Non-Tumor Cells within the Tumor Microenvironment-The "Eminence Grise" of the Glioblastoma Pathogenesis and Potential Targets for Therapy.

Glioblastoma (GBM) is the most common malignancy of the central nervous system in adults. GBM has high levels of therapy failure and its prognosis is usually dismal. The phenotypic heterogeneity of the tumor cells, dynamic complexity of non-tumor cell populations within the GBM tumor microenvironment (TME), and their bi-directional cross-talk contribute to the challenges of current therapeutic approaches. Herein, we discuss the etiology of GBM, and describe several major types of non-tumor cells within its TME, their impact on GBM pathogenesis, and molecular mechanisms of such an impact. We also discuss their value as potential therapeutic targets or prognostic biomarkers, with reference to the most recent works on this subject. We conclude that unless all "key player" populations of non-tumor cells within the TME are considered, no breakthrough in developing treatment for GBM can be achieved.

Humoral Immune Responses in Patients with Severe COVID-19: A Comparative Pilot Study between Individuals Infected by SARS-CoV-2 during the Wild-Type and the Delta Periods.

Since the onset of the COVID-19 pandemic, humanity has experienced the spread and circulation of several SARS-CoV-2 variants that differed in transmissibility, contagiousness, and the ability to escape from vaccine-induced neutralizing antibodies. However, issues related to the differences in the variant-specific immune responses remain insufficiently studied. The aim of this study was to compare the parameters of the humoral immune responses in two groups of patients with acute COVID-19 who were infected during the circulation period of the D614G and the Delta variants of SARS-CoV-2. Sera from 48 patients with acute COVID-19 were tested for SARS-CoV-2 binding and neutralizing antibodies using six assays. We found that serum samples from the D614G period demonstrated 3.9- and 1.6-fold increases in RBD- and spike-specific IgG binding with wild-type antigens compared with Delta variant antigens (p < 0.01). Cluster analysis showed the existence of two well-separated clusters. The first cluster mainly consisted of D614G-period patients and the second cluster predominantly included patients from the Delta period. The results thus obtained indicate that humoral immune responses in D614G- and Delta-specific infections can be characterized by variant-specific signatures. This can be taken into account when developing new variant-specific vaccines.

Recombinant Strains of Oncolytic Vaccinia Virus for Cancer Immunotherapy.

Cancer virotherapy is an alternative therapeutic approach based on the viruses that selectively infect and kill tumor cells. Vaccinia virus (VV) is a member of the Poxviridae, a family of enveloped viruses with a large linear double-stranded DNA genome. The proven safety of the VV strains as well as considerable transgene capacity of the viral genome, make VV an excellent platform for creating recombinant oncolytic viruses for cancer therapy. Furthermore, various genetic modifications can increase tumor selectivity and therapeutic efficacy of VV by arming it with the immune-modulatory genes or proapoptotic molecules, boosting the host immune system, and increasing cross-priming recognition of the tumor cells by T-cells or NK cells. In this review, we summarized the data on bioengineering approaches to develop recombinant VV strains for enhanced cancer immunotherapy.

Novel B-Cell Epitopes of Non-Neutralizing Antibodies in the Receptor-Binding Domain of the SARS-CoV-2 S-Protein with Different Effects on the Severity of COVID-19.

Antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein (RBD S-protein) contribute significantly to the humoral immune response during coronavirus infection (COVID-19) and after vaccination. The main focus of the studies of the RBD epitope composition is usually concentrated on the epitopes recognized by the virus-neutralizing antibodies. The role of antibodies that bind to RBD but do not neutralize SARS-CoV-2 remains unclear. In this study, immunochemical properties of the two mouse monoclonal antibodies (mAbs), RS17 and S11, against the RBD were examined. Both mAbs exhibited high affinity to RBD, but they did not neutralize the virus. The epitopes of these mAbs were mapped using phage display: the epitope recognized by the mAb RS17 is located at the N-terminal site of RBD (348-SVYAVNRKRIS-358); the mAb S11 epitope is inside the receptor-binding motif of RBD (452-YRLFRKSN-459). Three groups of sera were tested for presence of antibodies competing with the non-neutralizing mAbs S11 and RS17: (i) sera from the vaccinated healthy volunteers without history of COVID-19; (ii) sera from the persons who had a mild form of COVID-19; (iii) sera from the persons who had severe COVID-19. Antibodies competing with the mAb S11 were found in each group of sera with equal frequency, whereas presence of the antibodies competing with the mAb RS17 in the sera was significantly more frequent in the group of sera obtained from the patients recovered from severe COVID-19 indicating that such antibodies are associated with the severity of COVID-19. In conclusion, despite the clear significance of anti-RBD antibodies in the effective immune response against SARS-CoV-2, it is important to analyze their virus-neutralizing activity and to confirm absence of the antibody-mediated enhancement of infection by the anti-RBD antibodies.

