Monoclonal antibodies

How it's done

About one of the most modern and most promising areas in the treatment of cancer Saint-Petersburg.ru Timofey Nemankin, Head of the antibody Development Department of the Russian biotech corporation BIOCAD, told. Yuri Strofilov and Anna Kiktenko understood what was said.

The works that eventually led to the creation of anti-cancer drugs based on monoclonal antibodies were awarded eight Nobel Prizes.

A cancer cell has one unpleasant feature – when a disease occurs, the body does not consider it a stranger and therefore does not include an immune response. The idea was as follows: to mark cancer cells with antibodies so that they begin to be perceived as foreign and "turn on" the body's protective reaction, which will destroy them, as hundreds of infectious agents destroy every day. It's beautiful, isn't it? And how do you like the idea of removing rose-colored glasses from the cells of the immune system and letting them see a stranger among their own?

In these cases, there are practically no side effects, since the body copes with cancer on its own and there is no need for carpet bombing with chemicals. All that remained was to find antibodies that would selectively bind to cancer cells and trigger an immune response.

The general structure of antibodies. The Fab fragment is responsible for specificity,
Fc-fragment – for effector functions.

Antibodies are specific proteins, each of which binds to one specific antigen (target molecule). Antibody libraries created in research laboratories consist of ^10-12 proteins of various properties. A trillion molecules.

A trillion seconds is five hundred human lives in a row, the entire housing stock of Russia consists of less than a trillion bricks, and about a trillion fish swim in the oceans of our planet.

From this multitude of antibodies, it is necessary to select one molecule that will firmly bind to the cancer cell, will be stable, and the technology of its production will not cost astronomical money. While there is no way to calculate such a molecule from scratch, everything is done by selecting natural or artificially created (synthetic) libraries and sorting through options. But computer modeling is already actively used to improve the options found. That is, of all the fish in the ocean, we need to catch one individual that meets the strictest fitness criteria, and, if necessary, create a computer model of it and calculate possible ways to improve it.

onethousandninehundredone Emil Behring. Nobel Prize "for work on serum therapy".

The serums that Bering started developing are called polyclonal – they contain millions of different antibodies, some of which defeat the disease. Such serums have two significant drawbacks: the content of individual antibodies in a polyclonal preparation may vary from one batch to another and polyclonal antibodies cannot be used if it is necessary to distinguish two similar targets, that is, they are not specific. In the animal body, each of the B-lymphocyte cells produces antibodies of only one type (it is a mini-factory for their production, floating in the blood). Therefore, the task is to isolate the antibodies produced by the descendants of one cell. In this case, we will get absolutely identical molecules called monoclonal.

There are several thousand molecular varieties of cancer. It is currently not possible to create and produce thousands of different monoclonal antibodies (over the past 20 years, only a few dozen therapeutic antibodies have been created and produced in the world). This means that it is necessary to create antibodies with the most universal mechanism of action, effectively affecting a wide range of diseases, but minimally affecting healthy cells of the body. Therefore, the process of developing antibodies begins with the selection of targets on cancer cells or cells of the immune system (in the case of ultramodern immuno-oncological drugs) and the desired mechanism of their action. We need to determine which types of cancer we will fight. After the start of the drug development project, antibody libraries are selected (filtered) on selected targets, leaving millions of proteins capable of attaching to the target. Then, out of the millions of antibodies remaining in the postselection library, tens of thousands are analyzed individually, selecting only a few of the best candidates for the role of therapeutic agents.

MEGAN LIB – Monoclonal Antibody Library

The world's largest library of monoclonal antibodies has been created at BIOCAD Corporation. It contains billions of genes of various antibodies. The library contains native antibodies from 2000 donors. Native antibodies are proteins obtained from a healthy human donor, not from an infected animal. The probability of obtaining a drug created on the basis of human antibodies is higher than when using synthesized or humanized antibodies. The library looks like a small test tube with 2 ml of liquid, but it contains all the power of the human immune system.

The search for a candidate molecule looks like this: antibodies are produced in bacterial cells (used as mini-factories in laboratories), the resulting culture liquid is poured into 96-well plastic transparent plates with target molecules (or target cells) pre-fixed in the wells. After a series of reactions, a color reaction occurs in some wells. This means that antibodies of the desired specificity were found in them, that is, they bound to the target. Antibodies from such wells are repeatedly developed and subjected to further tests. The process goes on until the antibodies pass all the necessary tests, during which they are selected by the strength of binding to the target, by specificity (by the absence of binding to unnecessary targets), by the ability to perform the necessary functions, and so on. This is how we obtained candidate molecules for the role of therapeutic monoclonal antibodies. The entire cycle of searching for the right candidates takes several months, depending on the complexity of the target.

