Chimera against cancer

How leukocytes with chimeric receptors helped to achieve real success in cancer treatment

Oleg Lischuk, N+1

Medicine has begun to achieve real success in one of its most difficult areas – cancer treatment. Recently, the results of several clinical studies have been published at once, in which it was possible to achieve a complete cure of patients with malignant neoplasms. For example, the staff of the Fred Hutchinson Cancer Research Center in Seattle reported that during the experiment they managed to cure more than half of patients with leukemia, and some of them completely disappeared malignant B lymphocytes (Dramatic remissions seen in immunotherapy trial of blood cancer patients - VM). The technology of chimeric antigenic receptors helped to achieve such results. Read about what it is and whether cancer has finally become a curable disease in our partner material with the biotech company BIOCAD.

So, can we talk about a real breakthrough in cancer treatment?

The test results are impressive, but in order to achieve them, it took many years of work by research teams around the world. The idea of creating chimeric antigen receptors (CAR, from the English chimeric antigen receptor) belongs to chemist and immunologist Zelig Eskhar from the Weizmann Institute of Sciences in Israeli Rehovot. In his laboratory in 1989, the first transgenic T-lymphocytes with these receptors were obtained.

They were first used in small clinical trials for ovarian cancer already in 1996, however, with very modest results. The improvement of CAR technology has been going on for decades, and the number of laboratory, preclinical and clinical trials is currently growing exponentially. As of the end of 2014, 91 clinical trials using CAR-lymphocytes were registered in the world, 58 of them for the treatment of various types of blood cancer.

As can be seen from the news, several research teams have already managed to achieve success in the early stages of testing. The growing interest in CAR technology allows us to expect that there will be more and more such news.

What is the essence of such treatment?

The main feature of treatment with transgenic T-lymphocytes with CAR is its specificity. The chimeric receptor allows the lymphocyte to accurately recognize only the tumor cells to be destroyed. But just recognizing them is not enough, the lymphocyte must be activated in order to attack the target cell. To do this, CAR contains a fragment that activates a T-lymphocyte upon contact with a given cell type.

What are chimeric receptors? How does it even work?

To understand how transgenic lymphocytes work, you need to say a few words about ordinary ones. A natural T-lymphocyte has receptor complexes on the surface, which consist of the T-cell receptor itself (TCR) associated with the CD3 signaling module (it consists of three parts: CD3δε, CD3γε and cd3ζζ, aka CD247). All cells of the body have proteins of the main histocompatibility complex (MHC) on the surface, which "demonstrate" fragments of their proteins (antigens) to immune cells. TCR recognizes these antigens, and if they are foreign to the body, activates the lymphocyte via CD3. An activated lymphocyte kills an infected cell, or releases cytokines that attract other immune cells.

T-lymphocytes with normal (left) and chimeric (right) receptors
and their interaction with target cells (Srivastava, Riddell, Trends in Immunology, 2015)

Transgenic lymphocytes synthesize artificially created CAR instead of the usual receptor complexes. They are called chimeric because they are "assembled" into a single molecule from modules of different origin. In the simplest case, their set is as follows.

The extracellular antigen-recognizing domain is a single-stranded mutable fragment of a monoclonal antibody (scFv). It is responsible for binding to a specific antigen on the surface of target cells.

Flexible hinge area (the G1 immunoglobulin hinge is most often used). It is necessary to deflect the antigen-recognizing fragment to the sides to facilitate its binding to the antigen.

The transmembrane site serves to fix the CAR on the lymphocyte membrane and contact the extracellular and intracellular parts of the receptor. As a rule, it is part of the intracellular domain.

The intracellular signaling domain is taken from the T-receptor complex – this is one of the CD247 (cd3ζ) chains. Its function is to activate the lymphocyte when the antigen–recognizing fragment binds to the antigen on the surface of the desired cell.

