nanoantibodies: we optimize their structure (by introducing additional point nucleotide substitutions), we try to obtain their derivatives (dimers), we try to improve the binding properties and effectiveness of action. At least, we hope to approach, and maybe surpass Avastin in efficiency,“ notes Sergey Tillib, ”Then on the basis of these developments we will be able to apply for public funds at the R&D stage. And then, based on this, it will be possible to develop a specific drug for clinical trials. And this means that in a few years our country may have its own, and possibly more effective, analogue of Avastin."
Portal "Eternal youth" www.vechnayamolodost.ru08.04.2009