1972, Rodney Porter and Gerald Edelman. Nobel Prize "for discoveries concerning the chemical structure of antibodies".

Since the thirties of the XX century, mankind has actively begun to study the structure and properties of antibodies. Antibodies are huge, 150 thousand times larger than a carbon atom. It is impossible to understand their structure without disassembling the molecule into parts. And Rodney Porter did exactly that – split the rabbit antibody molecule into pieces. In 1969, scientists found out the sequence of all 1300 amino acids forming a protein chain. At that time, it was the largest decoded amino acid sequence.

So, we got the right candidate molecules. Now it is necessary to decipher their structure, determine the stability of the molecules and evaluate the manufacturability of their production in mammalian cell cultures. If the obtained antibodies do not meet the requirements, their computer models are created, and a complex self-learning system predicts the ways of their possible improvement. Based on these computational predictions for candidate improvement, de novo synthetic libraries of optimized antibodies are synthesized, and the selection cycle is repeated.

When the antibodies finally meet the strictest requirements, their genes are introduced into the producing cells (usually using cell culture from the ovary of a Chinese hamster). Each cell gets the ability to produce only one type of antibody. Next, each of the cells that have thus become a biofactory for the production of monoclonal antibodies is placed in separate wells with a volume of 2 ml and the one that is capable of producing the maximum amount is selected. In wells with a volume of 2 ml, you can accumulate several micrograms of antibodies. And to produce a drug for a global scale, we need kilograms. Therefore, the process of scaling begins – gradually bringing the volume of culture to several tons without loss of productivity. In addition, when developing antibodies, it is always mandatory to check their safety and effectiveness in animals and in clinical trials.

In order to be able to produce antibodies in a ton bioreactor, world science has passed a long and thorny path, overcoming obstacles laid down by nature. In nature, recall, antibodies are produced by lymphocytes. Lymphocytes divide a limited number of times. Leonard Haiflick proved that after about 50 divisions, cells die without leaving offspring, exhausting the potential of chromosome division. Dead end. We need a lot of descendants of one cell to develop industrially significant amounts of antibodies, and almost all cells stop dividing, reaching the Highflick limit. Wait, what does "almost" mean? Cancer, germ and stem cells do not have a Highflick limit, they divide indefinitely.

1984. Milstein, Koehler, Erne. Nobel Prize "for the discovery of the principle of production of monoclonal antibodies".

In the seventies of the XX century, the Argentine scientist Cesar Milstein and the German biologist Georges Koehler proposed a beautiful solution to the problem of the production of large volumes of cells. It is necessary to create a hybrid of a lymphocyte and a myeloma cancer cell. Such a cell will live forever, like a cancer cell, and produce antibodies, like a lymphocyte. Such a cell was called a hybridoma. All her descendants will produce the same antibodies and there will be a lot of them.

The first monoclonal antibodies were obtained from lymphocyte cells of mice immunized (vaccinated) with a target protein. However, mouse antibodies are poorly suited for use in clinical practice – they cause an immune response of the human body and are quickly eliminated before reaching the target. Therefore, genetic engineering came to the aid of scientists. It was necessary to replace parts of animal DNA with parts of human DNA so that the cell produces antibodies not of an animal, but of a human. Oh, how! It was necessary to intervene in the providence of God and create a new (recombinant) molecule, and also learn how to multiply mammalian cells in culture.

1980. Frederick Sanger, Walter Gilbert. Nobel Prize "for the method of DNA sequencing".

In the mid-seventies of the XX century, Frederick Sanger, the only winner of two Nobel Prizes in chemistry, unwound the double helix of DNA and, attaching short pieces ending in certain sequences to it, counted the entire chain of nucleotides. All first-generation sequencing machines are built on the principle of the Sanger method. As a result, we can recognize the sequence of nucleotides in the genes we need. A new generation of sequencers came to the aid of classical sequencers at the end of the XX century: now DNA decoding is routine laboratory work. In 2003, the Human Genome project, launched in 1990 by the US Department of Energy and the National Institutes of Health, was completed. He helped to establish the structure of human DNA.

In order to modify a gene, it needs to be cut in the right place and sew a new sequence of nucleotides into it. In which place to cut, we know thanks to the works of Sanger, about whom we have already told. What to insert is also clear, since the human genome has been decoded. It remains to do this technically. If all parts of the antibodies are replaced with human ones, then humanized antibodies are obtained, and if only small pieces, then chimeric ones.

1978. Smith, Arber, Nathans. Nobel Prize for "the discovery of restriction enzymes".

In the sixties of the XX century Hamilton Smith and Werner Arber found out the mechanism of introduction of bacteriophage DNA into bacterial DNA. When a bacteriophage penetrates a bacterial cell, it can be embedded in the genetic structure of a bacterial cell and transmitted to daughter cells during division. The study of this mechanism led scientists to the discovery of restrictases ("molecular scissors") – enzymes that can cut DNA in strictly defined places. If there is an enzyme that cuts, then there must be an enzyme that glues. Soon it was found and named ligase.

