EpCAM-targeting CAR-T cell immunotherapy is safe and efficacious for epithelial tumors.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Late-stage cascade of oxidation reactions during the biosynthesis of oxalicine B in Penicillium oxalicum.

Oxalicine B (1) is an α-pyrone meroterpenoid with a unique bispirocyclic ring system derived from Penicillium oxalicum. The biosynthetic pathway of 15-deoxyoxalicine B (4) was preliminarily reported in Penicillium canescens, however, the genetic base and biochemical characterization of tailoring reactions for oxalicine B (1) has remained enigmatic. In this study, we characterized three oxygenases from the metabolic pathway of oxalicine B (1), including a cytochrome P450 hydroxylase OxaL, a hydroxylating Fe(II)/α-KG-dependent dioxygenase OxaK, and a multifunctional cytochrome P450 OxaB. Intriguingly, OxaK can catalyze various multicyclic intermediates or shunt products of oxalicines with impressive substrate promiscuity. OxaB was further proven via biochemical assays to have the ability to convert 15-hydroxdecaturin A (3) to 1 with a spiro-lactone core skeleton through oxidative rearrangement. We also solved the mystery of OxaL that controls C-15 hydroxylation. Chemical investigation of the wild-type strain and deletants enabled us to identify 10 metabolites including three new compounds, and the isolated compounds displayed potent anti-influenza A virus bioactivities exhibiting IC50 values in the range of 4.0-19.9 µmol/L. Our studies have allowed us to propose a late-stage biosynthetic pathway for oxalicine B (1) and create downstream derivatizations of oxalicines by employing enzymatic strategies.

Distinct functions of CAR-T cells possessing a dectin-1 intracellular signaling domain.

Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated remarkable efficacies in treating hematopoietic malignancies, but not in the solid tumors. Incorporating costimulatory signaling domains, such as ICOS or 4-1BB, can positively influence CAR-T cell functions and then the immune responses. These CAR-engineered T cells have showed their enhanced persistence and effector functions with improved antitumor activities, and provided a new approach for the treatment of solid tumors. Here, we designed novel 2nd generation CARs with a costimulatory signaling molecule, dectin-1. The impacts of dectin-1 signaling domain on CAR-T cells were evaluated in vitro and in vivo. Our data show that in vitro cytokine secretions by HER2 or CD19 specific CAR-T cells increase significantly via incorporating this dectin-1 signaling domain. Additional properties of these novel CAR-T cells are affected by this costimulatory domain. Compared with a popular reference (i.e., anti-HER2 CAR-T cells with 4-1BB), in vitro T cell functions and in vivo antitumor activity of the dectin-1 engineered CAR-T cells are similar to the 4-1BB based, and both are discrete to the mock T cells. Furthermore, we found that the CAR-T cells with dectin-1 show distinct phenotype and exhaustion marker expression. These collective results suggest that the incorporation of this new signaling domain, dectin-1, into the CARs may provide the clinical potential of the CAR-T cells through this signaling domain in treating solid tumors.

Effects of carbon nanotube-mediated Caspase3 gene silencing on cardiomyocyte apoptosis and cardiac function during early acute myocardial infarction.

RNA interference (RNAi) technology can achieve efficient and specific silencing of Caspase3 gene expression, thus providing new options for anti-apoptosis treatment. However, delivering siRNA to specific cells and tissues in the body is a significant challenge. Therefore, we aim to construct a functionalized single-walled carbon nanotube (F-CNT) bound to siRNA from Caspase3. The obtained gene transfer carrier F-CNT-siCas3 not only demonstrated a good water solubility and biocompatibility, but also had a high transfection efficiency of up to 82%, which significantly downregulated the expression level of the Caspase3 gene miRNA and protein in primary cardiomyocytes. Furthermore, it was verified by in vivo experiments that Caspase3 gene silencing had obvious protective effects on myocardial cell apoptosis, ventricular remodeling, and cardiac function in Sprague-Dawley (SD) rats after coronary artery ligation. This study may provide an important theoretical basis for the application of F-CNT in vivo siRNA gene therapy to treat cardiovascular diseases.

