Pharmacologic Control of CAR T Cells

Chimeric antigen receptor (CAR) therapy is a promising modality for the treatment of advanced cancers that are otherwise incurable. During the last decade, different centers worldwide have tested the anti-CD19 CAR T cells and shown clinical benefits in the treatment of B cell tumors. However, despite these encouraging results, CAR treatment has also been found to lead to serious side effects and capricious response profiles in patients. In addition, the CD19 CAR success has been difficult to reproduce for other types of malignancy. The appearance of resistant tumor variants, the lack of antigen specificity, and the occurrence of severe adverse effects due to over-stimulation of the therapeutic cells have been identified as the major impediments. This has motivated a growing interest in developing strategies to overcome these hurdles through CAR control. Among them, the combination of small molecules and approved drugs with CAR T cells has been investigated. These have been exploited to induce a synergistic anti-cancer effect but also to control the presence of the CAR T cells or tune the therapeutic activity. In the present review, we discuss opportunistic and rational approaches involving drugs featuring anti-cancer efficacy and CAR-adjustable effect.     

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1β secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1β in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.     

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.     

Synthesis and molecular structure of the sodium arsanediyl zincate [(thf)Na]2[(EtZn)6(AsSiiPr3)4]

The reaction of NaAs(H)SiiPr₃ with ZnEt₂ in the presence of additional triisopropylsilylarsane yields the heterobimetallic complex [(thf)Na]₂ [(EtZn)₆(AsSiiPr₃)₄] (1) in the shape of red prisms. The Na–As and Zn–As bond lengths show values of 301/309 pm and between 247 and 260 pm, respectively.     

Activation Markers on B and T Cells and Immune Checkpoints in Autoimmune Rheumatic Diseases

In addition to identifying the major B- and T-cell subpopulations involved in autoimmune rheumatic diseases (ARDs), in recent years special attention has been paid to studying the expression of their activation markers and immune checkpoints (ICPs). The activation markers on B and T cells are a consequence of the immune response, and these molecules are considered as sensitive specific markers of ARD activity and as promising targets for immunotherapy. ICPs regulate the activation of the immune response by preventing the initiation of autoimmune processes, and they modulate it by reducing immune cell-induced organ and tissue damage. The article considers the possible correlation of ICPs with the activity of ARDs, the efficacy of specific ARD treatments, and the prospects for the use of activation molecules and activation/blocking ICPs for the treatment of ARDs.     

Crystallization of Lead Niobate Glass by Mechanical Activation

Mechanical activation-triggered crystallization in PbNb₂O₆-based glass was dependent on the initial presence of nuclei. The crystallization cannot be initiated by mechanical activation in a highly amorphous glass composition quenched from 1350°C where PbNb₂O₆ nuclei did not exist. The steady growth of nanocrystallites of PbNb₂O₆ was observed with an increasing degree of mechanical activation in the glass quenched from 1300°C, where a density of PbNb₂O₆ nuclei existed before mechanical activation. The inability to nucleate in the highly amorphous oxide glass by mechanical activation is consistent with the much higher structural stability as compared with that of metallic glasses, such as Fe-Si-B. The mechanical activation-grown PbNb₂O₆ nanocrystals were 10–15 nm in size as observed using HRTEM and their crystallinities were further improved by thermal aging at an elevated temperature in the range of 550° to 650°C.     

Properties of Cd-Based Binary Quasicrystals and Their 1/1 Approximants

Electronic and structural properties of Cd-based binary quasicrystals (QCs) and their 1/1 approximants (APs) without chemical disorder are overviewed in terms of electronic states, electronic transport, and phase transitions. A pseudogap in the electronic density of states (DOS) as well as hybridization effect between sp and d states are discussed for both the QCs and their 1/1 APs in comparison with theoretical calculations. In contrast with the cases of ternary stable QCs, the resistivity of the Cd_5.7Yb QC exhibits a positive temperature coefficient at low temperatures, which is regarded as an indication of an intrinsic metallic nature of the QC. A unique structural phase transition has been observed at low temperatures (LT) for a series of 1/1 Cd₆M APs, which is interpreted as an orientational transition of a tetrahedron residing at the center of the Tsai-type icosahedral cluster. In most of the cases, the LT superstructure is understood as an “antiferromagnetic” ordering of the tetrahedra along a <110> direction of the high-temperature bcc phase. The transition is classified as a non-diffusive order–disorder transition, which has not been observed in metallic alloys before.     

