The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer.

BACKGROUND: Chemotherapy and surgery have been the mainstays of epithelial ovarian cancer (EOC) treatment so far. Cellular immunotherapies such as CAR T cell therapy have recently given hope of a cure for solid tumors like EOC. However, extrinsic factors associated with the CAR T cell manufacturing process and/or intrinsic dysregulation of patient-derived T cells, which could be associated with cancer itself, cancer stage, and treatment regimen, may hamper the efficacy of CAR T cell therapy and promote their exhaustion or dysfunction. METHODS: To investigate the association of these factors with CAR T cell exhaustion, the frequency of T and CAR T cells expressing three immune inhibitory receptors (i.e., TIM3, PD1, A2aR) generated from T cells of EOC patients and healthy controls was measured during each stage of CAR T cell production. RESULTS: Our findings revealed that primary T cells from EOC patients show significantly elevated expression of immune inhibitory receptors, and this increase was more prominent in patients undergoing chemotherapy and those with advanced cancer. In addition, the CAR T cell manufacturing process itself was found to upregulate the expression of these inhibitory receptors and more importantly increase the population of exhausted mesoCAR T cells. CONCLUSIONS: Our observations suggest that intrinsic characteristics of patient-derived T cells and extrinsic factors in CAR T cell production protocols should be considered and properly counteracted during CAR T cell manufacturing process. In addition, mitigating the signaling of immune inhibitory receptors through pharmacological/genetic perturbation during CAR T cell manufacturing might profoundly improve CAR T cells function and their antitumor activity in EOC and other solid tumors.

Targeting hypoxia and hypoxia-inducible factor-1 in the tumor microenvironment for optimal cancer immunotherapy.

The development of new strategies of anticancer immunotherapies has provided promising approaches in the treatment of solid tumors. However, despite the improved survival in responders, most of the patients show incomplete responses with a lack of remarkable clinical improvement. Hypoxia has been identified as a common characteristic of solid tumors contributing to different aspects of tumor progression, including invasion, metastasis, and the creation of the immunosuppressive tumor microenvironment. Hypoxia, through its main mediator, hypoxia-inducible factor-1 (HIF-1) is also associated with the limited efficacy of immunotherapies. Therefore, designing new strategies for immunotherapy implicating therapeutic targeting of HIF-1 molecules may enhance the clinical effectiveness of immunotherapy. Here, we discuss the contribution of hypoxia to the development of the immunosuppressive tumor microenvironment. We will also outline different strategies for targeting hypoxia to provide insight into the therapeutic potential of the application of such strategies to improve the clinical benefit of cancer immunotherapy.

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Epigenetic strategies to boost CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has led to a paradigm shift in cancer immunotherapy, but still several obstacles limit CAR T cell efficacy in cancers. Advances in high-throughput technologies revealed new insights into the role that epigenetic reprogramming plays in T cells. Mechanistic studies as well as comprehensive epigenome maps revealed an important role for epigenetic remodeling in T cell differentiation. These modifications shape the overall immune response through alterations in T cell phenotype and function. Here, we outline how epigenetic modifications in CAR T cells can overcome barriers limiting CAR T cell effectiveness, particularly in immunosuppressive tumor microenvironments. We also offer our perspective on how selected epigenetic modifications can boost CAR T cells to ultimately improve the efficacy of CAR T cell therapy.

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option.

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSCenr -EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSCenr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSCenr -EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSCenr -EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSCenr lysate, CSCenr -EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSCenr -EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSCenr -EXOs disrupted spheroids' structure. Thus, CSCenr -EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.

CRISPR/Cas9-mediated TGFßRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro.

