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ВЕСТНИК РОССИЙСКОГО научного центра РЕНТГЕНОРАДИОЛОГИИ (ВЕСТНИК РНЦРР), 2023, Т. 2023, № 4
ОБЗОР
Использование мРНК-векторов для CAR-T терапии in vivo
Т.М. Кулинич 1, В.К. Боженко 1, Р. Ранджит 2'3, А.Д. Каприн 2,4
1 ФГБУ «Российский научный центр рентгенорадиологии» Минздрава России, Россия, 117997, г. Москва, ул. Профсоюзная, д. 86
2 ФГАОУ Российский университет дружбы народов (РУДН), Россия, 117198, г. Москва, ул. Миклухо-Маклая, д. 6
3 ГБУЗ города Москвы "Городская клиническая онкологическая больница №1 Департамента здравоохранения города Москвы", Россия, 117151, г. Москва, Загородное шоссе, д. 18А
4 ФГБУ МНИО им. П.А. Герцена - филиал ФГБУ «МНИЦ Радиологии» Минздрава России, Россия, 125284, г. Москва, 2-й Боткинский проезд, д. 3
Для цитирования: Кулинич Т.М., Боженко В.К., Ранджит Р., Каприн А.Д. Использование мРНК-векторов для CAR-T терапии in vivo. Вестник Российского научного центра рентгенорадиологии. 2023; 2023(4):31-44. EDN: wvxtzr
Адрес для корреспонденции: Владимир Константинович Боженко, [email protected]
Статья поступила в редакцию 27.11.2023; одобрена после рецензирования 06.12.2023; принята к публикации 11.12.2023.
Терапия CAR-T-лимфоцитами произвела революцию в иммунотерапии рака, поскольку генетически модифицированные Т-клетки позволили распознавать необходимые опухолевые антигены. Однако генетически модифицированные Т-клетки атакуют не только раковые клетки, но и другие физиологически функционирующие клетки, которые экспрессируют аналогичный антиген на своей клеточной поверхности. Еще одним недостатком терапии CAR-T-лимфоцитами является связанная с этим стоимость, поскольку массовое производство препарата невозможно из-за необходимости генетически модифицировать Т-клетки пациента. Чтобы решить эти проблемы, можно использовать мРНК для доставки генетического материала в Т-лимфоциты. Поскольку мРНК временно экспрессирует свой генетический материал, побочные эффекты можно контролировать, регулируя количество вводимого препарата. Кроме того, процесс производства не требует использования Т-клеток пациента, а значит, препарат можно производить массово, что снижает его стоимость.
Ключевые слова: САЯ-Т, Т-лимфоциты, мРНК, опухоли, иммунотерапия
The application of mRNA vectors for CART therapy in vivo T.M. Kulinich V.K. Bozhenko 1, R. Ranjit 2'3, A.D. Kaprin 2'4
1 Russian Scientific Center of Roentgenoradiology (RSCRR), 86 Profsoyuznaya St., Moscow, 117997, Russia
2 Рeoples' Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St., Moscow,
117198, Russia
Резюме
3 State Budgetary Healthcare Center "Municipal Clinical Hospital Number 1" of the Healthcare Department of Moscow City, 18A Zagorodnoye highway, Moscow, 117151, Russia
4 P.A. Hertzen Moscow Oncology Research Institute - branch of the National Medical Research Radiological Center, b. 3, 2nd Botkinsky proezd, Moscow, 125284, Russia
For Citation: Kulinich T.M., Bozhenko V.K., Ranjit R., Kaprin A.D. Utilization of mRNA vectors for CAR-T therapy in vivo. Vestnik of the Russian Scientific Center of Roentgenoradiology. 2023; 2023(4):31-44. (In Russ.). EDN: wvxtzr
Address for correspondence: Vladimir K. Bozhenko, [email protected]
The article was submitted on November 27, 2023; approved after reviewing on December 06, 2023; accepted for publication on December 11, 2023.
