"Self-guided" medicine

Molecular nanoconstructor for theranostics

Sergey Deev, "First-hand Science" No. 4(75), 2017
Published on the website "Elements"

The hero of ancient Greek myths Achilles was invulnerable because his mother, the goddess Thetis, dipped him after birth into the waters of the Styx, a river in the realm of the dead. She held the future warrior by the heel, which remained his only unprotected place – it was there that the arrow that struck the hero, directed by the god Apollo, hit. This story is a kind of metaphor to describe one of the most promising areas in modern medicine. Today we can create more and more new drugs that affect, for example, cancer cells, but the problem is not to create this or that compound: the main thing is that it does not affect the whole body, healthy organs and tissues, allowing the patient to survive the treatment itself. And this task leads us into the field of molecular physiology – to the creation of tools capable of targeting the Achilles heel of the disease and selectively influencing it.

At the beginning of the XX century, the German immunologist P. Ehrlich, who received the Nobel Prize in 1908 together with the Russian physiologist I. I. Mechnikov, in his works on the theory of immunity for the first time formulated the concept of a "magic bullet" – a medicine that would find and selectively affect the foci of the disease in the human body. We can say that this idea is "borrowed" from nature, which created "self–guided" drugs - protein antibodies (immunoglobulins) produced by immune cells in response to an alien invasion.

Antibodies, by targeting a specific antigen on the cell surface, can stop its reproduction or cause apoptosis (programmed cell suicide). Antibodies can also trigger an attack by immune killer cells. In addition, they cause a cascade of reactions of the complement system – a complex of enzymes that serve to protect the body from the action of foreign agents. This leads to the destruction of the cell membrane and the further development of the immune response. By: (Acta Naturae, 2009)

But the wonderful idea of the "magic bullet" began to take flesh only more than half a century later, when the production of so-called monoclonal antibodies capable of binding to only one specific antigen (a foreign or dangerous substance that causes an immune response) was established. Such antibodies were produced by cell lines from "hybrids" of healthy and tumor immune cells of mice, and for use in medicine, these proteins learned to "humanize" with the help of genetic engineering technologies, replacing fragments of mouse immunoglobulins with human ones.

Most natural mammalian antibodies are similar in structure and contain a pair of identical heavy and light chains. According to the functions, two sites are distinguished: variable, recognizing the antigen of the target cell, and constant, responsible for the development of the immune response of the body. Some animals (camel, llama, shark) have simpler antibodies consisting only of heavy chains. By: (Acta Naturae, 2009)

Both of these ideas formed the basis of one of the most rapidly developing areas of medicine of the XXI century – theranostics (from therapy and diagnostics). Thus, although this term itself was first used only in 2002, the idea of combining in "one bottle" an addressable component that recognizes the Achilles heel of the disease and a therapeutic one turned out to be far from new, but very promising!

The first drugs based on monoclonal antibodies appeared on the market back in 1986, and today about fifty similar drugs are used in medicine, mainly for the treatment of oncological diseases. Antibodies, specifically binding to a tumor cell, can play a dual role. They either carry a medicinal compound or a toxin, or they themselves serve as a medicine, since they are able to cause the "killing" of a pathological cell, attracting the attention of the body's immune system to it.

However, such immunoglobulins are expensive to produce, since they are obtained in mammalian cells, and not in "high-tech" cell cultures of microorganisms. At the same time, one course of treatment often requires at least a dozen grams of the drug. As a result, for example, a course of treatment with herceptin for breast cancer will cost about 100 thousand rubles.

Today, antitumor drugs occupy a large share of the market. The cost of entering the market of one drug in 1962 was $ 4 million, now it is more than $ 1 billion. If one person worked on the production of a medicine, he would spend 899 years to make one jar, i.e. go from idea to development. But all these are marketing problems. The real problem is cancer's resistance to drugs. More and more new drugs are being brought to the market, more and more advanced, but the resistance of oncological diseases to these drugs remains a serious problem.

The second problem is that any immunoglobulin is a large molecule that is not easy to pass through the pores of blood vessels, so it will circulate through the bloodstream for a long time (several weeks). This complicates the targeted delivery of medicines. A large molecular weight prevents such drugs from penetrating into tumors, so they linger on the periphery of the focus. In addition, full-sized monoclonal antibodies have constant sites responsible for interacting with cells of the body's immune system, which can provoke additional adverse reactions when antibodies are used to deliver therapeutic toxic agents.