Now we are able to replace the genes responsible for the synthesis of animal antibodies with similar human genes. Advances in understanding the work of various sections of DNA and a giant leap in mathematical modeling make it possible to improve antibodies. Let's say the antibodies bond perfectly with the target cell, but are not stable enough. No problem, computer modeling can calculate a similar molecule that will not only bond better, but also become more stable than the prototype. It remains a little bit – to synthesize the necessary sequences.

1968. Holly, Korana, Nirenberg. Nobel Prize "for deciphering the genetic code and its role in protein synthesis".

In the sixties of the XX century, the Indian biophysicist Khar Koran, in the course of work on deciphering the genetic code, came up with a method of gene synthesis. In fact, the Koran has learned to glue nucleotides in the right sequence. In 1976, two small full-sized genes were synthesized and cloned for the first time, and in 1977, a gene encoding the human protein somatostatin was synthesized and cloned.

1993. Carey Mullis. Nobel Prize "for the invention of the polymerase chain reaction method".

In 1983, American biochemist Cary Mullis invented the polymerase chain reaction. DNA is created by copying different pieces of source code with specified conditions into a new structure. The PCR reaction allows the routine laboratory method to synthesize very long sequences.

Now we have everything. We can synthesize the human genes we need, we can insert them into animal cells, we can get mammalian cells infinitely dividing in bioreactors and develop the right amount of antibodies with precisely defined properties. However, before it had time to develop, the hybrid technology had become obsolete. Modern methods of creating proteins look like this: a gene obtained in a test tube is implanted into an infinitely dividing mammalian cell.

onethousandninehundredfiftyfour Enders, Weller, Robbins. Nobel Prize "for the discovery of the ability of the polio virus to grow in cultures of various tissues."

In the middle of the XX century, more than 20 thousand people fell ill with polio in the USA alone. US President Franklin Delano Roosevelt also suffered from this disease. The polio virus, as it was believed at that time, multiplies only in the nervous tissue, which makes it impossible to produce any significant amounts of vaccine from weakened viruses. Maintaining a culture of nerve cells was extremely difficult. It was necessary to find a way to cultivate. Enders, Weller and Robbins found conditions under which poliovirus multiplied in the cell culture of human and monkey embryos. Thus, it was possible to obtain large amounts of poliovirus "in vitro".

Now humanity had a way of cultivating cells outside the body. In 1952, a well-known line of immortal human cells was obtained by NeLa.

The cells are cloned in small wells with a volume of 2 ml, then the contents are poured into cones, then into reactors. Cells divide in a solution of a very complex composition: 200 components, several gases, strictly maintained temperature, absolute sterility, controlled mixing.

Cells are cloned and cloned to volumes of one ton. After that, the producing cells are removed from the solution, the solution in which the cloning was performed is replaced with saline with special osmotic filters, complex purification of the finished protein is performed. The final product is two hundred kilograms of serum of monoclonal antibodies, which are poured into syringes. 200 kilograms is Russia's annual need for such a drug.

Several classes of medications are made on the basis of monoclonal antibodies. Firstly, for the treatment of cancer. We can mark a cancer cell with an antibody and the body will kill it with an immune response. Secondly, we can combine antibodies with a radioactive component, obtaining a method of high-precision diagnostics (we remember that the antibodies obtained are very specific and will deliver the label only to "their" cancer cell). Or we can kill the cell with a toxin attached to the antibody without touching the neighboring ones. Thirdly, we can remove the recognition block produced by the cancer cell, and then such cancer cells will be recognized and destroyed by the immune system. In addition, we can reduce the autoimmune response by blocking signaling molecules. In this case, we will receive drugs against psoriasis and arthritis.

Biotech Corporation "BIOCAD"
BIOCAD consists of two factories, two research centers, a department based on the Chemical and Pharmaceutical Academy, 1,000 employees, 350 of whom are engaged in the development of new drugs, and a turnover of 10 billion rubles in 2015. The company's offices and representative offices are located in the USA, Brazil, China, India, Singapore and other countries. The drugs are intended for the treatment of cancer, HIV, hepatitis, multiple sclerosis.
In the early 2000s, banker Dmitry Morozov became bored. I wanted to make money on something other than a hole in the ground. The myth of numerous Russian scientific developments turned out to be a myth, I had to do the whole chain myself, from the development of molecules to the packaging of finished drugs. It turned Out To Be A Corporation.

Portal "Eternal youth" http://vechnayamolodost.ru  12.04.2016