Summary
CAR-T-lymphocyte therapy has revolutionized cancer immunotherapy because genetically modified T cells have made it possible to recognize the necessary tumor antigens. However, genetically modified T cells attack not only cancer cells but also other physiologically functioning cells that express a similar antigen on their cell surface. Another disadvantage of CAR-T-lymphocyte therapy is the associated cost, as mass production of the drug is not possible due to the need to genetically modify the patient's T cells. To address these problems, mRNA can be used to deliver genetic material to T lymphocytes. Since mRNA temporarily expresses its genetic material, side effects can be controlled by adjusting the amount of drug administered. In addition, the manufacturing process does not require the use of the patient's T cells, which means that the drug can be mass produced, reducing its cost.
Keywords: CAR-T, T-lymphocytes, mRNA, tumours, immunotherapy
Introduction
Cancer immunotherapy is the process of stimulating the immune system to enhance its natural ability to treat cancer. It has long been known that cytotoxic innate and adaptive immune cells play a critical role in controlling tumor growth and development [1]. One of the emerging fields in immuno-oncology is related to T cells and their ability to exert antigen-directed cytotoxicity [2]. In particular, chimeric antigen receptor (CAR) T-cell therapy exploits the fact that cancer cells often have tumor antigens, molecules on their surface that can be recognized by the immune system's antibody proteins by binding to them. This therapy has made it possible to take advantage of the exclusive properties of T cells by artificially targeting specific cancer receptors [3].
History
The story begins with Eva Klein's experiment in 1960, which showed that immune cells can kill cancer in mice. For this reason, she is considered the founder of cancer immunology [4]. But until then, the cells responsible for immunity were unknown. It was not until the following year, 1961, that Jacques Miller discovered lymphocytes and the function of the thymus in the immune system [5]. Later, in 1973, the first bone marrow transplant was performed and it was found that the donor's T cells killed the recipient's cancer cells - this is considered the first step towards successful cancer immunotherapy. Another major advance was made in 1986, when tumor-infiltrating lymphocytes were removed from a tumor, expanded in the laboratory, and then returned to the patient in large numbers. The therapeutic potency of this therapy was found to be 50-100 times more than that of lymphokine-activated killer (LAK) [6]. And, finally, in 1993, Eshhar et al. successfully combined the cytotoxic potential of a T cell
with the specific targeting of an antibody in a single gene transfer. This marks the new era to developing CAR-T therapy [7].
Different methods of transfection
Prior to mRNA transfection, there were many methods to introduce genetic materials into the T-cells. They can be classified as viral and non-viral delivery system (Tabl. 1).
Tabl. 1. Classification of transfection methods
Viral Non-viral
Adenovirus-associated viruses physical technique chemical technique
lentivirus electroporation calcium phosphate
adenovirus needle injection or micro injection DEAE Dextran
bacteriophage laser irradiation or optical transfection lipofection (lipid-mediated or liposome transfection)
gene guns or biolistic transfection
Viral vectors have long been used to deliver genetic material due to their high transfection efficacy, but they have a high probability of suffering from immunogenicity and cellular toxicity [8]. Lentiviral gene transfer is a versatile and powerful method for genetic transduction of many cell lines and primary cells, including "hard-to-transfect" cells. Lentiviral vectors can carry constructs up to 10 kB in size, transduce non-dividing cells, and provide stable transgene expression by integrating into the human genome. However, lentiviral vectors have a low efficiency of gene transfer compared to other viral vectors. Among all viruses, adenovirus-associated viruses have the lowest toxicity and has the advantage of a strong and transient induction of expression of the gene of interest in a wide variety of cell types and organs [9], but it is a time-consuming method and expensive, and its use is limited by its limited capacity to transfer genetic material of about 5.0 kb [10].
The use of bacteriophages is another viral transfection method. In particular, filamentous M13 bacteriophages are being developed as a new type of vector for safe and targeted systemic delivery of transgenes for in vivo applications [11-14]. They have a number of advantages over the use of traditional viral and non-viral vectors; first, their protein coat consists of a repeating protein unit arranged in an alpha-helical array. This gives the phage unlimited DNA packaging capacity, as the capsid coat only needs to stretch to accommodate the transgene. As a result, the bacteriophage is efficient at condensing and packaging DNA. Second, the protein coat has a high tolerance for mutations, allowing easy introduction of peptide ligands to achieve ligand-directed transduction of the desired cell type. In addition, they are safe, as they have long been used in humans to treat bacterial infections and are approved by the Food and Drug Administration (FDA-USA) for use in food preparations. Finally, phage vectors are easy to produce at high titers and low cost, which is highly desirable for large-scale industrial processes[15,16].