Antibodies Capable of Enhancing SARS-CoV-2 Infection Can Circulate in Patients with Severe COVID-19.

Antibody-dependent enhancement (ADE) has been shown previously for SARS-CoV-1, MERS-CoV, and SARS-CoV-2 infection in vitro. In this study, the first monoclonal antibody (mAb) that causes ADE in a SARS-CoV-2 in vivo model was identified. mAb RS2 against the SARS-CoV-2 S-protein was developed using hybridoma technology. mAb RS2 demonstrated sub-nanomolar affinity and ability to neutralize SARS-CoV-2 infection in vitro with IC50 360 ng/mL. In an animal model of SARS-CoV-2 infection, the dose-dependent protective efficacy of mAb RS2 was revealed. However, in post-exposure prophylaxis, the administration of mAb RS2 led to an increase in the viral load in the respiratory tract of animals. Three groups of blood plasma were examined for antibodies competing with mAb RS2: (1) plasmas from vaccinated donors without COVID-19; (2) plasmas from volunteers with mild symptoms of COVID-19; (3) plasmas from patients with severe COVID-19. It was demonstrated that antibodies competing with mAb RS2 were significantly more often recorded in sera from volunteers with severe COVID-19. The results demonstrated for the first time that in animals, SARS-CoV-2 can induce antibody/antibodies that can elicit ADE. Moreover, in the sera of patients with severe COVID-19, there are antibodies competing for the binding of an epitope that is recognized by the ADE-eliciting mAb.

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response.

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

Alterations in the CD56- and CD56+ T Cell Subsets during COVID-19.

The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56- T cells during COVID-19 among intensive care unit (ICU) patients, moderate severity (MS) patients, and convalescents. A decreased proportion of CD56+ T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8+ T cells, mainly due to the CD56- cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8+ T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3+ and NKp30+ cells in the CD56+ T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D+ and NKG2A+ cells and increased PD-1 and HLA-DR expression levels were found in both CD56- and CD56+ T cells, and can be considered as indicators of COVID-19 progression. In the CD56- T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56-CD16+ T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56+ T cells in COVID-19.

Oncolytic Efficacy of a Recombinant Vaccinia Virus Strain Expressing Bacterial Flagellin in Solid Tumor Models.

Oncolytic viral therapy is a promising novel approach to cancer treatment. Oncolytic viruses cause tumor regression through direct cytolysis on the one hand and recruiting and activating immune cells on the other. In this study, to enhance the antitumor efficacy of the thymidine kinase-deficient vaccinia virus (VV, Lister strain), recombinant variants encoding bacterial flagellin (subunit B) of Vibrio vulnificus (LIVP-FlaB-RFP), firefly luciferase (LIVP-Fluc-RFP) or red fluorescent protein (LIVP-RFP) were developed. The LIVP-FLuc-RFP strain demonstrated exceptional onco-specificity in tumor-bearing mice, detected by the in vivo imaging system (IVIS). The antitumor efficacy of these variants was explored in syngeneic murine tumor models (B16 melanoma, CT26 colon cancer and 4T1 breast cancer). After intravenous treatment with LIVP-FlaB-RFP or LIVP-RFP, all mice tumor models exhibited tumor regression, with a prolonged survival rate in comparison with the control mice. However, superior oncolytic activity was observed in the B16 melanoma models treated with LIVP-FlaB-RFP. Tumor-infiltrated lymphocytes and the cytokine analysis of the serum and tumor samples from the melanoma-xenografted mice treated with these virus variants demonstrated activation of the host's immune response. Thus, the expression of bacterial flagellin by VV can enhance its oncolytic efficacy against immunosuppressive solid tumors.

Recent Developments in Glioblastoma Therapy: Oncolytic Viruses and Emerging Future Strategies.

