Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients.

Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases.

Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function.

Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions after transplantation and during autoimmune disease. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. It is therefore critical to scrutinize the molecular changes which occur during T cell product generation, and reexamine current manufacturing practices. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product that has proven safety and hints of therapeutic efficacy in kidney transplant patients. We found progressive DNA methylation changes over the duration of culture, which were donor-independent and reproducible between manufacturing runs. Differentially methylated regions (DMRs) in the final products were significantly enriched at promoters and enhancers of genes implicated in T cell activation. Additionally, significant hypomethylation did also occur in promoters of genes implicated in functional exhaustion in conventional T cells, some of which, however, have been reported to strengthen immunosuppressive effector function in Tregs. At the same time, a set of reported Treg-specific demethylated regions increased methylation levels with culture, indicating a possible destabilization of Treg identity during manufacturing, which was independent of the purity of the starting material. Together, our results indicate that the repetitive TCR-mediated stimulation lead to epigenetic changes that might impact functionality of Treg products in multiple ways, by possibly shifting to an effector Treg phenotype with enhanced functional activity or by risking destabilization of Treg identity and impaired TCR activation. Our analyses also illustrate the value of epigenetic profiling for the evaluation of T cell product manufacturing pipelines, which might open new avenues for the improvement of current adoptive Treg therapies with relevance for conventional effector T cell products.

Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo.

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.

CRISPR-Cas9-Edited Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in Transplant Recipients.

Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.

IL-6/IL-6R pathway is a therapeutic target in chemoresistant ovarian cancer.

INTRODUCTION:: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy worldwide and despite an initial response to therapeutic agents, the majority of patients have chemoresistant disease. There is no treatment strategy with proven efficacy against chemoresistant EOC and in this setting, overcoming therapy resistance is the key to successful treatment. METHODS:: This study aimed to investigate expression of interleukin-6 (IL-6) (IL-6) and IL-6 receptor (IL-6R) in a panel of the EOC cell lines. To achieve this, the expression of IL-6 and its receptor were compared in the EOC cells using quantitative reverse transcription polymerase chain reaction. MTT assay was performed to obtain chemosensitivity of the EOC cells. RESULTS:: In this report, we show that expressions of IL6 and IL6R are higher in therapy-resistant EOC cells compared to sensitive ones. Higher expression of IL6 and its receptor correlated with resistance to certain chemotherapeutic agents. Moreover, our findings showed that combination of tocilizumab (Actemra; Roche), an anti-IL-6R monoclonal antibody, with carboplatin synergistically inhibited growth and proliferation of the EOC cells and the most direct axis for IL-6 gene expression was NF-κB pathway. CONCLUSION:: Collectively, our findings suggest that blockade of the IL-6 signaling pathway with anti-IL-6 receptor antibody tocilizumab might resensitize the chemoresistant cells to the current chemotherapeutics.

Blockade of nuclear factor-κB (NF-κB) pathway inhibits growth and induces apoptosis in chemoresistant ovarian carcinoma cells.

Epithelial ovarian cancer (EOC) has exhibited marginal improvement in survival rate, despite advances in surgical debulking and chemotherapy regimens. Although the majority of EOC patients achieve a clinical remission after induction therapy, over 80% relapse and succumb to chemoresistant disease. In this regard, it is of paramount importance to elucidate molecular mechanisms and signaling pathways which promote therapy resistance in EOC in order to devise novel and more effective treatment strategies. In this study, we showed that activation of nuclear factor-κB (NF-κB) is significantly higher in therapy-resistant EOC cells compared to chemosensitive counterparts, which was positively associated with resistance to cisplatin, carboplatin, paclitaxel and erlotinib. Bay 11-7082, a highly selective NF-κB inhibitor, reduced cell proliferation, clonogenicity and anoikis resistance in the therapy-resistant EOC cells and induced apoptotic cell death. Moreover, Bay 11-7082 decreased the expression of pro-survival, inflammatory and metastatic genes and synergistically increased anti-proliferative efficacy of cisplatin, carboplatin, paclitaxel and erlotinib. Altogether, these findings suggest that NF-κB is an attractive therapeutic target in EOC to be exploited in translational oncology and Bay 11-7082 is a potential anti-cancer drug to overcome chemoresistance and inhibit proliferation of the EOC cells.