Physical methods include electroporation, needle injection, optical transfection, and biolistic transfection. Microinjection is a technique that uses a fine needle to inject nucleic acids directly into the cell nucleus. The advantages of microinjection are precision in dosage and timing of delivery, high transduction efficiency, and low cytotoxicity. However, manual microinjection is labor-intensive and time-consuming, which limits its application to large numbers of cells in a sample [17].
Optical transfection is a technique that uses laser beams to create transient holes in the cell membrane, allowing nucleic acids to enter [18]. It is highly compatible with standard microscopy optics; the most focused point of the laser is aligned with the image plane, allowing the operator to easily observe the cell during transfection. It is a non-contact and aseptic method. The cell culture configuration can therefore remain 'closed' to the external environment during dosing. In contrast, it is challenging to maintain an aseptic environment on a microscopy platform with an "open-top" tissue culture configuration; this configuration is often required for competing techniques that use micromanipulator arms, such as capillary microinjection or single-cell electroporation [19].
The next physical method is biolistic transfection. This is a technique that uses a device that shoots microscopic gold particles coated with nucleic acids into cells [20]. It was originally developed as a method for gene transfer into plants, as it allows transfer across cell walls [21,22], but it is now being recognized as a technique that is much more broadly applicable, and because it can be used for in vivo as well as in vitro transfections, it has tremendous potential for gene therapy [23,24]. It has the advantage of being able to overcome physical barriers (e.g. the stratum corneum of the epidermis), it can be used multiple times on the same sample, it is suitable for co-transfection of two or more DNAs in a single shot, it can be used on large numbers of cells, and it is fast and easy to use [25,26]. The major advantage is that it is highly efficient; a recent study in rat brain cultures showed that this method was 160-fold, 189-fold and 450-fold more efficient than lipofection, electroporation and calcium phosphate precipitation, respectively, in assaying luciferase activity [27]. The main drawback is that the gene gun itself is expensive to purchase, although the cost of consumables is relatively low.
Among the physical techniques, electroporation is the most widely used. In this method, electrical pulses are used to form cell membranes that are transiently permeable for the entry of genes into the cell membrane [28-30]. One of the main advantages of the physical technique is that it has low immunogenicity, since no biological element is used during the procedure [31]. In addition, it is versatile and can be used for all cell types and for transfection of DNA, RNA, mRNA, RNPs or proteins [32]. It is also cost-effective and reduces development costs and time compared to viral-based delivery methods [33].
Chemical transfection techniques include calcium phosphate transfection, DEAE-Dextran transfection, and lipofection.
Phosphate transfection is a method in which the DNA construct is mixed with calcium chloride in a buffered saline/phosphate solution. Since nucleic acids such as DNA and RNA are also negatively charged, they repel each other, inhibiting their uptake by the cell. One way to overcome this challenge is to use positively charged carrier molecules to deliver negatively charged substrates close enough to the cell membrane to be internalized via endocytosis. Calcium phosphate transfection is one of the chemical transfection methods using this principle. When the DNA construct is mixed with calcium chloride in a buffered saline/phosphate solution and incubated at room temperature, DNA-calcium phosphate coprecipitates are formed. Because these coprecipitates can adhere to the plasma membrane, they are thought to facilitate endocytosis. Unfortunately, this method can be toxic to cells (especially primary cells) and transfection efficiency is relatively low compared to most other methods. In addition, the reaction used to generate the coprecipitates is sensitive to slight changes in pH, temperature, and buffer salt concentrations, leading to unreliable results [34].
Another method of chemical transfection is DEAE-Dextran transfection. DEAE-dextran is a polycationic derivative of dextran (a carbohydrate polymer). When mixed with DNA, the resulting complex is brought into contact with the negatively charged plasma membrane by the polymer's excess positive charge. As with calcium phosphate, this proximity to the membrane is thought to facilitate endocytosis into the cell. The major advantages of this technique are its relative simplicity and speed, limited cost, and remarkably reproducible inter- and intraexperimental transfection efficiencies. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes
in cells. In addition, the serum concentration in the culture medium must be temporarily reduced during transfection [35].