All these modules are assembled into one amino acid sequence, that is, they are encoded by one gene. This receptor design makes it possible to purposefully activate cytotoxic lymphocytes upon contact with target cells (for example, tumor cells), does not depend on MHC and can recognize any antigens (not only peptide ones).

If this is the simplest case, then what are the complex ones?

The so-called first-generation cars have the described structure. In many cases, CD3ζ alone is not enough to activate a transgenic lymphocyte. To do this, a co-stimulating factor is added to the intracellular domain of the chimeric receptor. It can be a receptor that induces the production of interleukins, representatives of the superfamily of receptors for tumor necrosis factor, ensuring the proliferation and survival of T-lymphocytes and their production of cytokines, and others. Co-activator receptors belong to the second generation CAR. Third-generation cars contain several co-activators simultaneously.

The structure of cars of different generations (Dmitry Dzhagarov / Wikimedia Commons)

Such receptors provide more effective and stable activation of lymphocytes, enhance and prolong their cytotoxic effect, and therefore produce a more pronounced therapeutic effect.

In addition, additional transgenes can be introduced into lymphocytes, in addition to coding CAR, to give them additional properties. For example, in the work of the Fred Hutchinson Center for the treatment of various forms of B-cell leukemia, T-lymphocytes were used, in which, in addition to CAR, recognizing the universal antigen of B-lymphocytes, a gene of a shortened form of the human epidermal growth factor receptor (EGFRt) was expressed, deprived of the signaling intracellular domain, but retained the binding region of the therapeutic antibody –cetuximab. This modification makes it easy to sort and concentrate modified CAR-lymphocytes at the stage of preparation of a cellular drug for infusion, and, if necessary, will allow CAR-T to be removed from the patient's body quickly and specifically in case of side effects of cell therapy.

And where do these cells come from? Are they modified directly in the blood?

Of course not. A blood sample is taken from the patient and the necessary population of cytotoxic T-lymphocytes is isolated from it using sorter devices. They are cultured on a nutrient medium, after which a neutralized virus (vector) containing the CAR gene, which is specific to the patient's tumor cells, is added to it. In rare cases, other methods of gene delivery are used. The resulting cells expressing CAR undergo further cultivation, after which they are "turned on" with a set of cytokines and injected into the patient. Such individually produced ex vivo CAR-T lymphocytes are called autologous. They are used in current clinical trials.

Currently, work is underway to obtain allogeneic cells. These are the same T-lymphocytes, but taken from a healthy donor. They turn off their own T-cell receptor, which reduces the risk of their attack on the patient's cells carrying MHC molecules, to which this receptor may be specific. Before the procedure, the patient is given lymphodepletion – the elimination of his own cells that can reduce the activity of CAR-T-lymphocytes. It helps to temporarily get rid of the risk of ineffective therapy due to the attack of donor cells by the host immune system. This approach makes it possible not to make cells individually for each patient, but to have a ready-made cell preparation, which is much easier and more convenient.

For the introduction of transgenes into both auto- and allogeneic cells, in the vast majority of cases, lentiviruses devoid of virulence are used, which include, for example, HIV. They are convenient because they have a large capacity (CAR genes are very large), are able to embed transgenes into the DNA of cells, work well ex vivo and provide long-term expression of CAR. In addition, they are relatively simple to create, produce and clean.

Lentiviruses? HIV? Is it probably dangerous?

Any method of treatment is associated with risk, and the task of developers is to minimize it. When creating a vector, the virus is modified so that it cannot cause disease. Lymphocytes are treated with it outside the body, and when they are injected into the patient, the transgen is already embedded in the DNA. It cannot serve for the synthesis of viral proteins and nucleic acids, that is, for the production of viral particles capable of infecting other cells.

Is CAR treatment really completely harmless?

Based on the results of the conducted and ongoing clinical trials, it can be said that CAR-T-lymphocyte therapy is generally well tolerated. However, it was not without serious side effects, the main of which are cytokine release syndrome and a non–targeted effect.