Glioblastoma is the most aggressive form of malignant brain tumor. Standard treatment protocols and traditional immunotherapy are poorly effective as they do not significantly increase the long-term survival of glioblastoma patients. Oncolytic viruses (OVs) may be an effective alternative approach. Combining OVs with some modern treatment options may also provide significant benefits for glioblastoma patients. Here we review virotherapy for glioblastomas and describe several OVs and their combination with other therapies. The personalized use of OVs and their combination with other treatment options would become a significant area of research aiming to develop the most effective treatment regimens for glioblastomas.

SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines.

Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.

Coordinated Loss and Acquisition of NK Cell Surface Markers Accompanied by Generalized Cytokine Dysregulation in COVID-19.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is accompanied by a dysregulated immune response. In particular, NK cells, involved in the antiviral response, are affected by the infection. This study aimed to investigate circulating NK cells with a focus on their activation, depletion, changes in the surface expression of key receptors, and functional activity during COVID-19, among intensive care unit (ICU) patients, moderately ill patients, and convalescents (CCP). Our data confirmed that NK cell activation in patients with COVID-19 is accompanied by changes in circulating cytokines. The progression of COVID-19 was associated with a coordinated decrease in the proportion of NKG2D+ and CD16+ NK cells, and an increase in PD-1, which indicated their exhaustion. A higher content of NKG2D+ NK cells distinguished surviving patients from non-survivors in the ICU group. NK cell exhaustion in ICU patients was additionally confirmed by a strong negative correlation of PD-1 and natural cytotoxicity levels. In moderately ill patients and convalescents, correlations were found between the levels of CD57, NKG2C, and NKp30, which may indicate the formation of adaptive NK cells. A reduced NKp30 level was observed in patients with a lethal outcome. Altogether, the phenotypic changes in circulating NK cells of COVID-19 patients suggest that the intense activation of NK cells during SARS-CoV-2 infection, most likely induced by cytokines, is accompanied by NK cell exhaustion, the extent of which may be critical for the disease outcome.

Effect of vitamin D3 supplementation on cellular immunity and inflammatory markers in COVID-19 patients admitted to the ICU.

Vitamin D as an immunomodulator has not been studied in patients with severe COVID-19. This study aimed to estimate the efficacy of vitamin D3 supplementation on cellular immunity and inflammatory markers in patients with COVID-19 admitted to the intensive care unit (ICU). A single-center, double-blind, randomized, placebo-controlled pilot trial was conducted (N = 110). Patients were randomly assigned to receive a weekly oral dose of 60,000 IU of vitamin D3 followed by daily maintenance doses of 5000 IU (n = 55) or placebo (n = 55). Primary outcomes were lymphocyte counts, natural killer (NK) and natural killer T (NKT) cell counts, neutrophil-to-lymphocyte ratio (NLR), and serum levels of inflammatory markers on 7th day of treatment. On day 7, patients in the vitamin D3 group displayed significantly higher NK and NKT cell counts and NLR than those in the placebo group did. The mortality rate (37% vs 50%, P = 0.16), need for mechanical ventilation (63% vs 69%, P = 0.58), incidence of nosocomial infection (60% vs 41%, P = 0.05) did not significantly differ between groups. Vitamin D3 supplementation, compared with placebo, significantly increased lymphocyte counts, but did not translate into reduced mortality in ICU.Trial Registration: ClinicalTrials.gov Identifier: NCT05092698.

Corrigendum: Highly invasive fluorescent/bioluminescent patient-derived orthotopic model of glioblastoma in mice.

[This corrects the article DOI: 10.3389/fonc.2022.897839.].

Highly Invasive Fluorescent/Bioluminescent Patient-Derived Orthotopic Model of Glioblastoma in Mice.

Development of the novel diagnostic and therapeutic approaches in neuro-oncology requires tumor models that closely reproduce the biological features of patients' tumors. Patient-derived xenografts (PDXs) are recognized as a valuable and the most "close-to-patient" tool for preclinical studies. However, their establishment is complicated by the factors related to both the surgical material and technique of the orthotopic implantation. The aim of this work was to develop a patient-derived glioblastoma multiform (GBM) model that stably co-expresses luciferase and a far-red fluorescent protein for monitoring of tumor progression in the brain and, using this model, to validate new diagnostic methods-macroscopic fluorescence lifetime imaging (macro-FLIM) and cross-polarization optical coherence tomography (CP OCT). The established model was similar to the original patient's GBM in terms of histological and immunohistochemical features and possessed reproducible growth in nude mice, which could be observed by both fluorescence and bioluminescence imaging. Our results demonstrated the high potential of macro-FLIM and CP OCT for intraoperative differentiation of GBM from the white matter. Thus, the dual-labeled PDX model of GBM proved to be an excellent approach for observation of tumor development by optical methods.