IL-18R-dependent and independent pathways account for IL-18-enhanced antitumor ability of CAR-T cells.

Interleukin-18 (IL-18) has been demonstrated to augment the antitumor capacity of chimeric antigen receptor-T cells (CAR-T) but the underlying mechanisms are largely unknown. Here we explored the effects and mechanisms of exogenous IL-18 on the antitumor response of CAR-T cells. IL-18 boosted the cytotoxicity of human epidermal growth factor receptor-2 (HER2)-specific CAR-T cells ex vivo and enhanced the antitumor efficacy of the CAR-T cells in immunodeficient mice, moreover, IL-18 improved the antitumor capacity of OVA-specific T cells in immunocompetent mice, indicating the universal enhancing function of IL-18 for adoptive cell therapy. To address the roles of IL-18 receptor (IL-18R) in the enhancing function, we evaluated the effects of IL-18R knockout (IL-18R-/-) condition in immunocompetent host and CAR-T cells on the IL-18-enhanced antitumor activities. Interestingly, IL-18 persisted to improve the antitumor ability of IL-18R intact CAR-T cells in IL-18R-/- mice. For IL-18R-/- CAR-T cells, however, IL-18 still holds the enhancing ability to boost the antitumor efficacy in IL-18R-/- mice, albeit the ex vivo tumor-killing ability was lower than that of IL-18R intact CAR-T cells, indicating that IL-18R-independent pathway is involved in the enhancement. Furthermore, tagged IL-18 binded to the membrane of IL-18R-/- splenic and lymph node cells and IL-18R intact and IL-18R-/- CAR-T cells showed distinct transcriptomic profiles when stimulated by IL-18. These data demonstrate that IL-18R-independent pathways contribute to functions of IL-18.

An investigation into the rapid removal of tetracycline using multilayered graphene-phase biochar derived from waste chicken feather.

This study investigated the removal of tetracycline (TC) using multilayered graphene-phase biochar (MGB) derived from waste chicken feather. MGB was produced through a two-stage carbonization and KOH-activation method. MGB was characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), Fourier transform infrared (FT-IR), Raman spectra, Zeta potential and elemental analysis. Various chemical functional groups were demonstrated on the surface of MGB. MGB was featured by a very large BET surface area of 1838m2/g. A rapid equilibrium (within 30s) and an ultrahigh removal efficiency (up to 99.65%) were obtained when MGB was used in the adsorption of TCs. The adsorption processes were temperature-dependent and the maximum adsorption capacity of MGB was 388.33mg/g at 30°C. The data of adsorption isotherms and kinetics were represented well by the Langmuir and Elovich models, respectively. The chemical monolayer adsorption could play an important role in this process. Furthermore, the adsorption of MGB was tolerant with wide pH, high ionic strength and even co-existing anions. Regeneration experiments indicated the removal efficiency was still satisfied (96.61%) even after four cycles. These results have important implications for the future application of animal waste-derived adsorbents in the treatment of wastewater containing antibiotic residues.

Combination of human Fas (CD95/Apo-1) ligand with adriamycin significantly enhances the efficacy of antitumor response.

The prognosis of hepatocellular carcinoma (HCC) is poor, even with the combined treatment of curative resection and adjuvant chemoradiotherapy. To solve this problem, many biologic therapies have been investigated. Fas ligand (FasL, CD95L) is mainly expressed in activated T lymphocytes and natural killer (NK) cells, and plays a central role in both cell-mediated immunity and immune downregulation. Several studies have shown that FasL is expressed in HCC. In the present report, we prepared recombinant human pET-22b(+)/FasL protein and investigated the effect of FasL on HCC cells in vitro and on tumor growth in a murine HCC tumor model. The well-known cytotoxic chemotherapeutic reagent adriamycin (ADM) served as a control. We found that FasL effectively suppressed the viability of H22 tumor cells and significantly induced the apoptosis of H22 cells. The apoptotic levels of cells treated with FasL-ADM were significantly higher than those treated with FasL or ADM alone, and the FasL-ADM combination resulted in a more than additive effect on tumor growth delay in this model. The results suggested that combined treatment of FasL and other chemotherapeutic agents may be a new approach to improve the efficacy of chemotherapy for HCC.

