Vol 17, No 3 (2025)

The advent of the T-cell engineering technology using chimeric antigen receptors (CARs) has revolutionized the treatment of hematologic malignancies and reoriented the direction of research in the field of immune cell engineering and immunotherapy. Regrettably, the effectiveness of CAR T-cell therapy in specific instances of hematologic malignancies and solid tumors is limited by a number of factors. These include (1) an excessive or insufficient CAR T-cell response, possibly a result of both resistance within the tumor cells or the microenvironment and the suboptimal structural and functional organization of the chimeric receptor; (2) a less-than-optimal functional phenotype of the final CAR T-cell product, which is a direct consequence of the manufacturing and expansion processes used to produce CAR T-cells; and (3) the lack of an adequate CAR T-cell control system post-administration to the patient. Consequently, current research efforts focus on optimizing the CAR structure, improving production technologies, and further developing CAR T-cell modifications. Optimizing the CAR structure to enhance the function of modified cells is a primary strategy in improving the efficacy of CAR T-cell therapy. Since the emergence of the first CAR T-cells, five generations of CARs have been developed, employing both novel combinations of signaling and structural domains within a single molecule and new systems of multiple chimeric molecules presented simultaneously on the T-cell surface. A well thought-out combination of CAR components should ensure high receptor sensitivity to the antigen, the formation of a stable immune synapse (IS), effective costimulation, and productive CAR T-cell activation. Integrating cutting-edge technologies – specifically machine learning that helps predict the structure and properties of a three-dimensional biopolymer, combined with high-throughput sequencing and omics approaches – offers new possibilities for the targeted modification of the CAR structure. Of crucial importance is the selection of specific modifications and combinations of costimulatory and signaling domains to enhance CAR T-cell cytotoxicity, proliferation, and persistence. This review provides insights into recent advancements in CAR optimization, with particular emphasis on modifications designed to enhance the therapeutic functionality of CAR T-cells.

Calcium signaling ensures efficient cellular functioning; calcium homeostasis disruption leaves behind detrimental sequelae for the cell both under calcium excess and deficiency conditions. Malignant transformation is accompanied by significant alterations in the expression of the proteins critical for store-operated calcium entry, resulting in the dysregulation of calcium signaling. It is plausible that a remodeling of intracellular signal transduction pathways in cancer cells is required in order to accelerate metabolic processes, as well as fuel further tumor growth and invasion. Meanwhile, fine-tuning of calcium signaling is observed under both normal and pathological conditions. In this context, research into the changes accompanying signal transduction within the tumor microenvironment is a key aspect of the investigation of the role of calcium signaling in tumor development. Factors characteristic of the tumor microenvironment were shown to have a significant effect on the function of calcium channels and the proteins that regulate calcium signaling. Major, adverse microenvironmental factors, such as acidification, elevated levels of reactive oxygen species and hypoxia, have a bearing on the store-operated calcium entry. It is crucial to understand whether changes in the expression of the key SOCE components represent an adaptation to the microenvironment or a result of carcinogenesis.

Widespread environmental contamination with polychlorinated biphenyls (PCBs) leads to serious health problems for humans and animals. Our main focus should be on studying the negative effects of exposure to medium- and highly chlorinated PCBs in the human body. There is limited information on the impact of low-chlorinated biphenyls containing 1–2 substituents per molecule on the functions of mammalian organs and systems. Under natural conditions, PCBs can undergo bacterial degradation; the resulting compounds belong to a group of secondary pollutants and are considered hazardous to the environment. Because of limited research, the question regarding the impact of mono-substituted chlorobiphenyl congeners, as well as the products of their biotransformation, remains open. In the presented work, the effects of ortho- and meta-substituted monochlorinated biphenyls on the functions of immune system cells and the morphofunctional state of the liver of mammals in vivo are revealed for the first time. PCB 1 and PCB 2 were found to suppress humoral immunity and induce a productive inflammatory response, as well as widespread protein dystrophy with necrotic foci in the liver. The products of a aerobic bacterial transformation of PCB 1 and PCB 2 were shown to not have a negative effect on the mammalian immune system but proved toxic to hepatocytes, although to a lesser extent than the original chlorobiphenyls.