It is not surprising that scientists have an idea to develop further engineering solutions of nature. They began to modify full-size antibodies, including crushing them into pieces, creating mini-antibodies, etc. In parallel, intensive study and creation of completely new structures based on natural or artificially synthesized protein compounds began, which began to be called scaffolds (from the English "scaffolding"), or frame proteins.

The next task was to create multifunctional compounds that would simultaneously contain both the targeting part and diagnostic and/or therapeutic agents: fluorescent proteins, radioisotopes, protein toxins, antibiotics, enzymes, etc. Ideally, such a complex should still be able to "turn on/off" the action of the agent and thereby even more selectively control its action.

The Golden Mean

There are many options for modifying a full-sized human antibody consisting of two light and two heavy peptide chains. For example, we can cut off from a natural immunoglobulin molecule the site responsible for binding to a specific antigen. This fragment, in turn, can also be torn apart by enzymatic crushing or using genetically engineered methods. Scaffolds with an even smaller molecular weight are produced only by protein engineering design.

In this way, it is possible to obtain, for example, a so-called single-stranded antibody consisting only of antigen-binding variable sections of light and heavy chains of immunoglobulin connected by a flexible peptide bridge. Even more convenient designs for targeted delivery can be obtained from a sample of camel antibodies, most of whose immunoglobulins consist only of heavy chains that perform all the necessary functions.

But which of these molecular constructs will work most effectively? Here it is necessary to take into account the speed with which the body will be freed from all molecules. Tumor tissue is characterized by very tight contacts between cells. And in this context, it is advantageous to reduce the size of molecules, since they more easily penetrate dense tumors. But here we are faced with a dilemma.

S. M. Deev: "It's terrible when people with cancer come up and ask if we can help them. I do not know what to answer them... I'm a scientist, not a doctor. I have no right to treat."

In our kidneys, blood plasma is filtered through pores with a diameter of about 6 nm, as a result of which substances with a molecular weight less than 60-65 kDa are rapidly excreted through the kidneys. And, consequently, the smaller the size of the molecular structures, the greater the load on the kidneys. On the other hand, a cancerous tumor is all riddled with blood vessels, which can be called "leaky": they have large (compared to normal) pores, which ensures effective passive delivery of small molecules. And the smaller the molecule, the more likely it is that it will get into the tumor tissue.

As a result, a biotechnologist, choosing the size of his molecular structure, must always think about the balance between the effectiveness of the drug and its nephrotoxicity. We need to look for a middle ground.

To date, in addition to antibodies of various formats, a number of other natural and artificial biopolymers are used in the creation of drugs for theranostics: affibody, affitins, finomers, aptamers, etc.

An example of promising scaffold proteins are DARPins (DARPins) – small molecules in the structure of which there are "anchored" amino acid repeats that provide structural stability, and mutable amino acid residues that allow modulating binding to specific targets. At the same time, the binding strength of such molecules to the target is sometimes even higher than that of natural antibodies. But for biotechnology, the main thing is economic indicators. If we get ~ 5 mg of "shortened" immunoglobulins from one liter of E. coli culture, then in the case of darpins – 30-40 times more! In addition, darpins are very stable – they can almost be boiled. And even if you attach something sufficiently voluminous to them (toxin, nanoparticles), the size of the structure will still remain optimal for blood flow.

DARPins is an example of an artificial scaffold protein containing mutable ankyrin repeats that serve to bind to an antigen. By: (Stumpp, Binz, Plückthun, 2003)

All currently known alternative scaffold proteins have different structures and molecular weights. But in general, we can say that they are too large compared to small peptides and too small compared to conventional antibodies. Therefore, we cannot directly transfer to them the already existing knowledge about the distribution or pharmacokinetics of other protein molecules. You need to check everything: you can't mechanically replace immunoglobulin with darpin and expect it to work.