Decoy receptor 3 overexpression and immunologic tolerance in hepatocellular carcinoma (HCC) development.

The recently identified decoy receptor 3 (DcR3) inhibits FasL-induced apoptosis by binding to FasL, and it is considered to play a key role in the immune escape system of neoplastic cells. In order to examine the involvement of DcR3 in the immunologic tolerance of hepatocellular carcinoma (HCC), we investigated the amplification and expression of DcR3, FasL, and Fas in an HCC mice model using RT-PCR, western blotting, and ELISA, and analyzed the space-time relationship with various cytokines including the forkhead transcription factor forkhead/winged helix transcription factor gene (Foxp3), CTLA-4, TGF-beta, IL-10, TNF-alpha, and IFN-gamma. The RT-PCR results revealed that Fas expression preceded that of DcR3 during the early phases of tumorigenesis. Thereafter, the expression of DcR3 was up-regulated; however, the expression of Fas was down-regulated and eventually ceased. DcR3 and FasL were expressed and amplified simultaneously in muscle tumor. CTLA-4 expression was earlier than Foxp3, and both CTLA-4 and Foxp3 amplification and expression were consistent with that of DcR3. The results suggest that the elevated levels of DcR3, Foxp3, and CTLA-4 in tissue were positively correlated with tumor growth. The partial tumor immunoregulation inclined to negative modulation, and DcR3 may play an important role in inducing immunologic tolerance.

Effect of isoverbascoside, a phenylpropanoid glycoside antioxidant, on proliferation and differentiation of human gastric cancer cell.

AIM: To investigate the effects of phenylpropanoid glycoside antioxidant isoverbascoside on cell proliferation and differentiation of human gastric cancer cell line MGC 803. METHODS: MGC 803 cells were treated with isoverbascoside. Its effects on cell proliferation, tumorigenicity, enzymatic activities, cell cycles, and gene expression were respectively evaluated with cell counting, tumor formation assay, enzymatic assay, flow cytometer analysis, and Western blotting, with Me2SO as positive control. RESULTS: Isoverbascoside could markedly inhibit cell proliferation in dose- and time-dependent manner. Isoverbascoside 20 micromol/L strikingly suppressed cell tumorigenicity, activities of alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), and caused G0/G1 arrest. The expression of G1 S checkpoint related proteins, p53, p21/WAF1, and p16/INK4, were up-regulated after MGC 803 cells were treated with isoverbascoside 20 micromol/L for 4-8 h. Contrarily, the expression of C-myc protein was suppressed after 8 h treatment. CONCLUSION: Isoverbascoside inhibited cell proliferation, reversed cell malignant phenotypic characteristics, and consequently caused differentiation in MGC 803 cells. These effects might be associated with its activities of causing G0/G1 arrest and regulating the expression of cell cycle related proteins.

Sequencing of p53 mutation in established human hepatocellular carcinoma cell line of HHC4 and HHC15 in nude mice.

AIM:To set up cell lines of human hepatocellular carcinoma in nude mice for the research of cell biology and gene therapy.METHODS:Xenotransplantation of human hepatoma into nude mice was carried out and the growth rate, histopathology and immunology of the nude mice were studied. The DNA from xenografts were analyzed by HBV gen and PCR amplification of a fragment of p53 gene exon 7, which were identified by dot blot hybridization, restriction fragments length polymorphism and DNA sequencing.RESULTS:HHC4 and HHC15 cell lines could be successively transplanted in nude mice and the population doubling time was 7 and 5 days respectively. These strains retained the original characteristics of histopathology, secreting AFP and heteroploid karyotypes in human hepatocellular carcinoma. The fragment of HBV gene was detected in the genomic DNA of both hHCC4 and hHCC15, however only HHC4 secreted HBsAg.The mutation at 250 code (C A) and 249 code (G T) were detected respectively in the genomic DNA of HHC4 and HHC15.CONCLUSION:The two cell lines are useful material for the studying of cell biology and gene therapy in human hepatocellular carcinoma and provide molecular biological trace of relationship between high mortality of hepatoma and AFB1 severe pollution of the daily common foods in this district.