Identification of the FDA-Approved Drug Pyrvinium as a Small-Molecule Inhibitor of the PD-1/PD-L1 Interaction

Immune checkpoint blockade involving inhibition of the PD-1/PD-L1 interaction has provided unprecedented clinical benefits in treating a variety of tumors. To date, a total of six antibodies that bind to either PD-1 or PD-L1 protein and in turn inhibit the PD-1/PD-L1 interaction have received clinical approvals. Despite being highly effective, these expensive large biotherapeutics possess several inherent pharmacokinetic limitations that can be successfully overcome through the use of low-molecular-weight inhibitors. One such promising approach involves small-molecule induced dimerization and sequestration of PD-L1, leading to effective PD-1/PD-L1 inhibition. Herein, we present the discovery of such potential bioactive PD-L1 dimerizers through a structure- and ligand-based screening of a focused library of approved and investigational drugs worldwide. Pyrvinium, an FDA-approved anthelmintic drug, showed the highest activity in our study with IC₅₀ value of ∼29.66 μM. It is noteworthy that Pyrvinium, being an approved drug, may prove especially suitable as a good starting point for further medicinal chemistry efforts, leading to design and development of even more potent structural analogs as selective PD-1/PD-L1 inhibitors. Furthermore, the adopted integrated virtual screening protocol may prove useful in screening other larger databases of lead- and drug-like molecules for hit identification in the domain of small-molecule PD-1/PD-L1 inhibitors.     

稀土冠醚络合物的研究与进展

本文描述了稀土冠醚络合物的性质及应用，报道了稀土冠醚络合物的研究进展及前景，就稀土离子选择何种冠醚为配体最稳定及其影响因素做了介绍，最后对其荧光性及其应用给予了概述。     

检测硝基芳烃化合物的荧光聚合物薄膜的研究

介绍了爆炸物的检测技术和痕量检测技术,综述了荧光聚合物检测TNT的原理,以及检测硝基芳烃化合物荧光聚合物传感器的研究进展,对其发展方向和应用前景进行了展望。     

Preclinical Evaluation of CAR T Cell Function: In Vitro and In Vivo Models

Immunotherapy using chimeric antigen receptor (CAR) T cells is a rapidly emerging modality that engineers T cells to redirect tumor-specific cytotoxicity. CAR T cells have been well characterized for their efficacy against B cell malignancies, and rigorously studied in other types of tumors. Preclinical evaluation of CAR T cell function, including direct tumor killing, cytokine production, and memory responses, is crucial to the development and optimization of CAR T cell therapies. Such comprehensive examinations are usually performed in different types of models. Model establishment should focus on key challenges in the clinical setting and the capability to generate reliable data to indicate CAR T cell therapeutic potency in the clinic. Further, modeling the interaction between CAR T cells and tumor microenvironment provides additional insight for the future endeavors to enhance efficacy, especially against solid tumors. This review will summarize both in vitro and in vivo models for CAR T cell functional evaluation, including how they have evolved with the needs of CAR T cell research, the information they can provide for preclinical assessment of CAR T cell products, and recent technology advances to test CAR T cells in more clinically relevant models.     

Photocaged T7 RNA Polymerase for the Light Activation of Transcription and Gene Function in Pro‐ and Eukaryotic Cells

A light‐activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site‐specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho‐nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro‐ and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.     

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.     