Fluorescent models of human ovarian adenocarcinoma cells have been created at IBH RAS, which have preserved the cancer marker on their surface. These cells are inoculated with experimental animals, which makes it possible to trace the development of the disease in vivo. This greatly facilitates the work of the experimenter, since in some laboratories the tumor is still measured with a ruler, which leads to large errors. From above – fluorescent cancer cells of human ovarian adenocarcinoma SKOV-3 kat. Photo from the author's archive. At the bottom is the observation of tumor growth in control animals and those treated with DARPin29–PE40 (the positive dynamics of treatment in the first 30 days is clearly visible). © 2015 Zdobnova et al. Oncotarget

Toxin as medicine

A large number of studies in theranostics today are devoted to the creation of bifunctional compounds based on antitumor antibodies and powerful toxins of various nature to affect target cells. Almost half of the drugs currently undergoing clinical trials are such immunotoxins.

Back in the 1990s, attempts began to use ribonuclease enzymes (RNases) for this purpose, which catalyze the cleavage of ribonucleic acids (RNA), which perform a number of important functions in the cell, starting with participation in protein synthesis. These enzymes attract researchers with their availability, lack of mutagenic effect and low side toxicity to the body. In our country, a team of employees led by Academician A. A. Makarov performed a number of studies that proved the prospects of using ribonucleases for antitumor therapy.

To create our immunotoxin, we used barnase, a bacterial ribonuclease that is not inhibited in human cells. The design containing two barnase molecules and a mini-antibody specific to the tumor marker HER2 caused the death of cancer cells with such a marker in vitro and effectively suppressed tumor growth in vivo. At the same time, the cytotoxic effect of the drug with the antibody turned out to be about three orders of magnitude (!) higher than the individual barnase.

Pseudomonas toxin A, secreted by Pseudomonas aeruginosa, is considered the champion of toxicity today: 2-3 molecules of this protein are enough to kill a cell. However, such a toxin kills indiscriminately, so when creating a drug, one of the sections of this protein is replaced by an addressing module.

A number of immunotoxins of targeted action against malignant neoplasms carrying the HER2 / neu oncomarker, the presence of which suggests an unfavorable prognosis for the patient, have been created at IBH RAS

In the two immunotoxins we have created, a single-stranded mini-antibody and darpin act as a guiding module. In both cases, very good therapeutic results were obtained: the toxin killed only cancer cells and acted in very low concentrations. The effectiveness of the toxin has been shown not only in cell cultures, but also in experiments on laboratory animals.

Let there be light!

At the beginning of the new century, fluorescent proteins were created that, when irradiated with light, could produce reactive oxygen species that have a detrimental effect on cells, including tumor cells. The first example was Killer Red, a mutant hydroid jellyfish protein obtained at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences (Moscow). By attaching a specific anti-cancer mini-antibody to this protein, we obtained the first fully genetically encoded immunophototoxin that can be produced by genetic engineering.

Another amazing fluorescent protein is mini-SOG, a modified protein of a plant from the cabbage family. With this phototoxin, we also created two designs where either a single-stranded mini-antibody or darpin served as guiding modules. However, when checking the toxic effect of these drugs, strange facts were revealed: a larger design with a mini-antibody turned out to be almost 30 times more effective than with a small darpin. This went against the established canons of photodynamic therapy, according to which small phototoxins work best, which easily penetrate into the cell and into the cell nucleus.

The principle of operation of protein structures containing flavoprotein miniSOG is that when irradiated with blue light miniSOG promotes the formation of reactive oxygen species. The latter literally "burn through" the membrane, causing the death of the target cell, with which the guiding fragment (for example, DARPin) of the drug design binds

Further studies have shown that the design with a mini-antibody is really more effective, since it lingers longer on the surface of the target and literally "burns" holes in the cell membrane, causing its destruction. And the molecule with darpin immediately enters the cytoplasm, where there are many protective mechanisms that repair damage. Here is such a different functional activity with the similarity of the structure!

An example of designing mini-antibodies for targeted delivery of radioisotopes with different binding efficiency to the target cell and different molecular weight

Mini-SOG is ten times more active than Killer Red, but it is not easy to use it for medical purposes. To excite this protein, blue light is required, which does not penetrate well through the skin and other tissues. The problem was solved radically: the light source was delivered directly to the place where the phototoxin should work. For this purpose, a tandem genetic engineering design was created that provides simultaneous biosynthesis of mini-SOG and the NanoLuc luciferase enzyme, which, when a specific substrate is added, triggers the bioluminescence process, and the desired radiation is generated near or directly inside the target cell.