Thalidomide attenuates development of morphine dependence in mice by inhibiting PI3K/Akt and nitric oxide signaling pathways.

Morphine dependence and the subsequent withdrawal syndrome restrict its clinical use in management of chronic pain. The precise mechanism for the development of dependence is still elusive. Thalidomide is a glutamic acid derivative, recently has been reconsidered for its clinical use due to elucidation of different clinical effects. Phosphoinositide 3-kinase (PI3K) is an intracellular transducer enzyme which activates Akt which in turns increases the level of nitric oxide. It is well established that elevated levels of nitric oxide has a pivotal role in the development of morphine dependence. In the present study, we aimed to explore the effect of thalidomide on the development of morphine dependence targeting PI3K/Akt (PKB) and nitric oxide (NO) pathways. Male NMRI mice and human glioblastoma T98G cell line were used to study the effect of thalidomide on morphine dependence. In both models the consequent effect of thalidomide on PI3K/Akt and/or NO signaling in morphine dependence was determined. Thalidomide alone or in combination with PI3K inhibitor, Akt inhibitor or nitric oxide synthase (NOS) inhibitors significantly reduced naloxone induced withdrawal signs in morphine dependent mice. Also, the levels of nitrite in hippocampus of morphine dependent mice were significantly reduced by thalidomide in compared to vehicle treated morphine dependent mice. In T98G human glioblastoma cells, thalidomide alone or in combination with PI3K and Akt inhibitors significantly reduced iNOS expression in comparison to the morphine treated cells. Also, morphine-induced p-Akt was suppressed when T98G cells were pretreated with thalidomide. Our results suggest that morphine induces Akt, which has a crucial role in the induction of NOS activity, leading to morphine dependence. Moreover, these data indicate that thalidomide attenuates the development of morphine dependence in vivo and in vitro by inhibition of PI3K/Akt and nitric oxide signaling pathways.

Pharmacological evidence for the involvement of adenosine triphosphate sensitive potassium channels in chloroquine-induced itch in mice.

BACKGROUND: Chloroquine (CQ) evokes itch in human and scratching behavior in rodents through a histamine-independent pathway. Chloroquine directly excites peripheral sensory neurons which convey itch signals to the central nervous system. It has been revealed that ATP-sensitive potassium channels (KATP channels) are important in regulating neuronal excitability. Thus, we aimed to investigate the involvement of KATP channels in CQ-induced itch which may also reveal a linkage between metabolic state of cells and itch. METHODS: Intradermal (id) injection of CQ at dose of 400µg/site induces the scratching behavior. KATP channel openers, diazoxide (DZX) and minoxidil (MIN), and a KATP channel blocker, glibenclamide (GLI), were administered intraperitoneally (ip) before CQ. Then the behavior was recorded for 30min, in an unmanned condition, and the scratching bouts were counted by an expert observer who was blinded to the experiments. Furthermore, quantitative reverse transcription-PCR (qRT-PCR) was used to investigate the possible changes in dermal expression of Kcnj8 and Kcnj11, the genes encoding the KATP channels. RESULTS: Our results show that either DZX (10mg/kg, ip) or MIN (10mg/kg, ip) significantly attenuated CQ-induced scratching behavior in mice. Moreover, pretreatment with GLI (3mg/kg, ip) significantly reversed the anti-pruritic effects of DZX and MIN. Our finding of qRT-PCR analysis also show that the expression of Kcnj8 is decreased after CQ injection. CONCLUSIONS: We suggest that KATP channels are possibly involved in CQ-induced itch. While, further studies will be significant to better elucidate the association of metabolic state of cells and itch.

Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells.

Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy worldwide. Development of chemoresistance and peritoneal dissemination of EOC cells are the major reasons for low survival rate. Targeting signal transduction pathways which promote therapy resistance and metastatic dissemination is the key to successful treatment. Members of the ErbB family of receptors are over-expressed in EOC and play key roles in chemoresistance and invasiveness. Despite this, single-targeted ErbB inhibitors have demonstrated limited activity in chemoresistant EOC. In this report, we show that dacomitinib, a pan-ErbB receptor inhibitor, diminished growth, clonogenic potential, anoikis resistance and induced apoptotic cell death in therapy-resistant EOC cells. Dacominitib inhibited PLK1-FOXM1 signalling pathway and its down-stream targets Aurora kinase B and survivin. Moreover, dacomitinib attenuated migration and invasion of the EOC cells and reduced expression of epithelial-to-mesenchymal transition (EMT) markers ZEB1, ZEB2 and CDH2 (which encodes N-cadherin). Conversely, the anti-tumour activity of single-targeted ErbB agents including cetuximab (a ligand-blocking anti-EGFR mAb), transtuzumab (anti-HER2 mAb), H3.105.5 (anti-HER3 mAb) and erlotinib (EGFR small-molecule tyrosine kinase inhibitor) were marginal. Our results provide a rationale for further investigation on the therapeutic potential of dacomitinib in treatment of the chemoresistant EOC.

Anti-tumour activity of tivozanib, a pan-inhibitor of VEGF receptors, in therapy-resistant ovarian carcinoma cells.

Epithelial ovarian cancer (EOC) is the most fatal gynaecological malignancy. Despite initial therapeutic response, the majority of advanced-stage patients relapse and succumb to chemoresistant disease. Overcoming drug resistance is the key to successful treatment of EOC. Members of vascular endothelial growth factor (VEGF) family are overexpressed in EOC and play key roles in its malignant progression though their contribution in development of the chemoresistant disease remains elusive. Here we show that expression of the VEGF family is higher in therapy-resistant EOC cells compared to sensitive ones. Overexpression of VEGFR2 correlated with resistance to cisplatin and combination with VEGFR2-inhibitor apatinib synergistically increased cisplatin sensitivity. Tivozanib, a pan-inhibitor of VEGF receptors, reduced proliferation of the chemoresistant EOC cells through induction of G2/M cell cycle arrest and apoptotic cell death. Tivozanib decreased invasive potential of these cells, concomitant with reduction of intercellular adhesion molecule-1 (ICAM-1) and diminishing the enzymatic activity of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-2 (MMP-2). Moreover, tivozanib synergistically enhanced anti-tumour effects of EGFR-directed therapies including erlotinib. These findings suggest that the VEGF pathway has potential as a therapeutic target in therapy-resistant EOC and VEGFR blockade by tivozanib may yield stronger anti-tumour efficacy and circumvent resistance to EGFR-directed therapies.

Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells.

Glioblastoma (GBM) remains one of the most fatal human malignancies due to its high angiogenic and infiltrative capacities. Even with optimal therapy including surgery, radiotherapy and temozolomide, it is essentially incurable. GBM is among the most neovascularised neoplasms and its malignant progression associates with striking neovascularisation, evidenced by vasoproliferation and endothelial cell hyperplasia. Targeting the pro-angiogenic pathways is therefore a promising anti-glioma strategy. Here we show that tivozanib, a pan-inhibitor of vascular endothelial growth factor (VEGF) receptors, inhibited proliferation of GBM cells through a G2/M cell cycle arrest via inhibition of polo-like kinase 1 (PLK1) signalling pathway and down-modulation of Aurora kinases A and B, cyclin B1 and CDC25C. Moreover, tivozanib decreased adhesive potential of these cells through reduction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Tivozanib diminished GBM cell invasion through impairing the proteolytic cascade of cathepsin B/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase-2 (MMP-2). Combination of tivozanib with EGFR small molecule inhibitor gefitinib synergistically increased sensitivity to gefitinib. Altogether, these findings suggest that VEGFR blockade by tivozanib has potential anti-glioma effects in vitro. Further in vivo studies are warranted to explore the anti-tumour activity of tivozanib in combinatorial approaches in GBM.

The inhibitory effect of disulfiram encapsulated PLGA NPs on tumor growth: Different administration routes.