BACKGROUND: CAR T-cell therapy has been recently unveiled as one of the most promising cancer therapies in hematological malignancies. However, solid tumors mount a profound line of defense to escape immunosurveillance by CAR T-cells. Among them, cytokines with an inhibitory impact on the immune system such as IL-10 and TGFß are of great importance: TGFß is a pleiotropic cytokine, which potently suppresses the immune system and is secreted by a couple of TME resident and tumor cells. METHODS: In this study, we hypothesized that knocking out the TGFß receptor II gene, could improve CAR T-cell functions in vitro and in vivo. Hereby, we used the CRISPR/Cas9 system, to knockout the TGFßRII gene in T-cells and could monitor the efficient gene knock out by genome analysis techniques. Next, Mesothelin or Claudin 6 specific CAR constructs were overexpressed via IVT-RNA electroporation or retroviral transduction and the poly-functionality of these TGFßRII KO CAR T-cells in terms of proliferation, cytokine secretion and cytotoxicity were assessed and compared with parental CAR T-cells. RESULTS: Our experiments demonstrated that TGFßRII KO CAR T-cells fully retained their capabilities in killing tumor antigen positive target cells and more intriguingly, could resist the anti-proliferative effect of exogenous TGFß in vitro outperforming wild type CAR T-cells. Noteworthy, no antigen or growth factor-independent proliferation of these TGFßRII KO CAR T-cells has been recorded. TGFßRII KO CAR T-cells also resisted the suppressive effect of induced regulatory T-cells in vitro to a larger extent. Repetitive antigen stimulation demonstrated that these TGFßRII KO CAR T-cells will experience less activation induced exhaustion in comparison to the WT counterpart. CONCLUSION: The TGFßRII KO approach may become an indispensable tool in immunotherapy of solid tumors, as it may surmount one of the key negative regulatory signaling pathways in T-cells.

Prediction of anti-CD25 and 5-FU treatments efficacy for pancreatic cancer using a mathematical model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with rising incidence and with 5-years overall survival of less than 8%. PDAC creates an immune-suppressive tumor microenvironment to escape immune-mediated eradication. Regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSC) are critical components of the immune-suppressive tumor microenvironment. Shifting from tumor escape or tolerance to elimination is the major challenge in the treatment of PDAC. RESULTS: In a mathematical model, we combine distinct treatment modalities for PDAC, including 5-FU chemotherapy and anti- CD25 immunotherapy to improve clinical outcome and therapeutic efficacy. To address and optimize 5-FU and anti- CD25 treatment (to suppress MDSCs and Tregs, respectively) schedule in-silico and simultaneously unravel the processes driving therapeutic responses, we designed an in vivo calibrated mathematical model of tumor-immune system (TIS) interactions. We designed a user-friendly graphical user interface (GUI) unit which is configurable for treatment timings to implement an in-silico clinical trial to test different timings of both 5-FU and anti- CD25 therapies. By optimizing combination regimens, we improved treatment efficacy. In-silico assessment of 5-FU and anti- CD25 combination therapy for PDAC significantly showed better treatment outcomes when compared to 5-FU and anti- CD25 therapies separately. Due to imprecise, missing, or incomplete experimental data, the kinetic parameters of the TIS model are uncertain that this can be captured by the fuzzy theorem. We have predicted the uncertainty band of cell/cytokines dynamics based on the parametric uncertainty, and we have shown the effect of the treatments on the displacement of the uncertainty band of the cells/cytokines. We performed global sensitivity analysis methods to identify the most influential kinetic parameters and simulate the effect of the perturbation on kinetic parameters on the dynamics of cells/cytokines. CONCLUSION: Our findings outline a rational approach to therapy optimization with meaningful consequences for how we effectively design treatment schedules (timing) to maximize their success, and how we treat PDAC with combined 5-FU and anti- CD25 therapies. Our data revealed that a synergistic combinatorial regimen targeting the Tregs and MDSCs in both crisp and fuzzy settings of model parameters can lead to tumor eradication.

Predictive and therapeutic biomarkers in chimeric antigen receptor T-cell therapy: A clinical perspective.

The adoptive transfer of genetically engineered T cells modified to express a chimeric antigen receptor (CAR) has shown remarkable activity and induces long-term remissions in patients with advanced hematologic malignancies. To date, little is known about predictive indicators of therapeutic efficacy or serious toxicity after CAR T-cell therapy in clinical practice. Biomarkers are not only potentially able to inform physicians and researchers of immunotherapy targets in particular but could also be used to monitor the effectiveness of treatments and to predict incidence of side effects in some circumstances. Identification of new biomarkers can therefore not only contribute to the development of new therapeutic and prognostic strategies for CAR T-cell therapy for cancer but also help to generate improved clinical practices for early recognition and minimization of adverse effects while preserving the antitumor activity of the CAR T cells. Herein, we will consider a variety of predictive and therapeutic biomarkers in CAR T-cell therapy and the state of current understanding of their clinical utility. The incorporation of biomarker studies in CAR T-cell clinical trials and practice will help to realize the potential clinical benefit of biomarker-guided therapy.