Lipofection is the most commonly used chemical transfection method. The approach involves combining cationic lipids with other molecules to form unilamellar liposomal vesicles that carry a positive charge. The exact molecular mixture has varied over time as lipofection methods have improved. Regardless of the composition of the vesicles, lipofection is designed to package negatively charged molecules such as nucleic acids in a positively charged vesicle so that they can get closer to the cell membrane (where they are presumably taken up by endocytosis). Therefore, you need to mix your construct or molecule of interest with the vesicle mixtures before transfecting your cells. This approach can be successfully used to transfect a wide range of cell types at relatively low cost (although cost and reaction conditions may increase if other polymers and antibody conjugates are added). In addition, lipofection can transfect cells with DNA of any size to achieve both stable and transient transfection, as well as deliver RNA and proteins into cells. The main drawback of the technique is the low transfection efficiency achieved when used for primary cell transfection, stem cell transfection, and when working with suspension cell lines (mainly due to the fact that it can be cytotoxic and relies on cell division for success) [36].
Among all transfection methods, studies have shown that transfection of immune cells in particular is more challenging than transfection of most other primary mammalian cells [37]. The low transfection efficiency in monocytes/macrophages can be attributed to the following reasons. First, there is a very limited chance for pDNA to freely enter the nucleus due to nuclear envelope breakdown during mitosis, as macrophages do not or hardly proliferate [38,39]. Second, these immune cells are equipped with pattern recognition receptors that can recognize nucleic acids as potential foreign and dangerous viral invaders and initiate the inflammatory signaling cascade leading to pDNA degeneration or macrophage apoptosis [40]. Therefore, finding a robust transfection approach to address these issues is highly desirable.
Transfection of mRNA is a promising alternative to pDNA or viral vectors for achieving target protein expression, especially in non-proliferative cells such as primary human cells [41,42]. An advantage of mRNA transfection is that there is no need for the mRNA to enter the nucleus, nor is there a possibility of integration into the host genomic DNA [43]. Thus, this method can be a suitable alternative for transfection of non-proliferative cells [44], including cells of the immune system. In addition, it avoids genotoxicity issues associated with chromosomal insertion of DNA vectors in clinical gene transfer applications. Unlike most pDNA transfection and viral transduction protocols, mRNA transfection results in transient, unstable gene expression. However, transient expression is advantageous for several hit-and-run applications, including current differentiation protocols [45,46].
Structure of car-t cells
These are the recombinants of different parts of the several receptors. Its major components are CD3 Z of T-cell receptor (TCR) and single-chain variable fragment (scFv) of antibody variable heavy (VH) and variable light (VL) chains, fused by a peptide linker [47] (Fig. 1).
A A
Single chain variable fragment
Antibody
CAR
Fig. 1. Structure of CAR-T cells.
As shown in Fig. 1, the extracellular antigen-identifying domain consists of fragments of monoclonal antibodies (variable zones of light and heavy chains linked to a single chain) that are programmed to identify a specific protein on the cell membrane of cancer cells (e.g., CA-125 for ovarian cancer [48]). This part of the CAR is responsible for recognizing and binding specific tumor-related antigens independent of major histocompatibility complex (MHC) molecules [49,50]. Next, there is a linker protein that crosses the cell membrane and connects the extracellular domain to the intracellular domain. The linker protein is usually derived from CD8, CD3-Z, CD4, 0X40, and H2-Kb [51]. Finally, there is an intracellular domain that consists of a signal transduction component of a T cell receptor (TCR) that directs the signal to the TCR and triggers CAR T cell activation and function [52,53].