When irradiated with blue light, reactive oxygen species and the familiar riboflavin (vitamin B2) and its derivatives are released. If tumor cells are saturated with this vitamin (and they have the corresponding receptor) and then irradiated, then their degradation can be achieved. But it is possible to do this again either on an open operating field or in a "test tube".

A non-standard solution has been found for this problem. Red light, unlike blue, penetrates very deeply. And there are very interesting inorganic photoluminescent nanoparticles with good biocompatibility – nanophosphores, which, when irradiated with red light, begin to re-emit in the blue part of the spectrum. It remains only to deliver these nanoparticles to the lesion site, which was done together with Professor A.V. Zvyagin and a group of Russian scientists led by Academician V. Ya. Panchenko.

Molecular LEGO

As you know, the future belongs to personalized medicine. And in this sense, it is very important that we learn how to "assemble" the molecular theranostic constructions necessary for a particular patient immediately before use from ready-made blocks. In other words, universalization is needed.

Our favorite pair of barnaza-barstar proteins can serve as such a universal frame module. The already mentioned barnase is a bacterial enzyme, and barstar is its natural inhibitor. These small, highly soluble and stable proteins form a surprisingly strong complex by self-assembly with simple mixing. At the same time, the ends of both proteins remain free, and everything you need can be attached to them: from the addressing mini-antibody to toxins. Important advantages of such a module are the exact (strictly 1:1) the ratio of components, which is normally not present in the cell of higher organisms, and the exceptionally high specificity of interaction, which eliminates the problem of the formation of "wrong" pairs.

The barnaza-Barstar universal structural module is a complex of bacterial proteins: the barnase enzyme and its natural inhibitor barstar. One barnase molecule binds strictly to one barstar molecule. The N- and C-terminal sections of proteins remain free and convenient for modification

There are many options for using this complex. For example, by attaching the addressing part of the antibody to two barnase molecules, and an exotoxin to the barstar, we obtained a well–functioning therapeutic construct against tumor cells with the HER2 marker.

The barnaza-barstar system provides the possibility of two-stage delivery of the agent to the pathological focus. For example, to visualize a tumor, a drug based on a barstar is first injected, coupled with a mini-antibody that recognizes the cancer surface antigen HER2 / neu. Then barnase is introduced, to which the fluorescent protein EGFP is attached. Self-assembly of the barnaza-barstar module occurs only on the surface of cancer cells, which allows you to "see" the pathogenic focus. Such separate delivery can also be used in the case of the use of radioisotopes and other highly toxic compounds to reduce the risk of damage to healthy tissues.

The barnaza-barstar module can serve as the basis of hybrid structures for theranostics with high specificity of interaction with target cells. As a therapeutic agent, toxins of various nature, radioisotopes, etc. can be attached to them.

Both barnase and barstar can be covalently bound not only to proteins, but also to colloidal structures. The interaction force of barnase and barstar is sufficient to combine and hold in a single complex various micro- and nanoparticles of organic and inorganic origin ("quantum dots", magnetic nanoparticles, colloidal gold, nanodiamonds, nanophosphores, polymer nanoparticles). The surface of the nanoparticles themselves with an extremely large specific area can serve as a "platform" for binding to various molecules, which allows them to be equipped with additional functional modules, creating superstructures. This not only expands the possibilities of molecular design, but also allows you to directly influence target cells with the help of external (thermal, optical, electromagnetic, acoustic) effects.

An example of the effectiveness of molecular structures based on nanoparticles: cancer cells labeled with trifunctional nanobioconjugates line up in a magnetic field along a given contour. On the left is a self–assembly diagram of such a bioconjugate based on the barnaza-barstar module. © 2010 Nikitin et al. PNAS

Most of the developments discussed above are potential medicines of the future. However, some of them have already passed preclinical trials, including the already mentioned immunotoxin against breast cancer, more specifically, against a widespread and one of the most malignant types of cancer characterized by the HER2 cancer marker.

The immunotoxin with the help of a mini-antibody recognizes cells carrying HER2, and the enzyme barnase suppresses them. The uniqueness of this drug is that it has the ability to cancel the cytotoxic effect. To do this, just add barstar, a natural barnase inhibitor, and the drug completely loses its cytotoxic activity.