Cytokine release syndrome ("cytokine storm") is the simultaneous release of a large number of immune mediators in response to the introduction of transgenic cells. It is manifested by a sharp increase in temperature, chills, vomiting and diarrhea. This condition requires intensive therapy, but in a clinic, as a rule, it is curable.

The non-targeted effect is the attack of CAR-T lymphocytes on healthy cells. It occurs due to the fact that tumor antigens can be expressed to one degree or another by other tissues of the body. To overcome this effect, several approaches have been developed, which were described by Natalia Belozerova, head of the BIOCAD Gene Therapy laboratory.

One of them is the use of multi-specific CAR cells. They express not one chimeric receptor, but two or more that bind to different tumor antigens. Activation of a lymphocyte occurs only if the target cell has a complete set of such antigens. The opposite is also possible – if the antigen of the main CAR is present in both the tumor and in any kind of healthy cells, an additional receptor (in this case it is called an inhibitory one) recognizes the protein marker of normal tissue and blocks the activation of the lymphocyte.

Another technology for reducing non–targeted cytotoxicity is the creation of CAR, the activation of which leads not to its direct attack on the cell, but to the expression of transgenic cytokines that attract various immune cells of the body to the tumor tissue.

It is also possible to reduce the risk of CAR-T-lymphocyte treatment by embedding an expression regulator (promoter) in the receptor gene, which is activated only in the presence of a certain low-molecular substance (most often a drug) or optical radiation with a given wavelength.

In addition, there are technologies for "emergency shutdown" of transgenic lymphocytes in case of side effects. For example, the introduction of an additional transgene expressing the protein of apoptosis – caspase, which causes cell death when a low-molecular activator is administered to a patient.

Other technologies are also being developed to increase the effectiveness and reduce the risk of CAR-T-lymphocyte therapy.

And what, in the future, any malignant tumor can be cured with such cells?

Theoretically, CAR technology can be used to treat any form of cancer. The main task is to find highly specific targets, that is, antigens inherent only in a given type of tumor cells. The search for such targets continues constantly.

Currently, most of the CAR is being created and tested for the treatment of various forms of blood cancer. This is due to the fact that leukocytes have a relatively small number of highly specific antigens, that is, it is easier to develop an effective and safe chimeric receptor for them. B-lymphocytes, for example, express different markers at different stages of development, which makes therapy even more targeted.

With various types of cancer of other tissues, it is more difficult – they have more antigens, and many of them are present on healthy cells. In addition, such tumors are often heterogeneous in antigenic composition due to the high level of cell mutations. Nevertheless, cars are being created for them (melanomas, breast, lung, brain and other tumors), and many of them are already undergoing preclinical and clinical trials.

According to Alexander Karabelsky, head of the Advanced research department of the biopharmaceutical company BIOCAD, the treatment of non–hematological tumors is likely to be combined - in addition to CAR, at least additional transgenes or antibodies will be required.

Well, the transgenic lymphocytes destroyed the tumor, and what will they do next?

The same as ordinary lymphocytes – to be in the body and wait in the wings. Some of them, of course, will eventually die, but, judging by current observations, they are found in patients decades after administration. And while they are there, a person will no longer get sick with the form of cancer from which he was cured.

Great, and when will every hospital be treated with CAR?

Unfortunately, not soon. The technology, although rapidly developing, is still at the stage of early experiments and needs further improvement with subsequent long-term tests.

We should not forget that CAR technology is protected by a number of patents. Moreover, they are compiled in such a way that it is not very clear how it will be possible to sell developments based on it at all. As Alexander Karabelsky noted, it is likely that many large biopharmaceutical companies will challenge the validity of such a wide range of patents in court.

It is also important that while the use of CAR is very expensive. Treatment of one patient with autologous cells costs more than 100 thousand dollars. Of course, with the development of technology and an increase in the scale of its application, it will become cheaper, but it will definitely not become widely available in the near future.

Portal "Eternal youth" http://vechnayamolodost.ru  20.04.2016