Depending on the construction of the intracellular domain, CARs can be classified into 4 different generations. The first generation of CARs was developed in Israel in 1989 and contained only the TCR CD3 Z chain as an intracellular domain [54]. The main drawback of this generation was that it didn't often respond to antigen stimulation. Therefore, the main difference between the first-generation CARs and others is that the first-generation CARs could not promote CAR T-cell expansion in vivo after reinfusion. On the other hand, subsequent generations contain additional co-stimulatory intracellular domains besides the TCR CD3 Z chain, which allowed them to enhance the potential of CAR T cells to grow, expand, and ultimately persist in the patient's body [55-57]. To solve this problem, researchers developed the second generation of CARs, where they added an additional co-stimulatory molecule, usually CD28/4-1BB (CD137)/OX40, into the endo-domain with the pre-existing TCR CD3 Z-chain.The new modification increased the CAR T response to antigen, but inadvertently also increased cell apoptosis [58]. To prevent unanticipated apoptosis of the CAR-T after stimulation, the third generation of CARs was developed that contained a molecule such as CD137 (41BB) as a "survival" factor (Fig. 2). This
allowed the CARs to proliferate up to 1000-fold and prolonged their survival up to 3 years in vivo [59]. The latest 4th generation of CARs are called TRUCKs (T cells redirected for universal cytokine killing). In this generation, cytokine genes are transferred into the CAR T design. In this way, CAR-redirected T cells are used as vehicles to produce and release a transgenic product that accumulates in the targeted tissue [60,61].
First generation
Second generation
Third generation
Fourth generation
c
"c3
o
m
Q
O
Cell membrane
IL12
Fig. 2. Structures of different generations of CARs.
One of the major drawbacks of CAR-T therapy is its toxicity, especially cytokine release syndrome (CRS) and neurological toxicity [62]. CRS is manifested by an inflammatory response with an unprecedented increase in cytokine levels [63] associated with T-cell activation and proliferation. Symptoms include fever, myalgias, vascular leakage, hypotension, respiratory/renal insufficiency, cytopenias, and coagulopathy [64]. Neurotoxicity is another serious side effect, but the mechanisms involved are poorly understood [65]. CAR T-cell related encephalopathy syndrome (CRES) is a common neurotoxic manifestation that includes a range of symptoms from mild confusion to fatal cerebral edema [66]. However, these side effects are negligible compared to conventional chemotherapy, while the clinical effects are dramatic.
The next problem with CAR T-cell therapy is its cost, which is about half a million dollars [67]. The high cost is associated with the tedious process of introducing genetic material into the T cells and purifying the final product.
Another concern with CAR-T is its on-target, off-target effect. This is related to the fact that tumor-associated antigens (TAAs) are present not only on tumor cells but also on other normal cells. For this reason, other normally functioning cells that express these TAA are also targeted by CAR-T lymphocytes [68].
Benefits of mRNA car-t therapy
The transient effect of mRNA transfection may seem unattractive at first glance, but the technique is capable of solving several serious problems associated with CAR-T therapy. First, in vivo modification of T cells using mRNA technology bypasses the tedious task of PBMC extraction, chemotherapy, and reinfusion of genetically modified T cells. Similarly, the structure of the mRNA is highly customizable, and thus appropriate mRNA for targeted tumors can be rapidly and cost-effectively designed to maximize transfection and translation [69,70]. Another advantage of the mRNA genome delivery system is that the delivered genome is not integrated into the host cell genome, so the side effects associated with it are also transient [71]. Nevertheless, it has been shown that mRNA transfection has the potential to cause fewer on-target/off-target side effects with less toxicity [72-76]. In addition, the level of CAR-T expression and its toxicity is directly proportional to the amount of mRNA delivered to the T cells, so that toxicity such as cytokine release syndrome can be tuned simply by regulating the amount of mRNA injected [72,73].
In a recent study by Rurik et al [77], researchers took the mRNA delivery technology seen in current COVID-19 vaccines and applied it to the basic design of CARs to treat cardiac fibrosis. The mRNA did not integrate into the genome of the T cells, but allowed transient transcription of the mRNA and transient expression of the novel receptors for targeting fibroblast activation protein (FAP) (a marker of activated fibroblasts). The mRMA was then packaged into lipid nanoparticles (Fig. 3), and the surfaces of the lipid nanoparticles were decorated with CD5 targeting antibodies so that they could be taken up by T cells.
Fig. 3. Schematic diagram of temporary integration of mRNA into the T-cells [77].
Once inside the T cells, the mRNA remained in the T cell cytoplasm but began to express receptors for targeting FAP. The researchers concluded that more than 80% of the T cells expressed the desired CAR after treatment with the tissue culture and could effectively kill target cells with fibroblast activation protein [77].