Functional Profiling of In Vitro Reactivated Memory B Cells Following Natural SARS-CoV-2 Infection and Gam-COVID-Vac Vaccination.

Both SARS-CoV-2 infection and vaccination have previously been demonstrated to elicit robust, yet somewhat limited immunity against the evolving variants of SARS-CoV-2. Nevertheless, reports performing side-by-side comparison of immune responses following infection vs. vaccination have been relatively scarce. The aim of this study was to compare B-cell response to adenovirus-vectored vaccination in SARS-CoV-2-naive individuals with that observed in the COVID-19 convalescent patients six months after the first encounter with the viral antigens. We set out to use a single analytical platform and performed comprehensive analysis of serum levels of receptor binding domain (RBD)-specific and virus-neutralizing antibodies, frequencies of RBD-binding circulating memory B cells (MBCs), MBC-derived antibody-secreting cells, as well as RBD-specific and virus-neutralizing activity of MBC-derived antibodies after Gam-COVID-Vac (Sputnik V) vaccination and/or natural SARS-CoV-2 infection. Overall, natural immunity was superior to Gam-COVID-Vac vaccination. The levels of neutralizing MBC-derived antibodies in the convalescent patients turned out to be significantly higher than those found following vaccination. Our results suggest that after six months, SARS-CoV-2-specific MBC immunity is more robust in COVID-19 convalescent patients than in Gam-COVID-Vac recipients. Collectively, our data unambiguously indicate that natural immunity outperforms Gam-COVID-Vac-induced immunity six months following recovery/vaccination, which should inform healthcare and vaccination decisions.

Superior infectivity of the fiber chimeric oncolytic adenoviruses Ad5/35 and Ad5/3 over Ad5-delta-24-RGD in primary glioma cultures.

Ad5-delta-24-RGD is currently the most clinically advanced recombinant adenovirus (rAd) for glioma therapy. We constructed a panel of fiber-modified rAds (Ad5RGD, Ad5/3, Ad5/35, Ad5/3RGD, and Ad5/35RGD, all harboring the delta-24 modification) and compared their infectivity, replication, reproduction, and cytolytic efficacy in human and rodent glioma cell lines and short-term cultures from primary gliomas. In human cells, both Ad5/35-delta-24 and Ad5/3-delta-24 displayed superior infectivity and cytolytic efficacy over Ad5-delta-24-RGD, while Ad5/3-delta-24-RGD and Ad5/35-delta-24-RGD did not show further improvements in efficacy. The expression of the adenoviral receptors/coreceptors CAR, DSG2, and CD46 and the integrins αVß3/αVß5 did not predict the relative cytolytic efficacy of the fiber-modified rAds. The cytotoxicity of the fiber-modified rAds in human primary normal cultures of different origins and in primary glioma cultures was comparable, indicating that the delta-24 modification did not confer tumor cell selectivity. We also revealed that CT-2A and GL261 glioma cells might be used as murine cell models for the fiber chimeric rAds in vitro and in vivo. In GL261 tumor-bearing mice, Ad5/35-delta-24, armed with the immune costimulator OX40L as the E2A/DBP-p2A-mOX40L fusion, produced long-term survivors, which were able to reject tumor cells upon rechallenge. Our data underscore the potential of local Ad5/35-delta-24-based immunovirotherapy for glioblastoma treatment.

Isolation of a panel of ultra-potent human antibodies neutralizing SARS-CoV-2 and viral variants of concern.

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

Low Circulating Vitamin D in Intensive Care Unit-Admitted COVID-19 Patients as a Predictor of Negative Outcomes.