To test this medicine, we once received a grant from the Ministry of Industry and Trade of the Russian Federation. And then came 2008, the economic crisis... Now someone has to pick up this project and promote it further. Clinical trials are needed, and this is a completely different kind of money.

Nanostructures made of magnetic and fluorescent particles constructed at IBH RAS using the barnaza-barstar module. Electron microscopy. Photo from the author's archive

We have repeatedly had emissaries from abroad, offering to buy our developments. These were, as a rule, small traveling salesmen who seek to buy a patent for relatively little money, and then resell it to a large firm. But the latter often buy patents to put them "under the cloth". And I am sure there are many such patents. Large firms spend hundreds of millions on the creation of medicines, including marketing. For example, the introduction of the same herceptin into clinical practice cost tens of millions of dollars. This is a lot of money, and if a company has invested it, then it is determined to recoup it, even if scientists will have more effective developments.

But it's not just that. There are no revolutionary things in the field of oncology: it is impossible to cure everyone of everything and forever. We can make the drug 10% better, 70% better... But cure? No one is cheating, but there is a big and a small truth.

The main problem with cancer is that it often cannot be cured "permanently". Often we see that the tumor disappears, but after a few months it reappears. There may be several mechanisms here. Academician G. I. Abelev once compared cancer to a racing SUV: it accelerates quickly and goes where it wants. Tumor cells divide and adapt quickly. For example, they can drop or "hide" their cancer markers (like airplanes dropping aluminum foil so that radars can't see them). But our HER2 antibody binds primarily to such markers.

Perhaps it's all about cancer stem cells, which give rise to a generation of new pathological cells. We are not engaged in this area yet, but perhaps there will be a revolution in the future. If we can kill not only the tumor itself, but also potential cancer cells – that would be a breakthrough. Everything else, including what we do, is evolution.

Here we can slow down the process, and here the main question is: at what stage, what are the risk factors? They say that the best therapy is early diagnosis. In the West, early–stage breast cancer therapy is the key to 98% of treatment success. I am sure that the sooner you start using our drug, the higher the chances of getting rid of cancer, if not a full recovery, then at least a long remission.

Professor on the rent

I defended my PhD thesis in chemical sciences, my doctoral thesis in biological sciences, and became a member of the faculty in nanobiotechnology, i.e., in scientific terms, I am multifunctional.

And it all started with enzymology, which studies the activity of enzymes. In 1971, while still a student of the Chemistry faculty of Moscow State University, I came to the Institute of Molecular Biology named after V. A. Engelhardt in the laboratory of Academician A. E. Braunstein, who was nominated for the Nobel Prize in 1952 (I think he would have received it if not for the then "Iron Curtain"). At that time, enzymology was the pinnacle of molecular biology, because then the first Nobel Prizes for the discovery of DNA and the basics of heredity had just begun to be awarded. PCR, genetic engineering – all this was not in sight.

After defending my PhD in 1977, I realized that with the departure of the great Braunstein, this field of science would no longer be so "hot". At our institute, they began to study DNA, and at the age of 25 I completely changed the direction of research.

Then there were the works of the Japanese molecular biologist S. Tonegawa, who in 1987 received the Nobel Prize for discovering the mechanism of rearrangement of genes encoding immunoglobulins. I became interested in the genetics of the immune response, and my doctoral thesis, defended in 1990, was already called "Immunoglobulin genes. Structure and rearrangements". I would like to note that we managed to clarify the mechanism of Tonegawa regrouping in some details. Of course, this is a "grain of sand", but still we have made our contribution to understanding the gene mechanism responsible for the diversity of antibodies.

The 1990s are not only a hard hungry time for our country. At this time, genetic engineering continued to develop rapidly. From this "hot area" a technological direction branched off – "antibody engineering", which also began to develop actively. There were two directions that competed with each other to some extent: the creation of full-sized antibodies, which have now firmly entered the market, and the creation of mini-antibodies.

I got a tiny laboratory, where first we dealt with full-sized immunoglobulin molecules. With the help of genetic engineering methods, we managed to change the isotype of these molecules, determined by the structure of heavy chains. But full-size immunoglobulins are expensive, and at that time it was unrealistic to establish such production. That's why we started to deal with small molecules – it was a purely practical choice (in recent years this direction has been actively developing in the world, and I am sure that it is the future).