Short-term CAR-T therapy can be achieved not only by mRNA, but also by other means. When genetic material is delivered by a non-viral method, it can be encoded so that it is not incorporated into
the host cell's DNA. In fact, preclinical studies have successfully demonstrated that with electroporation, the introduced genetic material is expressed for only 10 days, after which it disappears, along with all the associated side effects [30].
Conclusion
CAR-T lymphocyte therapy has revolutionized the treatment of cancer because of its ability to specifically target cancer cells. However, their cost and tedious manufacturing process have made them less attractive for use in clinical practice. Similarly, the side effects associated with its use can sometimes be dreadful. To solve both of these problems, the temporary expression of the injected genetic material can kill two rabbits with a single stone-as it would be cost effective and reduce the side effects that occur during the treatment process.
Author Contributions. V.K. Bozhenko: planning and supervision of the project; T.M. Kulinich: collecting data, analyzing and interpreting results, and preparing the manuscript.; R. Ranjit: analyzing the results and writing the manuscript; A.D. Kaprin: the concept and design of the study. All authors have read and agreed with the version of the manuscript submitted for publication.
Foundation. The publication was supported by the RUDN Strategic Academic Leadership Program. The support had no role in the study, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with patients' rights and bioethics regulations. This review study was based on published papers and therefore did not require ethics committee approval.
Conflict of interest. The authors declare that there is no conflict of interest.
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Информация об авторах
Татьяна Михайловна Кулинич - к.м.н., заведующая лабораторией иммунологии и онкоцитологии, ФГБУ «Российский научный центр рентгенорадиологии» Минздрава России. ORCID: https://orcid.org/0000-0003-2331-5753
Владимир Константинович Боженко - д.м.н., профессор, заслуженный врач РФ, заведующий отделом молекулярной биологии и экспериментальной терапии опухолей ФГБУ «Российский научный центр рентгенорадиологии» Минздрава России. ORCID: https://orcid.org/0000-0001-8351-8152
Раджеш Ранджит - аспирант и ассистент кафедры, ФГАОУ Российский университет дружбы народов (РУДН); ФГБУ «Российский научный центр рентгенорадиологии» Минздрава России; врач онколог, ГБУЗ ГКОБ № 1 ДЗМ. ORCID: https://orcid.org/0000-0002-4255-4197 Андрей Дмитриевич Каприн - д.м.н., профессор, академик РАН, заслуженный врач РФ, генеральный директор ФГБУ «НМИЦ радиологии» Минздрава России, директор МНИОИ имени П.А. Герцена, заведующий кафедрой онкологии и рентгенорадиологии им академика ВП Харченко медицинского института РУДН, главный внештатный онколог Минздрава России. ORCID: https://0000-0001 -8784-8415
Information about the authors
Tatyana M. Kulinich - Candidate of Medical Sciences, Head of the Laboratory of Immunology and Oncocytology, Russian Scientific Center of Roentgenoradiology. ORCID: https://orcid.org/ 0000-00032331-5753
Vladimir K. Bozhenko - Doctor of Medical Sciences, Professor, Honored Doctor of the Russian Federation, Head of the Department of Molecular Biology and Experimental Tumor Therapy, Russian Scientific Center of Roentgenoradiology. ORCID: https://orcid.org/0000-0001-8351-8152 Rajesh Ranjit - Oncologist, PhD student and assistant at the Department of Oncology, Radiology and Nuclear Medicine, Peoples' Friendship University of Russia; Russian Scientific Center of Roentgenoradiology; Municipal Clinical Oncology Hospital No. 1 of the Moscow City Health Department. ORCID: https://orcid.org/0000-0002-4255-4197
Andrey D. Kaprin - Doctor of Medical Sciences, Professor, Academician of the Russian Academy of Sciences, Honored Physician of the Russian Federation, General Director of FGBU "NMRC Radiology" of the Ministry of Health of Russia, Director of the P.A. Herzen Medical Research Institute, Head of the Department of Oncology and Radiology named after Academician V.P. Kharchenko of the RUDN Medical Institute, Chief Outside Oncologist of the Ministry of Health of Russia. ORCID: https://0000-0001-8784-8415