Mitochondrial Glycerol-3-Phosphate Acyltransferase-Dependent Phospholipid Synthesis Modulates Phospholipid Mass and IL-2 Production in Jurkat T Cells

Changes in glycerophospholipid metabolism with age and disease can have a profound effect on immune cell activation and effector function. We previously demonstrated that glycerol‐3‐phosphate acyltransferase‐1, the first and rate limiting step in de novo glycerophospholipid synthesis, plays a role in modulating murine T cell function. The resultant phenotype is characterized by decreased IL‐2 production, increased propensity toward apoptosis, and altered membrane glycerophospholipid mass similar to that of an aged T cell. Since T cells in previous experiments were harvested from GPAT‐1^?/? mice, questions remained as to what extent the macro environment of the model influenced the observed cellular phenotype. Therefore, we generated and phenotypically characterized a mitochondrial glycerol‐3‐phosphate acyltransferase (GPAM) deficient Jurkat T cell. Furthermore, this line was used to probe possible mechanisms by which GPAT‐1/GPAM regulates T cell function. We report here that many of the key dysfunctional characteristics of murine GPAT‐1^?/? T cells are recapitulated in the GPAMKD Jurkat T cell. We found striking decreased IL‐2 production along with altered phospholipid mass and increased incidence of apoptosis. Since PtdOH is an indirect downstream product of GPAM, we attempted to rescue IL‐2 production with PtdOH supplementation; however, this addition did not return IL‐2 production to normal levels. Interestingly, we did find significantly decreased Zap‐70 phosphorylation following stimulation, suggesting that GPAM deficiency may alter membrane based stimulatory signaling. These data show for the first time that GPAM deficiency results in an inherent defect in Jurkat T cell function and glycerophospholipid composition and that this defect cannot be rescued by addition of exogenous PtdOH.     

Reactive Oxygen Species: Modulators of Phenotypic Switch of Vascular Smooth Muscle Cells

Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism in the cell. At physiological levels, they play a vital role in cell signaling. However, high ROS levels cause oxidative stress, which is implicated in cardiovascular diseases (CVD) such as atherosclerosis, hypertension, and restenosis after angioplasty. Despite the great amount of research conducted to identify the role of ROS in CVD, the image is still far from being complete. A common event in CVD pathophysiology is the switch of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. Interestingly, oxidative stress is a major contributor to this phenotypic switch. In this review, we focus on the effect of ROS on the hallmarks of VSMC phenotypic switch, particularly proliferation and migration. In addition, we speculate on the underlying molecular mechanisms of these cellular events. Along these lines, the impact of ROS on the expression of contractile markers of VSMCs is discussed in depth. We conclude by commenting on the efficiency of antioxidants as CVD therapies.     

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide

Naїve CD4⁺ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4⁺CD45RA⁺ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFβ mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC₂ receptor increased significantly with respect to the VPAC₁ receptor from day 4 of CD4⁺CD45RA⁺ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4⁺CD45RA⁺ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.     

Cell Therapy for Colorectal Cancer: The Promise of Chimeric Antigen Receptor (CAR)-T Cells

Colorectal cancer (CRC) is a global public health problem as it is the third most prevalent and the second most lethal cancer worldwide. Major efforts are underway to understand its molecular pathways as well as to define the tumour-associated antigens (TAAs) and tumour-specific antigens (TSAs) or neoantigens, in order to develop an effective treatment. Cell therapies are currently gaining importance, and more specifically chimeric antigen receptor (CAR)-T cell therapy, in which genetically modified T cells are redirected against the tumour antigen of interest. This immunotherapy has emerged as one of the most promising advances in cancer treatment, having successfully demonstrated its efficacy in haematological malignancies. However, in solid tumours, such as colon cancer, it is proving difficult to achieve the same results due to the shortage of TSAs, on-target off-tumour effects, low CAR-T cell infiltration and the immunosuppressive microenvironment. To address these challenges in CRC, new approaches are proposed, including combined therapies, the regional administration of CAR-T cells and more complex CAR structures, among others. This review comprehensively summarises the current landscape of CAR-T cell therapy in CRC from the potential tumour targets to the preclinical studies and clinical trials, as well as the limitations and future perspectives of this novel antitumour strategy.