The hippocampus is a key component of the brain that is associated with the formation of long-term memory, the energy metabolism of neurons playing a pivotal role in its mechanisms. The P2X3 receptor in the hippocampus is considered an attractive target when searching for novel biologically active substances that could work to reduce anxiety, epileptic conditions, and improve cognitive functions. In this work, the intensity of mitochondrial respiration, the glycolytic capacity, and the energy phenotype of hippocampal neurons were studied in p2rx3 knockout mice. The p2rx3 knockout mice were engineered by genome editing using the CRISPR/Cas9 system. The primary mixed culture of hippocampal neurons was derived from two-day-old newborn mice with the p2rx3^-/- and p2rx3^+/- genotypes. Mitochondrial respiration was measured on a Seahorse Bioscience HS mini Cell Metabolism Analyzer (Agilent, USA) using the appropriate kits for the Mitostress test, glycotest, and energy phenotype assessment test. The transgenic mice with the p2rx3^-/- genotype were characterized by an aerobic type of mitochondrial respiration, an increase in ATP production by 84.4% (p < 0.05), an increase in maximum respiration by 72.3% (p < 0.05), and a 36% (p < 0.05) increase in the respiratory reserve. Meanwhile, the spare respiratory capacity of mitochondria, the rate of glycolysis, and the glycolytic capacity in these mice were reduced by 36.6, 75.7 and 78.6% (p < 0.05), respectively. Our findings indicate that mitochondria work at close to maximum energy capacity. The p2rx3 knockout animals are a unique model for the search for pharmacological targets that can help correct the energy metabolism of brain cells and eliminate cognitive dysfunctions.

This paper presents a new bioinformatics tool to meet the needs of researchers in the search for short (≥ 3) amino acid subsequences in protein sequences annotated in public databases (UniprotKB, SwissProt) and illustrates its efficacy with the example of a search for the EVHH tetrapeptide in the human proteome, which is a molecular determinant of amyloid beta and is involved in interactions that are crucial in Alzheimer’s disease pathogenesis. The topicality of developing such a tool is, on the one hand, supported by experimental data on the role of short tetrapeptide motifs in the architecture of intermolecular interfaces. On the other hand, there are currently no software products for efficient search for short (≥ 3) amino acid sequences in public databases, which drastically limits researchers’ ability to identify proteins with exact matches of short subsequences. This tool (PepString server, http://pepstring.eimb.ru/) allows one to use intuitive queries to retrieve information about all the proteins that contain sequences of interest, as well as their combinations.

Boron neutron capture therapy (BNCT) is a rapidly developing field of radiation therapy for cancer that is based on the accumulation of the radiosensitive ¹⁰B isotope in cancer cells, followed by tumor irradiation with thermal neutrons. Widespread use of BNCT in clinical practice remains limited because of the poor accumulation of boron-containing (¹⁰B) drugs in the tumor or their high toxicity to the body. This study focuses on the engineering of tumor-specific liposomes loaded with 4-L-boronophenylalanine (4-L-¹⁰BPA) for application in boron neutron capture therapy. According to the spectrophotometry and ICP-mass spectroscopy data, the 4-L-¹⁰BPA-to-liposome molar ratio is ~ 120,000. Liposomal targeting of human epidermal growth factor receptor 2 (HER2) was determined by HER2-specific designed ankyrin repeat protein (DARPin)_9-29 on the outer surface of liposomes. DARPin-modified liposomes were found to bind to HER2-overexpressing cells and be effectively internalized into the cytoplasm. The ability of DARPin-functionalized liposomes to precision-deliver large quantities of 4-L-¹⁰BPA into cancer cells may open up new prospects for BNCT.

OrthopoxVac, a fourth-generation smallpox vaccine, was the first of its kind registered worldwide in 2022, and it has been shown to be both safe and to induce only a mild reaction. A six-month clinical study confirmed its immunogenicity as compared to the first-generation live smallpox vaccine. Our study aimed to determine the levels of specific humoral and T-cell immune responses in volunteers following intradermal OrthopoxVac vaccine administration either in a single dose of 10⁷ PoFU or in two doses of 10⁶ PoFU, at 1.5, 3, and 5 years after initial vaccination. Following the immunization of volunteers with the OrthopoxVac vaccine at a dosage of 10⁷ PoFU, the T-helper response remained at a relatively high level for three years, before it significantly dropped. Administration of the same vaccine twice at a dose of 10⁶ PoFU resulted in a considerable decrease in the level of T-helpers, after 1.5 years. Additionally, some patients exhibited a reduction in viral neutralizing antibody (VNA) titers after 1.5 years of OrthopoxVac vaccine administration. When OrthopoxVac was administered at a dosage of 10⁷ PoFU, no substantial differences were noted between groups at the 1.5-, 3-, and 5-year marks. In contrast, in the groups receiving two doses of 10⁶ PoFU, VNA titers showed a significant reduction after 1.5 years. These findings indicate that a single intradermal dose of 10⁷ PoFU of the OrthopoxVac vaccine elicits a significant and lasting immune response involving both antibodies and T-cells for a minimum of three years.