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells.

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells-thereby reducing the risk of tumor relapse-but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19+ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA+ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.

Sodium Butyrate as a Histone Deacetylase Inhibitor Affects Toll-Like Receptor 4 Expression in Colorectal Cancer Cell Lines.

We assessed the effect of sodium butyrate (SB) as a histone deacetylase inhibitor (HDACi) on Toll-like receptor 4 (TLR4) gene expression levels, in low TLR4 expressing (HCT116) and high TLR4 expressing (SW480) colorectal cancer cells. The cytotoxic effect of SB was assessed by culturing SW480 and HCT116 cell lines using a broad spectrum of times and concentrations of SB. The MTT assay was done to check the cytotoxic properties of different SB concentrations. Gene expression levels of TLR4 was then evaluated for non-cytotoxic SB concentrations. Morphological analysis and MTT assay confirmed that SB concentrations equal to or less than 5mM were not cytotoxic for both cell lines. At 5mM concentration of SB in SW480 cell line and 1mM concentration of SB in HCT116 cell line, TLR4 gene expression level significantly increased from 24 to 48 hrs and decreased significantly from 48 to 72 hrs with an "early increased and late decreased pattern". At 1mM concentration of SB in SW480 cell line and 5mM concentration of SB in HCT116 cell line, TLR4 expression had a "gradually increased pattern". This study focuses on the dose-time-effect of SB in the pathogenesis of colorectal cancer. SB alters the expression level of TLR4 in colorectal cancer cells. This effect may depend on the cell type, treatment duration and SB concentration. The alterations in TLR4 expression may be due to the direct effect of SB on TLR4 and/or the expression changes of in other genes which may indirectly affect the TLR4 expression. Abbreviations: TLR4: Toll-like receptor 4; HDACi: histone deacetylase inhibitor; SB: sodium Butyrate; CRC: colorectal cancer; SCFA: short-chain fatty acid; hrs: hours.

Anti-angiogenic effects of CD73-specific siRNA-loaded nanoparticles in breast cancer-bearing mice.

CD73 facilitates tumor growth by upregulation of the adenosine (immunosuppressive factor) in the tumor microenvironment, however, its precise molecular mechanisms is not precisely understood. Regarding the importance of angiogenesis in tumor development and spreading, we decided to assign the anti-angiogenic effects of CD73 suppression. We used chitosan lactate (ChLa) nanoparticles (NPs) to deliver CD73-specific small interfering RNA (siRNA) into cancer cells. Our results showed that treatment of the 4T1 cells with CD73-specific siRNA-loaded NPs led to potent inhibition of cancer cell proliferation and cell cycle arrest, in vitro. This growth arrest was correlated with downregulation of angiogenesis-related molecules including vascular endothelial growth factor (VEGF)-A, VEGF-R2, interleukin (IL)-6, and transforming growth factor (TGF)-ß. Moreover, administration of NPs loaded with CD73-siRNA into 4T1 breast cancer-bearing mice led to tumor regression and increased mice survival time accompanied with downregulation of angiogenesis (VEGF-A, VEGF-R2, VE-Cadherin, and CD31) and lymphangiogenesis (VEGF-C and LYVE-1)-related genes in the tumor site. Furthermore, the expression of angiogenesis promoting factors including IL-6, TGF-ß, signal transducer, and activator of transcription (STAT)3, hypoxia inducible factor (HIF)-1α, and cyclooxygenase (COX)2 was decreased after the CD73 suppression in mice. Moreover, analysis of leukocytes derived from the tumor samples, spleen, and regional lymph nodes showed that they had lower capability for secretion of angiogenesis promoting factors after CD73-silencing. These results indicate that suppression of tumor development by downregulation of CD73 is in part related to angiogenesis arrest. These findings imply a promising strategy for inhibiting tumor growth accompanied with suppressing the angiogenesis process.

Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model.