The strong anticancer activity of disulfiram is hindered by its rapid degradation in blood system. A novel folate-receptor-targeted poly (lactide-co-glycolide) (PLGA)-polyethylene glycol (PEG) nanoparticle (NP) is developed for encapsulation and delivery of disulfiram into breast cancer tumor using passive (EPR effect) and active (folate receptor) targeting. The anticancer activity of disulfiram and its effect on caspase-3 activity and cell cycle are studied. The administration of encapsulated PLGA NPs using intra-peritoneal, intravenous and intra-tumor routes is investigated using animal model. Disulfiram shows strong cytotoxicity against MCF7 cell line. The activity of caspase-3 inhibited with disulfiram via dose dependent manner while the drug causes cell cycle arrest in G0/G1 and S phase time-dependently. The encapsulated disulfiram shows higher activity in apoptosis induction as compared to free drug. In nontoxic dose of encapsulated disulfiram, the highest and lowest efficacy of NPs in tumor growth inhibition is observed for intravenous injection and intraperitoneal injection. It is suggested that administration of disulfiram by targeted PLGA nanoparticles using intravenous injection would present an alternative therapeutic approach for solid tumor treatment.

NMDA receptor antagonists attenuate the proconvulsant effect of juvenile social isolation in male mice.

Experiencing psychosocial stress in early life, such as social isolation stress (SIS), is known to have negative enduring effects on the development of the brain and behavior. In addition to anxiety and depressive-like behaviors, we previously showed that juvenile SIS increases susceptibility to pentylenetetrazole (PTZ)-induced seizures in mice through enhancing the nitrergic system activity in the hippocampus. In this study, we investigated the possible involvement of N-methyl-D-aspartate (NMDA) receptors in proconvulsant effects of juvenile SIS. Applying 4 weeks of SIS to juvenile male mice at postnatal day 21-23, we observed an increased susceptibility to PTZ as well as anxiety and depressive-like behaviors in adult mice. Intraperitoneal (i.p.) administration of NMDA receptor antagonists, MK-801 (0.05 mg/kg) and ketamine (0.5mg/kg), reversed the proconvulsant effects of SIS in Isolated (and not social) housed animals. Co-administration of non-effective doses of nitric oxide synthase (NOS) inhibitors, 7NI (25mg/kg) and L-NAME (10mg/kg), with NMDA receptor antagonists, MK-801 (0.01 mg/kg) and ketamine (0.1mg/kg) attenuated the proconvulsant effects of juvenile SIS only in isolated housed mice. Also, using real time RT-PCR, we showed that hippocampal upregulation of NR2B subunit of NMDA receptor may play a critical role in proconvulsant effects of juvenile SIS by dysregulation of NMDA/NO pathway. In conclusion, results of present study revealed that experiencing SIS during adolescence predisposes the co-occurrence of seizure disorders with psychiatric comorbidities and also, alteration of NMDA receptor structure and function in hippocampus plays a role in proconvulsant effects of juvenile SIS through enhancing the NMDA/NO pathway.

Effects of silibinin on growth and invasive properties of human ovarian carcinoma cells through suppression of heregulin/HER3 pathway.

Epithelial ovarian cancer (EOC) is the most fatal gynecological malignancy due to its high proliferative and invasive capacities. A heregulin (HRG)/HER3 autocrine loop increases proliferative and metastatic properties of EOC cells, suggesting that modulators of this signaling pathway may prove effective to trammel growth and motility of these cells. This study aimed to evaluate the effects of multi-tyrosine kinase inhibitor silibinin on proliferative and invasive characteristics of EOC cell lines OVCAR8 and SKOV3 through suppression of the HRG/HER3 pathway. To achieve this, the effects of silibinin on proliferation, DNA synthesis, clonogenicity, cell cycle progression, cathepsin B enzymatic activity, and migration and invasion were explored in vitro. Silibinin suppressed proliferation, DNA synthesis, and clonogenic abilities of OVCAR8 and SKOV3 cells through inhibition of the autocrine HRG/HER3 circuit. Silibinin-mediated attenuation of the HER3 signaling disabled the HER3/AKT/survivin axis and thereby, induced G1/S cell cycle arrest. Furthermore, silibinin reduced invasive potentials of the EOC cells through quelling the HRG/HER3 pathway and suppression of cathepsin B activity. Altogether, these results suggest that silibinin is a potential anti-cancer drug to inhibit proliferative and invasive characteristics of the EOC cells that exhibit an autocrine HRG/HER3 pathway.