BACKGROUND: Vitamin D deficiency has been associated with an increased risk of respiratory infections. OBJECTIVES: The study aimed to evaluate the serum 25-hydroxyvitamin D [25(OH)D] concentration in patients admitted to the intensive care unit (ICU) as a predictor of coronavirus disease 2019 (COVID-19) mortality. METHODS: A single-center retrospective observational study was conducted. Forty adult patients (50% men) with confirmed COVID-19 who were admitted to the ICU were enrolled. The primary endpoint was mortality at day 60. Serum 25(OH)D concentration was measured on the day of admission to the ICU. We used the Mann-Whitney test, Fisher's exact test, Kaplan-Meier analysis, and receiver operator characteristic (ROC) analysis to assess serum 25(OH)D concentration as a predictor of COVID-19 mortality. RESULTS: All 40 patients had a low median (IQR) serum 25(OH)D concentration at admission [12 (9-15) ng/mL]. The median (IQR) serum 25(OH)D concentration was greater in survivors [13.3 (10.0-17.1) ng/mL, n = 22] than in nonsurvivors [9.6 (7.9-14.2) ng/mL; n = 18], P = 0.044. The area under the ROC curve was 0.69 (95% CI: 0.52, 0.86; P = 0.044). The 60-d mortality rate of those with serum 25(OH)D concentrations ≤9.9 ng/mL (n = 14, 71%) tended to be greater than that of those with concentrations >9.9 ng/mL (n = 26, 31%) (P = 0.065), and they had a 5.6-fold higher risk of death (OR: 5.63; 95% CI: 1.35, 23.45; P = 0.018). CONCLUSIONS: The ICU patients had a low serum 25(OH)D concentration. Serum 25(OH)D concentrations ≤9.9 ng/mL on admission can be used to predict in-hospital mortality in patients with COVID-19.This trial was registered at clinicaltrials.gov as NCT04450017.

Pattern of circulating SARS-CoV-2-specific antibody-secreting and memory B-cell generation in patients with acute COVID-19.

OBJECTIVES: To predict the spread of coronavirus disease (COVID-19), information regarding the immunological memory for disease-specific antigens is necessary. The possibility of reinfection, as well as the efficacy of vaccines for COVID-19 that are currently under development, will largely depend on the quality and longevity of immunological memory in patients. To elucidate the process of humoral immunity development, we analysed the generation of plasmablasts and virus receptor-binding domain (RBD)-specific memory B (Bmem) cells in patients during the acute phase of COVID-19. METHODS: The frequencies of RBD-binding plasmablasts and RBD-specific antibody-secreting cells (ASCs) in the peripheral blood samples collected from patients with COVID-19 were measured using flow cytometry and the ELISpot assay. RESULTS: The acute phase of COVID-19 was characterised by the transient appearance of total as well as RBD-binding plasmablasts. ELISpot analysis indicated that most patients exhibited a spontaneous secretion of RBD-specific ASCs in the circulation with good correlation between the IgG and IgM subsets. IL-21/CD40L stimulation of purified B cells induced the activation and proliferation of Bmem cells, which led to the generation of plasmablast phenotypic cells as well as RBD-specific ASCs. No correlation was observed between the frequency of Bmem cell-derived and spontaneous ASCs, suggesting that the two types of ASCs were weakly associated with each other. CONCLUSION: Our findings reveal that SARS-CoV-2-specific Bmem cells are generated during the acute phase of COVID-19. These findings can serve as a basis for further studies on the longevity of SARS-CoV-2-specific B-cell memory.

VAV1-overexpressing YT cells display improved cytotoxicity against malignant cells.

Immunotherapy based on adoptive transfer of genetically engineered T- and NK-cells is an area of active ongoing research and has proven highly efficacious for patients with certain B-cell malignancies. Use of NK cells and NK cell lines as carriers of chimeric antigen receptors (CARs) appears particularly promising, as this opens an opportunity for moving the therapy from autologous to the allogeneic (universal) format. This "off-the-shelf" approach is thought to significantly reduce the price of the treatment and make it available to many more patients in need. Yet, the efficacy of CAR-NK cells in vivo presently remains low, and boosting the activity of CAR NK cells via stronger tumor homing, resistance to tumor microenvironment, as well as greater cytotoxicity may translate into improved patient outcomes. Here, we established a derivative of a human NK cell line YT overexpressing a positive regulator of cytotoxicity, VAV1. Activity of YT-VAV1 cells obtained was assayed in vitro against several cancer cell lines and primary patient-derived cancer cells. YT-VAV1 cells outperform parental YT cells in terms of cytotoxicity.