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Increased efficacy of a dendritic cell-based therapeutic cancer vaccine with adenosine receptor antagonist and CD73 inhibitor.

Dendritic cells are important in initiating immune responses; therefore, a range of dendritic cell-based approaches have been established to induce immune response against cancer cells. However, the presence of immunosuppressive mediators such as adenosine in the tumor microenvironment reduces the efficacy of dendritic cell-based cancer immunotherapy. In this study, we investigated whether blockade of the A2A adenosine receptor with a selective antagonist and a CD73 inhibitor may increase the efficacy of a dendritic cell-based cancer vaccine. According to the findings, this therapeutic combination reduced tumor growth, prolonged survival of tumor-bearing mice, and enhanced specific antitumor immune responses. Thus, we suggest that targeting cancer-derived adenosine improves the outcomes of dendritic cell-based cancer immunotherapy.

Study of the tumor microenvironment during breast cancer progression.

BACKGROUND: Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy. METHODS: In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4+ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7. RESULTS: The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression. CONCLUSION: We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Downregulation of CD73 in 4T1 breast cancer cells through siRNA-loaded chitosan-lactate nanoparticles.

The immunosuppressive factors in tumor microenvironment enhance tumor growth and suppress anti-tumor immune responses. Adenosine is an important immunosuppressive factor which can be secreted by both tumor and immune cells trough action of two cell surface ecto-nucleotidase molecules CD39 and CD73. Blocking the adenosine generating molecules has emerged as an effective immunotherapeutic approach for treatment of cancer. In this study, CD73-siRNA encapsulated into chitosan-lactate (ChLa) nanoparticles (NPs) was employed to suppress the expression of CD73 molecule on 4T1 breast tumor cells, in vitro. ChLa NPs were generated through ionic gelation of ChLa by tripolyphosphate (TPP). Small interfering RNA (SiRNA)-loaded NPs had about 100 nm size with a polydispersive index below 0.3 and a zeta potential about 13. Our results showed that ChLa NPs with Ch 50 kDa exhibit the best physicochemical features with the high siRNA encapsulation capacity. Synthesized NPs were able to fully bind with siRNA, protect them against serum and heparin degradation, and promote the transfection process. While the NPs exhibited low toxicity during 72 h cell culture, the transfection of Ch-plasmid expressing green fluorescent protein (pEGFP) NPs was efficient in 4T1 cells with a transfection rate of 53.6 % as detected by flow cytometry. In addition, CD73-siRNA-loaded ChLa NPs could efficiently suppress the expression of CD73 as assayed by real-time polymerase chain reaction and flow cytometry. As a conclusion, CD73-siRNA-loaded ChLa NPs may be considered as a promising therapeutic tool for cancer therapy; however, further in vivo investigations are necessary.

Low Noncytotoxic Concentrations of 5-Fluorouracil Have No Adverse Effects on Maturation and Function of Bone Marrow-Derived Dendritic Cells in vitro: A Potentially Safe Adjuvant for Dendritic Cell-Based Cancer Therapy.

BACKGROUND: Low, noncytotoxic concentrations of various chemotherapeutic drugs like 5-fluorouracil (5-FU) induce antitumor immune responses by selectively depleting tumor-induced immunosuppressive cells, and could therefore be used in combination with dendritic cell (DC) vaccines in order to enhance their immunotherapeutic efficacy. However, the likely negative influences of low, noncytotoxic doses of 5-FU on bone marrow-derived (BM)-DCs in vitro have not yet been investigated. METHODS: The effects of low, noncytotoxic concentrations of 5-FU on mouse BM-DC differentiation and maturation markers (CD11c, MHC class II and CD80) as well as antigen-presenting capacity and cytokine production (IL-12p70 and IL-10) have been assessed. RESULTS: Different low doses of 5-FU had no significant effect on the expression of DC differentiation and maturation or on costimulatory markers (p = 0.5). Furthermore, suboptimal doses of 5-FU did not affect the immunostimulatory functions of DCs such as antigen presentation (p = 0.6) and cytokine production (p = 0.9). CONCLUSIONS: These data suggest that low doses of 5-FU have no adverse effects on DC maturation and function, and the efficacy of DC-based cancer immunotherapy may be greatly enhanced by combining it with suboptimal doses of 5-FU.

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.