My son was growing up, he was playing LEGO, and the thought struck me: it would be nice to learn how to create the right structure from blocks synthesized in advance. After all, it's easier and faster to work with ready–made "bricks": one brick is guiding, the second is therapeutic. As a platform, I chose two small molecules that form a solid complex: barnazu and barstar.

The idea is good, but there were no opportunities for its implementation. In the late 1990s, the situation in science became very difficult. By hook or by crook, I got to a conference in San Diego on antibody engineering. There I chose Professor A. Plyuktun from all those who were engaged in such things, who, in my opinion, was "the best of the best". I approached him and offered myself to work together in any capacity (by that time I was already a professor in my homeland).

Plyuktun himself was engaged in the technology of "leucine zippers" – amino acid "fasteners". It is interesting, although a completely different story, how he came up with these "fasteners". As far as I remember from his stories, it was based on a graduate student's mistake in the construction of DNA, which led to the fact that other amino acids formed into a "fastener". Nine out of ten scientists would have passed by, but the great Pluctun realized that it could be used, and developed the technology of "zippers", which he initially suggested that I do. But I wanted to develop my own idea of assembly based on a pair of "barnaza-barstar".

When academician V. A. Engelhardt founded his institute, we did not have a domestic "molecular". Engelhardt said: "Take any work from the American PNAS and repeat it, then you can get your own laboratory." His approach is a great thing: he made it possible to lay the foundation of the institute and achieve success (his second equally successful idea was that a chemist, a physicist and a biologist should work at the same table, but not in one person). Yes, first you need to use someone else's experience, but then – then you need to start something of your own! Of course, you can take any existing successful technology and develop it, create on this basis a new (similar) version of a drug for different forms of cancer, and this is likely to be doomed to success. But it won't be something unique, it won't be a "first time".

..As a result, Plyuktun still invited me to his laboratory and offered me a student scholarship so that I could work in Switzerland. For this I will always be grateful to him. And this concerns not only the opportunity to work in one of the best and well-equipped laboratories in the world, but also many interesting and fruitful discussions that helped me form an adequate view of the problem of bioengineering and my place in this field. By the way, it is appropriate to note that the new breakthrough technology of darpins was created and is successfully developing in the laboratory of Professor Plyuktun. He is indeed, and without any exaggeration, a "classic" of protein engineering.

But let's go back to the late 1990s. For the first time I lived in the hostel of evangelical youth "Oasis". It wasn't bad there, but it was so cold that I secretly had to buy a heater. The shower in the corridor could not be used from 10 pm to 6 am, so as not to wake up other guests. I returned from the laboratory just at this forbidden time and on winter nights, so as not to be heard, I quietly washed under a thin trickle of hot water to somehow warm up. Now it all seems funny and cute...

In the first three months of work, nothing worked out for me. The scholarship was extended for another three months – and it went! I began to divide my life between Moscow and Zurich. I began to receive not 1600 Swiss francs, but 10 thousand. In parallel with his wife and son, he built a house in Moscow, mastered construction specialties and even welding, which I consider no less an achievement than a professorship. It wasn't easy. At some point I realized that, just as peasants used to go to the city for rent, so I go to work in Switzerland. Of course, conditions have changed. I became an honorary visiting professor, I had visa-free entry, and instead of the hostel "Professor Sergei" was waiting for a specially rented comfortable apartment...

All this work ended with an article in Nature Biotechnology (2003), and my trips stopped for family reasons. The idea of the universal platform "barnaza-barstar" has worked and has Russian-Swiss citizenship, confirmed by a common patent. The following article was published in Nature Nanotechnology 11 years later. By that time, talented employees had appeared, and this work had a completely domestic "registration". But that's another story. Between these dates, about two dozen articles were published both from our laboratory and other authors who began to develop this topic.

About the author
Sergey Mikhailovich Deev – Corresponding Member of the Russian Academy of Sciences, Candidate of Chemical Sciences, Doctor of Biological Sciences, Professor, Head of the Laboratory of Molecular Immunology of the Institute of Bioorganic Chemistry named after Academicians M. M. Shemyakin and Yu. A. Ovchinnikov of the Russian Academy of Sciences (Moscow). Laureate of the I. I. Mechnikov Prize (2014), the M. M. Shemyakin Prize (2016). Author and co-author of more than 200 scientific papers and 10 patents.

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